The smooth identification and low-cost production of highly specific agents that interfere with signaling cascades by targeting an active domain in surface receptors, cytoplasmic and nuclear effector proteins, remain important challenges in biomedical research. We propose that peptide aptamers can provide a very useful and new alternative for interfering with protein–protein interactions in intracellular signal transduction cascades, including those emanating from activated receptors for growth factors. By their targeting of short, linear motif type of interactions, peptide aptamers have joined nucleic acid aptamers for use in signaling studies because of their ease of production, their stability, their high specificity and affinity for individual target proteins, and their use in high-throughput screening protocols. Furthermore, they are entering clinical trials for treatment of several complex, pathological conditions. Here, we present a brief survey of the use of aptamers in signaling pathways, in particular of polypeptide growth factors, starting with the published as well as potential applications of aptamers targeting Epidermal Growth Factor Receptor signaling. We then discuss the opportunities for using aptamers in other complex pathways, including Wnt/β-catenin, and focus on Transforming Growth Factor-β/Smad family signaling. This structural feature depends on the primary sequence and its length  and even the experimental conditions  [2+) [Nucleic acid aptamers are short, single-stranded RNAs and DNAs that represent high-affinity and highly selective ligands for different targets, ranging from large proteins to peptides, nucleotides, drugs and small compounds, and even metal ions. Such aptamers can be isolated from combinatorial libraries by SELEX  . The SELactions) ,8. The sns) [2+)  and smalns) [2+) . SELEX wns) [2+) . In a sins) [2+) .Peptide aptamers are combinatorial proteins consisting of a variable peptide inserted into a scaffold protein . ConceptN- and C-termini are fused to the scaffold. In general, constrained peptides possess several advantages compared to flexible peptides with free ends. First, even in the unbound state, the constrained peptide insert usually has higher affinity for its target than its flexible counterpart. For example, within the scaffold, the affinities of the variable peptide moieties to their target proteins decreased from 10 to 1000-fold when they were taken out from their scaffold and tested as such [In addition, peptide aptamers are dually constrained because their  as such . Second, as such , which i as such .E. coli (Perhaps the most popular scaffold protein for displaying constrained peptides is Thioredoxin-A (TrxA) of E. coli . It has Alternative scaffold proteins see  include in vitro, the selected positive aptamer R5G42 was subsequently used as bait in a yeast two-hybrid screening and identified Calcineurin, known to activate lymphocytes and is, in fact, a protein phosphatase, i.e., of Tau, linked to Alzheimer’s disease, and of BAD, a member of the Bcl2 family of apoptosis regulator factors, where BAD–Bcl2 binding has been documented. R5G42 binds to and activates the Calcineurin phosphatase activity and hence provokes dephosphorylation of BAD, and even can re-establish the effects on proliferation in cultured cells treated with inhibitors of Calcineurin [The action mechanisms underlying bioactive peptide aptamers (from here we use “peptide aptamer” as the combination of the scaffold protein with its peptide insert) have been investigated in different studies. In most of these, peptide aptamers have been shown or tested for inhibiting protein–protein interaction ,19,28,29cineurin .Collectively, these studies illustrate that aptamer-induced perturbations can resemble those caused by small molecules, including some small molecules that are already used in therapy. These results make peptide aptamers useful tools in discovery of small molecule drugs, due to the ability of aptamers to bind to various polymorphic protein surfaces. When the binding site of peptide aptamers is mapped on their target protein, structural studies can subsequently be performed on these surfaces. In most cases, the latter will turn out “druggable,” meaning that they are accessible to small molecules and two complementary approaches can then be followed. In the first approach, a virtual screening for target-based pharmacological designs can be performed against these surfaces. In the second approach, the aptamer can be used in high-throughput screening protocols to identify small molecules that disrupt the interaction between target protein and peptide aptamer .http://clinicaltrials.gov/ct2/results?term=aptamer; see In recent years aptamers have also moved from basic research (see section 1) to the clinic. Concerning their use in therapy, a major breakthrough was the approval for using Pegaptanib/Macugen©, a RNA aptamer directed against VEGF165 . This aAmongst these, of particular interest are the ones using nucleic aptamers that (i) target platelet-derived growth factor (PDGF) for inducing neovascular regression in AMD (E10030 aptamer); (ii) modulate hemostasis, via targeting selected factors of the coagulation pathway, thus making such aptamers new anti-coagulants for e.g., post-surgery interventions  or for treatment of hemophilia (aptamer ARC19499); (iii) provide a treatment for diabetes mellitus (aptamer NOX-E36); and (iv) specifically target tumor cells, as in the case of the AS1411 aptamer, which binds to nucleolin, a protein overproduced by cancer cells, and that plays a role in promoting cell survival and proliferation. AS1411 is currently in an advanced Phase 2 stage for treatment of acute myeloid leukemia (AML) see , but alsper se but sometimes require chemical modifications for increasing their resistance to endonucleases, increasing their half-life, as well as controlling their renal clearance .versus repeated doses. Toxicological and pharmacological information has been reported on antisense oligonucleotides (ASOs). The conclusions are, in the latter case, that three main effects should be carefully considered when designing ASOs or other oligonucleotides for drug purposes, i.e., the polyanion effect, the tissue accumulation of the oligonucleotides, and their stimulation of innate immunity, respectively , using single iew, see ). The poi.e., epidermal growth factor (EGF) and Wnt signaling. Thereafter, we will focus on TGFβ family signaling.Advances in aptamer chemistry and optimized ways of delivery of aptamers into cells cleared the way to use peptide aptamers as candidate therapeutic molecules against intracellular targets, including effector proteins of growth factor receptor initiated signaling. We have selected examples of the application of aptamers in two intensively studied signaling cascades relevant to embryogenesis, as well as cancers of different histo-pathological classes, HER2/ERBB2) are often associated with a poor prognosis in cancer patients. ErbB2 is considered a positive regulator (via amplifying and prolonging) of signaling, partly based on its deviant biochemistry, e.g., binding a larger set of phospho-Tyr binding proteins than the other ErbB receptors, and its over-activity directly correlates with poor prognosis. Activating mutations in the receptor kinase domain can also result in ligand-independent signaling.In EGF signaling, peptide aptamers have been selected based on their ability to bind to the intracytoplasmic domain of the EGF receptor (EGFR) or to the downstream TF Stat3. Sustained activation of EGF family receptors  is a coLigand binding to the ectodomain of the receptor chains induces a conformational change in the receptor within the receptor complex, resulting in auto-phosphorylation of the respective intracytoplasmic domains in the receptor complex. These phospho-Tyr residues enable docking of several cytoplasmic signaling proteins and activation of signaling via Sos to Ras, and then via Raf–Mek–Erk and PI3K–PDK–AKT pathways see . Thus, tAdministration of anti-EGF or optimized anti-Her2/ErbB2 monoclonal antibody (Herceptin/Trastuzumab) are the most widely used—but also very expensive—inhibitors of EGF signaling for the treatment of cancer. Furthermore, such therapeutic protocols can still coincide with severe collateral side effects. Trx-based peptide aptamers against EGFR that complex with its intracytoplasmic domain, and that provoke slower proliferation in a dose-dependent fashion and reduce soft agar colony formation by the tumor cells, have been obtained. Specifically, the KDI1 aptamer does not block the the receptor’s kinase activity, but affects the activation of downstream p46 Shc and Stat3, including the transcription activation by the latter . PeptideKd: 10−9 M), whereas it does not bind to other ErbB-family members. EGFR-overproducing cells treated with CL4 show a block in EGFR-mediated signaling and induce a cell death program. At low doses, this aptamer is able to induce apoptosis even in cells that are resistant to some of the commonly used EGFR inhibitors . It also slows down tumor growth in mice xenografted with human non-small-cell lung cancer (NSCLC) cells. CL4 can also induce apoptosis in EGFR-producing cells in a synergistic manner with Cetuximab [More recently, a nuclease-resistant RNA-aptamer, named CL4, was produced based on SELEX . It specetuximab . Thus, Ci.e., the large family of Frizzled (Frz) seven-pass transmembrane receptors and two members of the LDL receptor-related protein family  (The Wnt family of secreted (glycolipo)protein ligands are critical in embryonic development, in adult stem/progenitor cells and during regeneration after acute injury, where in particular the “canonical” signaling (implicating β-catenin) regulates cell fate, cell proliferation and stem cell self-renewal . The Wntnd LRP6) . Howevernd LRP6) . In addind LRP6)   GSK3 and (then) CK1. Thus, the phosphorylation of this motif is reversed—in terms of sequential order—compared to β-catenin phosphorylation, but primes the requirement and functionality of the same GSK3–Axin complex. Hence, the same kinase combination is used for positive as well as negative regulation of signaling.The key co-operation between the receptors, for which most data in cell culture support the model wherein Wnt induces the formation of the Frz–LRP complex, is essential to keep Wnt signaling within homeostatic range . In the In one more detailed model, recruitment of the Axin–GSK3 complex by Frz–Dvl initiates LRP6 phosphorylation by GSK3. Dvl and Axin would then form aggregates  that facilitate protein interactions that are weak and dynamic. This would result in Dvl-mediated receptor clustering and formation of a Wnt-loaded Frz-LRP-Axin-Dvl-GSK3-CK1 “signalosome” platform see . HoweverN-terminal segment to a region encompassing 12 copies of 42 amino acids each (named Armadillo repeats) present within β-catenin. These 12 repeats form a highly closed “superhelix” that, together with the binding site of Tcf-1, form a large, positively charged groove that cannot easily be displaced by chemical compounds or altered by mutagenesis [i.e., PKF115-854 and CGP049090) have been produced that inhibit or disrupt β-catenin/Tcf-1 binding, thus opening new ways to tackle protein–protein interaction in Wnt signaling, but the specificity and affinity for this target complex remain to be precisely documented [Targeting the Wnt pathway for therapeutic purposes, despite its conceptually straightforward and linearity towards β-catenin and Tcf-1/Lef-1 in canonical signaling, is not an easy task seen in the complex regulations that were identified recently (see above). Valid recent approaches may be those that lead to an increase in the stability of Axin, thus promoting desphosphorylation of β-catenin. Axin is, for example, stabilized by ADP-ribosylation, which is mediated by Tankyrase enzymes. Targeting of Tankyrase-1 and/or -2 with the chemical XAV939 inhibitor increases Axin levels . A well-agenesis . Small mcumented .N-terminal β-catenin-interacting domain of Tcf-1, affecting the binding of Tcf-1 to β-catenin and hence inhibiting their complex formation [Jeong and co-workers ,57 were ormation , especiaormation .versus differentiation, survival versus apoptosis, cell migration and shape changes, and cell adhesion to other cells and extracellular matrix. Deregulation of this signaling system leads to disease, e.g., fibrosis, cancer progression and metastasis, and disorders of multiple systems, including the immune system [TGFβ family signaling controls cell proliferation e system –61. The e system ). The ace system –65 formee system ,67). DesTGFβs themselves, like other members of the family such as bone morphogenetic proteins (BMPs), are multi-potent cytokines involved in many cellular processes. In normal, non-transformed cells, and depending on cell type and context, TGFβ can function as an inducer of apoptosis, control cell proliferation, and strongly induce in epithelial cells a transition towards mesenchymal cells. TGFβ is also implicated in many aspects of tumorigenesis by directly acting on cells of the tumor as well as influencing their environment. During early stages of tumorigenesis TGFβ acts as a tumor suppressor, but at later stages it promotes tumor growth and enhances tumor invasion and metastasis, tumor-induced angiogenesis, and systemic and local tumor immunosuppression.i.e., the co-receptor endoglin  cannot be explained by this. Marfan-related disorders also exist, including some in which mutations in TGFβ family system components have been identified [Aberrant TGFβ signaling causes diseases other than cancer. In hereditary hemorrhagic telangiectasia  mutatiod BMP10; ) and thed BMP10; ), causind BMP10; ). Marfanentified ,73. Despentified .Many antagonists of TGFβ signalling are being developed and are at a pre-clinical or clinical stage . The majKd : 10−5 M). These peptides seem to prevent receptor complex assembly rather than occupying the two ligand-binding sites in the ligand-induced tetrameric receptor complex. Hence, the activation of an acknowledged TGFβ-Smad reporter transgene (based on the reiterated Smad-binding element (SBE) CAGA, i.e., SBE12) in TGFβ-stimulated target cells was not affected. Neither of the two peptides could bind to e.g., BMPR–IA (Alk3), ActRII  and the co-receptors β-glycan and endoglin [in vitro to investigate the TGFβ-induced myofibroblast transdifferentiation of human Tenon’s fibroblasts [de novo expression of α-Smooth Muslce Actin (α-SMA), which is downregulated by S58 [One of the most successful approaches to pharmacological inhibition of TGFβ signaling is to use monoclonal antibodies with high affinity for the receptors. Li and co-workers, using phage display, identified two peptides that bind to the ectodomain of both TβRI (Alk5), but also to TβRII, with similar affinity . They pt in CBP ,84.Cui and colleagues  produced12, TGFβ-responsive elements of the genes PAI-1, Smad7 and p15, and the synthetic reporter 3TP-lux), only the FoxH1 peptide aptamer was able to inhibit 3TP-lux. Focusing on the Trx-based FoxH1 and Lef-1D peptide aptamers, the authors concluded that the Smads in the Smad complex expose different sites for the SBD of FoxH1 and Lef-1, and—importantly—that the binding of one aptamer to one site of the Smad target does not influence the binding to the second site.The produced set of aptamers was used in luciferase (lux)-based, TGFβ-sensitive promoter–reporter assays. The FoxH1 aptamer inhibits the A3-lux reporter, which contains three copies of an activin-responsive element. The Lef-1 aptamer inhibits TwntTop, a dual reporter of Smad signaling (via 2 SBEs) and Lef-1/Tcf (via 3 TBEs). In a second series of reporter assays  is bound by Smad4, which triggers c-Myc transcription in the absence of TGFβ. Upon addition of TGFβ, Smad4 dissociates from TBE1 and c-Myc is no longer transcribed. Thus, Smad4 can work in a TGFβ-independent fashion in c-Myc transcription, and the TrxLef-1D aptamer is able to block this. This makes Smad4-Lef1 interaction a potential therapeutic target when aiming at reduced proliferation of tumor cells.Transfection of an expression construct for the Trx-based Lef-1D aptamer slows down cell proliferation, even regardless of stimulation of the cells with TGFβ . In a fopromoter . TrxLef-C-terminal phophorylation in PTD-SARA transfected cells after stimulation with TGFβ [The Hoffmann team also produced Trx-based peptide aptamers based on the SBD of SARA . SARA  Smad [in vivo. Zeb1 and Zeb2 bind to CtBP co-repressors [Sip1 was identified in yeast two-hybrid screening using the MH2 domain of Smad1 as bait and a E12.5 mouse embryo cDNA library as source of prey fusion proteins . AmongstMP) Smad . Hence, pressors  and the pressors , and botpressors   that, based on amino acid changes as compared to cognate DNA-binding HDs, does not bind to DNA, unlike the true HD present in the Zeb-related ein Zfh1 , which lion, see ). While ZFHX1B (chr2q22) cause Mowat–Wilson syndrome (MWS), a single-gene disorder (MIM#235730) characterized by various malformations that do not all appear in every patient. The major defects occur in the central nervous system and cause intellectual disability, absence of corpus callosum, microcephaly, epilepsy, and delayed motor development. These combine with neural crest defects  and with a heterogeneous spectrum of other defects [Mutations in  defects –107. HapN- or C-terminal deletions thereof, enabled us to re-define the SBD as a 14aa-long shorter linear sequence within the SBD, serving binding with both TGFβ/Activin/Nodal and BMP Smads. This interaction occurs only in cells stimulated with the respective ligands [in vivo in knockout and knockin mice.Recently, a set of Trx-based peptide aptamers containing the entire Zeb2 SBD or  ligands . FurtherS-transferase, GST) and a DNA library of random 18-mers, allowed the identification of a palindromic consensus sequence composed of a half site GCACCC, a spacer of 2 bp (A/TA/T), and an inverted half-site GGGTGC; it also identified an imperfect inverted half-site with a single nucleotide substitution at one of the last two positions [While the zinc finger TF Zeb2 possesses a short, linear non-zinc-finger SBD, the multi-zinc finger TF OAZ/Zfp423  associates with Smad4 and the BMP–Smad Smad1 via its clustered zinc fingers 14–19, only in the presence of BMP2, while it binds DNA via a neighboring zinc finger cluster –111. OAZositions .Xenopus, OAZ binds to the promoter of XVent2, driving the expression of this gene during patterning of the lateral and ventral mesoderm. Activation of XVent2 is strictly dependent on the binding of OAZ to BMP-activated Smad1. Therefore, OAZ is a SIP-TF and, interestingly, the binding of OAZ with phospho–Smad1 and Olf-1/Ebf-1 is mutually exclusive, and each complex binds to different promoters and other regulatory regions [OAZ can bind the palindromic sequence via 7 zinc fingers, with a very low affinity for the half-site as monomer, while when as homodimer as well as heterodimer with Olf-1/Ebf-1 the affinity for this OAZ target DNA increases. Thus, the TF OAZ can form dimers on both the inverted and direct repeats, but not on the half-site. When in heterodimer complex with Olf-1/Ebl-1, OAZ eliminates transcriptional activation at the Olf-1/Ebf-1 binding site by sequestering the bHLH partner and preventing it from binding to DNA . In Xeno regions   and Smad2/3 . The SkiTMEPAI is overexpressed in patient specimens of breast, lung and prostate cancers. This makes TMEPAI an attractive new protein for aptamer studies in the field. Furthermore, such aptamers could be coupled with sequences that allow localization to the plasma membrane, e.g., via lipid modification, since only a small fraction of TMEPAI was found to co-localize with SARA in early endosomes.Transmembrane prostate androgen-induced (TMEPAI) is a mRNA induced by TGFβ and encodes a single-pass transmembrane protein. It negatively regulates Smad signalling: TMEPAI binds to TGFβ/Activin/Nodal Smads regardless of their activation status, via the Smad Interaction Motif (SIM) Pro–Pro–Asn–Arg, which in TMEPAI is similar to the SIM in the TFs Milk and Mixer  Figure . More stSMAD4, and in SMAD2 and SMAD3[i.e., L1 loop), the second affects the SSXS activation motif. Missense mutations in SMAD3 have been observed to lead to aortic aneurysma, and this is also the case for other genes encoding components of the TGFβ family system [TGFβ/BMP signaling is deregulated in cancers by various mechanisms, including mutations of R-Smad and Smad4 encoding genes. Recently, in a study comprising more than 700 primary colorectal cancer (CRC) tumors and 36 CRC cell lines, missense mutations have been mapped in the co-Smad encoding gene and SMAD3; these my system –120. We y system  for mutai.e., isoform or mutated) target protein, the targeting of the according specific variant-expressing cells—even in a mixed cell population—becomes conceptually possible.Over the last 20 years, aptamers have been developed as novel tools to interfere with protein–protein interaction. Many nucleic acid aptamers and peptide aptamers, directed against various types of targets, initially often including proteins involved in EGFR signaling, have been selected, produced and validated. Their low cost, ease of production and high affinity are features that make aptamers tempting tools for basic research, as well as for future therapeutic purposes. Interestingly, as it has emerged that aptamers can be directed towards even a certain variant  are not easy targets for a classical inhibitory treatment with small molecules. Moreover, a certain basal or homeostatic level of their signaling must often be maintained, making effective dosage with ligand-sequestering compounds or monoclonal antibodies very difficult. Aptamers provide also in such case a valuable alternative, including those that act at the level of the downstream-executing TFs or their complexes, whose composition and target gene dynamics start to be mapped intensively using various “omics” approaches.Our increasing interest in aptamers comes from the successful use of aptamers for affecting growth factor receptor signal transduction, in particular hyper-activation of EGFR signaling in tumorigenesis and tumor progression. We feel that the time has come to identify aptamers that can interfere with signaling cascades exerted by Wnt and TGFβ family factors. These pathways are fine-tuned at different levels in various ways, including by protein–protein interactions, and the often multi-functional effector proteins on which these pathways converge (


Pharmacological block of inhibition is often used to determine if inhibition contributes to spike selectivity, in which a preferred stimulus evokes more spikes than a null stimulus. When inhibitory block reduces spike selectivity, a common interpretation is that differences between the preferred- and null-evoked inhibitions created the selectivity from less-selective excitatory inputs. In models based on empirical properties of cells from the inferior colliculus (IC) of awake bats, we show that inhibitory differences are not required. Instead, inhibition can enhance spike selectivity by changing the gain, the ratio of output spikes to input current. Within the model, we made preferred stimuli that evoked more spikes than null stimuli using five distinct synaptic mechanisms. In two cases, synaptic selectivity (the differences between the preferred and null inputs) was entirely excitatory, and in two it was entirely inhibitory. In each case, blocking inhibition eliminated spike selectivity. Thus, observing spike rates following inhibitory block did not distinguish among the cases where synaptic selectivity was entirely excitatory or inhibitory. We then did the same modeling experiment using empirical synaptic conductances derived from responses to preferred and null sounds. In most cases, inhibition in the model enhanced spike selectivity mainly by gain modulation and firing rate reduction. Sometimes, inhibition reduced the null gain to zero, eliminating null-evoked spikes. In some cases, inhibition increased the preferred gain more than the null gain, enhancing the difference between the preferred- and null-evoked spikes. Finally, inhibition kept firing rates low. When selectivity is quantified by the selectivity index , inhibitory block reduced the SI by increasing overall firing rates. These results are consistent with inhibition shaping spike selectivity by gain control. To determine the role of inhibition in sensory processing, iontophoretic application of pharmacological agents during extracellular recording has been used for decades Sillito, . TypicalA limitation is that extracellular electrodes only measure output spikes, and therefore the underlying mechanisms must be inferred. For example, blocking inhibition in the auditory midbrain nucleus, the inferior colliculus (IC), reduces spike selectivity for frequency, intensity, frequency modulations (FMs), duration, binaural sensitivity, and conspecific communication calls  to input (current)  and in a range with high gain, where small input selectivity translates into large spike selectivity  functions were the number of spikes evoked in the first 50 ms during prolonged (200 ms) current step injections, as functions of current level. The eight conductance sets were derived from five sustained cells in which we recorded spikes and postsynaptic potentials (PSPs) evoked by a pair of FMs, a preferred and null FM that differed only in direction. In two cells we used a single FM pair in each cell to derive one conductance set per cell. In three cells we used two FM pairs in each cell to derive two conductance sets per cell, for a total of eight conductance sets (16 conductance pairs).Membrane properties and FM-evoked conductance analyses from the cells presented here were reported previously  and t-type calcium currents . This equation simulated the effect of delayed KLT activation because t < Tdelay and t > Tdelay. The timing and shape of W(t) determined the number of spikes in the burst before KLT activated sufficiently to curtail firing. We found a good match to the empirical data with Tdelay = 5 ms. While perhaps not biophysically realistic, implementing W(t) achieved the goal of matching the measured onset-burst function. In our view, the results in this study are not affected by how the I/O function was achieved.We matched the average measured I/O functions of sustained and onset-burst cell types  were systematically varied until the model spike counts matched the empirical spiking responses to FM sweeps and current steps. To simulate measured spike variability, we scaled the derived synaptic conductances by factors chosen randomly from a Gaussian distribution . Scaling the conductances would represent variation in the size of the postsynaptic currents, possibly due, for example, to variation in the afferent firing or in vesicle release. Models were then stimulated with 25 independent pairs of scaled synaptic conductances. Model spike probability, the average spikes/trial, was calculated and compared to measured spike probability.The average sustained model was adjusted to match empirical measurements from the individual sustained-type cells Figures , 4. FirsE(t) and gI(t) respectively] were simulated as the difference of two exponentials, i.e., E is the peak conductance, t is time (ms), τE and τE_rise are the decay and rising time constants (ms), respectively, with τE>τE_rise. The constant gnorm was chosen so that the maximum value of the conductance was GE. Specifically, the peak value of gE(t) occurs at time tp, where norm is the value of the difference in the exponentials at time tp, gnorm = exp(−tp/τE) − exp(−tp/τE_rise). The conductance parameters that we varied to generate selective responses were relative peak times, the conductance sizes (the integrated conductance functions) and the shapes (the time constants). Each conductance parameter was varied separately, once for excitation and once for inhibition. The integrated conductance was held fixed by adjusting the maximum conductance.In Figure in vivo from 20 IC cells that were representative of the larger data set in , and are shown as input/output (I/O) functions Figure . AlthougA–E) and onset  cell models from Figure We next created spike selectivity with five different mechanisms of synaptic selectivity Figure  using thIn the first example, the mechanism underlying synaptic selectivity was entirely excitatory  of the preferred and null excitation, with no differences in the size  of excitation, and no differences between the preferred and null inhibitions . In two cases the synaptic selectivity was entirely inhibitory; the preferred and null inhibitions differed either in size  . In other words, whatever contribution inhibition made to synaptic selectivity in this example, that contribution was undetectable in the spike counts.We next assessed the extent to which inhibition's role in modulating input strength affected spike selectivity by asking how eliminating inhibition affected the difference between the preferred and null spikes. Without inhibition, the preferred excitation evoked an average of 1.76 spikes per stimulus, whereas the null firing count increased to 0.96, reducing the SI to 0.29 Figure . Note thin vivo. Although, this example is representative of most of our data, there are cells in which inhibition's contribution to synaptic selectivity does affect spike selectivity, as shown in the next section.We conclude that in this example, inhibition enhanced spike selectivity by controlling the gain and by limiting firing rates rather than by its contribution to synaptic selectivity. Furthermore, the effects of removing inhibition on spike rate and spike selectivity are consistent with the effects commonly observed when inhibition is blocked A summary of the eight conductance sets from the five cells is shown in Figure In terms of directional selectivity, the model cells and measured data were also similar Figure . We showTo determine the extent to which inhibition enhanced spike selectivity by contributing to synaptic selectivity or gain control, we computed firing probabilities and SIs using either the average of the preferred and null inhibitions or by eliminating inhibition. We first show the effect of eliminating inhibition on firing probability Figures .Eliminating inhibition increased spike probability in almost every case Figure , and inhWe next assessed the effect on spike selectivity of inhibition's contribution to synaptic selectivity compared with inhibition's contribution to gain control Figure . For eacin vivo whole-cell recordings, we investigated the role of inhibition in shaping neuronal response selectivity in the IC. The first result is that changes in spike selectivity following inhibitory block do not necessarily distinguish amongst potential underlying mechanism(s) of spike selectivity. This was shown in two distinct cell types, sustained cells and onset cells, which represent the extremes of the cell types found in the IC in terms of input/output functions. Using empirically derived synaptic inputs and modeling, we found that when inhibitory block reduced or eliminated spike selectivity, inhibition sometimes enhanced spike selectivity by contributing to synaptic selectivity, but always by modulating the firing rates and gain. These inhibitory mechanisms are not mutually exclusive. We first discuss the simplicity of the model cells, then briefly review the terms gain and gain control in the context of enhancing spike selectivity.Using modeling and results from t-type calcium current , and therefore, the gain changes with input strength. At the far left of the curve, when the output is zero, the gain is zero. As the current strength increases, the I/O curve enters a rising phase during which output increases approximately in proportion to input. During this rising phase, the gain increases rapidly at first, but then more slowly as saturation is approached. During the saturation phase (far right), gain decreases as input strength increases, but does not return to zero.One definition of gain control, termed additive/subtractive, is a horizontal shift of the I/O curve , i.e., the proportional change of output with respect to input . Kopp-Scheinpflug et al.  showed iHaving noted that inhibition enhances spike selectivity by contributing to synaptic selectivity in some situations, several studies in which spike selectivity was measured in the IC before and after inhibitory block using extracellular electrodes have proposed that inhibition increases selectivity by gain control (Ingham and McAlpine, Evidence from whole-cell recordings in the IC suggests it is very rare for a sound to evoke excitation without evoking inhibition. As noted above, sometimes inhibition is evoked in the absence of excitation (Xie et al., in vivo have found evidence that inhibition enhances synaptic selectivity in the cortex in some cells and for some stimuli (Anderson et al., Finally, we note that inhibition performs these same functions in the cortex (see Isaacson and Scanziani, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Mycobacterium tuberculosis. In this study, we show thatthe lone Ser/Thr phosphatase (PstP) is regulated through phosphorylation bySTPKs.The integrated functions of 11 Ser/Thr protein kinases (STPKs) and onephosphatase manipulate the phosphorylation levels of critical proteins in2+-ions and inorganic phosphate (Pi). PstPis differentially phosphorylated on the cytosolic domain withThr137, Thr141, Thr174 andThr290 being the target residues of PknB whileThr137 and Thr174 are phosphorylated by PknA. TheMn2+-ion binding residues Asp38 andAsp229 are critical for the optimal activity of PstP andsubstitution of these residues affects its phosphorylation status. NativePstP and its phosphatase deficient mutant PstPcD38Gare phosphorylated by PknA and PknB in E. coli and additionof Zn2+/Pi in the culture conditions affect thephosphorylation level of PstP. Interestingly, the phosphorylated phosphataseis more active than its unphosphorylated equivalent.PstP is phosphorylated by PknA and PknB and phosphorylation is influenced bythe presence of ZnThis study establishes the novel mechanisms for regulation of mycobacterialSer/Thr phosphatase. The results indicate that STPKs and PstP may regulatethe signaling through mutually dependent mechanisms. Consequently, PstPphosphorylation may play a critical role in regulating its own activity.Since, the equilibrium between phosphorylated and non-phosphorylated statesof mycobacterial proteins is still unexplained, understanding the regulationof PstP may help in deciphering the signal transduction pathways mediated bySTPKs and the reversibility of the phenomena. Mycobacterium tuberculosis has an array of proteins to ensure itsexistence during the course of infection. In order to thrive and maintain itshomeostasis, the pathogen continuously influences its surroundings mainly throughsurface-located sensor proteins. Extracellular signals are communicated through thesensors to the cytosol leading to the appropriate cell responses. Apparently, alarge number of pathogens employ reversible phosphorylation of proteins by kinasesand phosphatases as a way of transmitting the signals from extracellular milieuwhich helps in their survival and pathogenicity M. tuberculosis is known to have 11 Ser/Thr protein kinases, one tyrosine kinase (PtkA), one Ser/Thr phosphatase (PstP)and two tyrosine phosphatases (PtpA and PtpB) 2+-ion for its activity et al. determined the structure of the catalytic phosphatasedomain of PstP 2+. Similarities between Human Ser/Thrphosphatase PP2Cα and the mycobacterial enzyme have been explained on the basisof structural folds, metal binding and conserved residues Apart from the well recognized two component systems targeting His/Asp residues inbacteria, Ser, Thr and Tyr residues are also the major targets for phosphorylation.M. tuberculosis Ser/Thr signalingmolecules is that both the essential STPKs, PknB (Rv0014c) and PknA (Rv0015c) andthe only Ser/Thr phosphatase PstP (Rv0018c) are located in the same genomic clusterwhich is conserved in several mycobacterial species in vitro and in the surrogate host Escherichiacoli. Additionally, we found that zinc ions (Zn2+) andinorganic phosphate (Pi) can inhibit the activity of PstP which in turn affects thephosphorylation status of both the kinases and phosphatase.The interesting feature of E. coli DH5α strain (Novagen) was used for cloning and BL21(DE3) (Stratagene) was used for the expression of recombinant proteins.E. coli cells were grown and maintained with constantshaking (220 rpm) at 37°C in LB medium supplemented with 100 µg/mlampicillin.c  andPstP  were PCR amplified usingM. tuberculosis H37Rv genomic DNA. Resulting PCR productswere digested with corresponding restriction enzymes and ligated into thevectors pProEx-HTc (Invitrogen) and/or pGEX-5X-3 previously digested with the same enzymes. cHtc-PknBand cHtc-PstP were obtained as described earliercpGEX-PknB was sub-cloned fromcHtc-PknB using standard protocols under thesame restriction sites. For cloning in dual-expression vector pETDuet-1(Novagen), genes coding for PstPc or PstPcD38Gwere inserted in MCS1 having N-terminal His6-tag while kinases PknAand PknB (full length) were cloned in MCS2 with N-terminal MBP-tag. MBP-alone (without kinase)was taken as control vector having PstPc orPstPcD38G in MCS1. The protocols used for cloning inpETDuet-1 have been discussed earlier The genes coding for PknAc were created as R20G, D38Gand D229G using Htc-PstP andcHtc-PstP as templates.cHtc-PstP was utilized for the generation ofct5aHtc-PstP andct141eHtc-PstP.cHtc-pknB was employed as template forgeneration of double mutantct171/173dHtc-pknB. The details ofall the primers and clones are provided in Mutagenesis of specific residues was carried out using the QuikChange XLsite-directed mutagenesis kit (Stratagene) as per manufacturer'sinstructions. Mutants of PstP and PstPE. coli as describedpreviously Proteins were expressed and purified from In vitro phosphorylation of PstPc or its mutants(0.5–3 µg) by PknAc (0.5–1 µg) orPknBc (1–3 µg) was carried out in kinase buffer containing 2 µCi [γ-32P]ATP  followed by incubation at 25°C for 20 min. Reactions were terminatedby 5X SDS sample buffer followed by boiling at 100°C for 5 min. Proteinswere separated by 12% SDS-PAGE and analyzed by PhosphorImager . Zn2+ and Pi were added to the kinase assay reactions asper requirement of the assay. For the visualization of phosphorylation signal oncleaved proteins, removal of recombinant tags was achieved by addition of TEVprotease (for His6-tagged PstP/PstPc and their mutants) inTEV buffer after the kinase reaction followed by an additional incubation for 2 hr at20°C. For phosphoamino acid analysis, PstPcD38G wasphosphorylated by PknBc and PknAc and cleaved with TEVprotease as mentioned above, separated by SDS-PAGE and electroblotted ontoImmobilon PVDF membrane (Millipore). Phosphoamino acid analysis bytwo-dimensional thin layer electrophoresis was performed as described earlierc and PknAc were autophosphorylated by invitro kinase assays using [γ-32P]ATP. 1µg of purifiedPstPc/PstPcD38G/PstPcR20G/PstPcD229Gwere added in four sets of reactions and incubated at 25°C for increasingtime points up to 30 min to measure the dephosphorylation potential ofPstPc and its mutants. For auto-dephosphorylation assays,PknBc and PknBcT171/173D (2 µg each)were autophosphorylated by in vitro kinase assays and exposedto dephosphorylation by PstPc and PstPcD38G (1µg). Reactions were stopped by adding 5X SDS sample buffer and boiled for5 min at 100°C. The samples were separated by 12% SDS-PAGE andphosphorylated bands were observed and analysed by PhosphorImager.PknBpNPP hydrolysis assay was performed as a measure of phosphataseactivity. PstPc was added to a reaction mixture containingphosphatase assay buffer and 10 mM pNPP in a 96-well plate and incubated at 37°C forindicated time points and absorbance was read at 405 nm . To assay the relative activity of PstPc and itsphosphatase-deficient variants, increasing concentrations of enzymes were addedto the reaction mix and processed as above. Alkaline phosphatase (Roche) andPknBc were taken as positive and negative controls, respectively,for the pNPP hydrolysis assays. Variations of PstPcactivity by addition of Zn2+ and Pi was assessed by addingZnCl2 or sodium phosphate [pH 7.2] to the reactionmixture as above, to achieve the indicated final concentrations. pETDuet-1purified PstPc and PstPcD38G, co-expressed withor without kinases, were employed for pNPP-assays to measurethe effect of phosphorylation on their activities.et al. was followed formetabolic labeling E. coli (BL21-DE3) transformantsharbouring eitherc/PstPcD38G-mbppETDuet-PstPorc/PstPcD38G-mbpPknApETDuet-PstPorc/PstPcD38G-mbpPknBpETDuet-PstPwere grown in 5 ml LB medium containing 100 µg/ml ampicillin to anO.D600 of ∼0.6. The cells were induced with 1 mM IPTG andfurther grown for 4 hr at 16°C. Cultures were harvested, washed with 5 ml ofM9 medium [pH 7.0] without phosphate salts . The cells were resuspended in 2 ml of M9 media supplemented with 1 mCi of[32P]orthophosphoric acid , 100µg/ml ampicillin and 1 mM IPTG and further grown at 16°C for 4 hr.Under specific conditions, Sodium phosphate [pH 7.2] (2 mM) orZnCl2 (4 mM) were added to M9 media and subsequent processingsteps of metabolic labeling. The cells were harvested and lysed by sonication inthe lysis buffer containing phosphate-buffered saline, 5% glycerol andprotease inhibitor cocktail. The cell lysate was clarified and the lysatescontaining His6-fusion protein were incubated with lysis bufferequilibrated Ni2+-NTA affinity beads for 2 hr at 4°C. Thebeads were then thoroughly washed with lysis buffer containing 20 mM imidazoleand resuspended in 5X SDS sample buffer followed by boiling for 15 min. Thesamples were resolved on SDS-PAGE followed by autoradiography.The procedure described by Kumar c and PknAc were employed for in vitrokinase assay using 50 µM cold ATP and PstPcD38G. Thesamples were run on 12% SDS-PAGE, stained with Coomassie Brilliant Blueand de-stained. Bands corresponding to PstPcD38G wereexcised from the gel and washed with MilliQ water. The samples were processedfor identification of phosphorylation sites by using Thermo-Finnagen LTQelectrospray instrument . The detailed protocol of sample processing for identification ofphosphorylation sites has been provided in PknBc were generated in rabbit. Toconfirm the presence of PstPc/PstPcD38G inNi2+-NTA pulled-out proteins after metabolic labeling bywestern blot analysis, the samples were resolved by SDS-PAGE along with positive(purified PstPc) and negative controls (GST-PknBc) andtransferred onto nitrocellulose membrane (Bio-Rad). Standard procedure forimmunoblotting was followed R West Pico Chemiluminescent Substrate kit (PierceProtein Research Products) according to manufacturer's instructions.Polyclonal antibodies against PstPc mutantswere generated using site-directed mutagenesis. These residues include theMn2+-ion binding sites-Asp38 andAsp229 and phosphate (Pi) binding residue-Arg20 . Theactivity of these mutants was compared using chromogenic substratepNPP. To confirm the authenticity of the assay, increasingconcentrations of alkaline phosphatase were utilized as a positive control whilePknBc was used as negative control  in PstPc were mutagenized to generatePstPcT5A and PstPcT141E. Therelative activities of these mutants were compared with the native enzymethrough pNPP-assay . Surprisingly, in this assay, additional phosphorylated bandscorresponding to the size of PstPcD38G were observed whenincubated with kinase for longer time. No such bands were observed withPstPc, PstPcR20G andPstPcD229G at the given concentrations.The dephosphorylation potential of PstPrylation  and pNPPs assays .PstPcRPcD229G . The actP-assays  and S3. c or PknBc wereincubated with increasing concentrations of PstPc. Interestingly,PstPc was phosphorylated by PknAc at higher kinase tophosphatase ratio  was not observed by in vitro kinase assay as itcompletely dephosphorylated PknAc and PknBc, making theminactive (heat-inactive PstPc was found to be phosphorylated-data notshown). To confirm that the observed phosphorylation is onPstPc-mutants and not on the N-terminally attachedHis6-tag, TEV-protease cleavage of the tag was performed after thekinase assays. Phosphorylation was confirmed to be specifically localized on thecleaved substrate protein (data not shown). Additionally, the R20G, D38G andD229G mutants were also created in full length PstP construct andpNPP-hydrolysis assays and phosphorylation reactions werealso confirmed using full length PstP and its mutants (data not shown).After identification of the residues critical for PstPnd PknBc , whereasc  while PknA phosphorylated PstPcD38Gon two residues (Thr137 and Thr174)  cells transformed withc/PstPcD38G-MBPpETDuet1-PstPorc/PstPcD38G-MBP-kinasepETDuet1-PstP were metabolically labelled with[32P]orthophosphoric acid. Phosphorylation ofPstPc and PstPcD38G could only be detectedwhen PknA or PknB were co-expressed .To further substantiate our results, the phosphorylation status ofPstPxpressed , suggestc and PstPcD38Gco-expressed with and without PknA/PknB, were evaluated. According to thepNPP assays, the activity of phosphorylatedPstPc (co-expressed with PknA or PknB) was higher than that ofunphosphorylated phosphatase   . The pheobserved .Also, tnassays .in vitro autophosphorylatedPknAc was assessed by PstPcD38G+MBP,PstPcD38G+MBP-PknA andPstPcD38G+MBP-PknB. As expected, due to higheractivity of phosphorylated PstPcD38G, intensity ofphosphorylation on PknAc was low as compared to the reactioncontaining unphosphorylated PstPcD38G+MBP  andATP/ADP ratio. PstP mM Zn2+  and 0.5  mM Zn2+ . Maximumc in the presence of Zn2+ and Piprovided a condition that could favour the phosphorylation of PstPcby STPKs. PstPc was indeed phosphorylated by PknAc andPknBc in presence of 0.2 mM Zn2+ or 0.5 mM Pi and Pi (2 mM) (c was indeed enhanced in thepresence of Zn2+ by ∼40%-50%. The enhancementin phosphorylation in the presence of Pi was not as prominent(∼10%–20%), possibly due to competition of phosphateions with [32P]orthophosphoric acid. Nevertheless, as aproof of principle, Zn2+ and Pi were identified as the novelregulators which can inhibit the activity of PstPc and facilitate itsphosphorylation.The inhibition of PstP.5 mM Pi, underiSDS-PAGE ,the assi (2 mM) . PhosphoM. tuberculosis where one Ser/Thrphosphatase PstP is accountable for the effects caused by 11 STPKs. In general, theprocesses involved in regulating the phosphatases include some external signals,variation in pH 2+, Zn2+, Mg2+,Ca2+2+-dependent protein phosphatase α (MPPα) by caseinkinase II Oryzasativa KAPP The coordinated regulation of Ser/Thr protein kinases and phosphatases is essentialfor maintaining the appropriate equilibrium of protein phosphorylation. Membraneassociated kinases and phosphatases are known or hypothesized to be regulated byexternal stimulus. It is of great relevance to decipher the regulatory mechanismsespecially in the systems like et al. resolved the crystal structure of PstPcatalytic domain and described the most important features of this molecule havingcharacteristic PP2C-fold along with three-metal binding centers that associate withMn2+Streptococcus agalactiae andThermosynechococcus elongatus have been shown to have a similarthird-metal binding centre PstP has conserved domain architecture of PP2C-phosphatases (PPM family). PPM familyphosphatases play an imperative role in a number of systems described earlier c, on the basis of similarity with Human phosphatasePP2Cα 20 (PP2Cα Arg33) is responsible for hydrolysis ofphosphate moiety from pSer/pThr residues in target proteins. Asp38(PP2Cα Asp60) and Asp229 (PP2Cα Asp282)constitute a part of Mn2+-metal centers and coordinate with the twocritical Mn2+. Mutations of Asp38 and Asp229affected the activity of PstP rendering it active to minimal level, though R20Gmutant retained about 40% activity. Thus, the residues that are involved inMn2+-ion binding and hydrolysis of phosphate are deciphered tobe critical for its activity. Accordingly, the extent of phosphorylation of eachmutant was dependent on the remaining dephosphorylation activity, so thatPstPcD38G and PstPcD229G wereefficiently phosphorylated by PknA and PknB.In this study, three conserved residues were selected for generation of site-directedmutants in PstPc activity is the presence of Mn2+2+/Mg2+ can inhibit their activity bycompetitively replacing the Mn2+ in the core enzyme structure 2+ are the most potent regulators, having comparable ionicradii with that of Mn2+. PstPc was partially inactive inthe presence of 0.2 mM ZnCl2 and displayed lower activity on increasingthe Zn2+-ion concentration upto 2 mM, as observed bypNPP assays. In vitro kinase assays withPknAc and PknBc in presence of Zn2+resulted in phosphorylation of PstPc. Also, there was increase inphosphorylation of PstPc during metabolic labeling by PknA and PknB inthe presence of Zn2+ added in the E. coli culture.These results indicate that in mycobacterial cell, if cytosolic Zn2+concentration increases, it may inhibit PstP perhaps leading to its phosphorylation.In an elaborative elemental analysis, Wagner et al. have reportedthat during infection, intravacuolar Zn2+-ion concentrationincreases from 0.037 mM to 0.46 mM in macrophages infected with M.tuberculosis2+-ionconcentration, it can only be speculated that if these changes in vacuolar ionicconcentrations alter the mycobacterial ionic profile, a condition may develop wherethe enzymes that respond to Zn2+ (like PstP) can be activated ordeactivated.Association with metals is crucial for PP2C phosphatases and any perturbation withinherently associated metals may lead to altered functional profile. The minimumrequirement for PstPc mediated pNPP hydrolysis is inhibited by Pi.To confirm that this effect is not limited to pNPP, invitro kinase assays and metabolic labeling in E. colishowed PstPc to be phosphorylated by PknA and PknB in presence of Pibecause of its inhibition. Pi content is indicative of nutrient availability andenergy status of the cell. In general, high Pi is associated with energy-starvedconditions, when all the ATP is depleted and metabolite homeostasis is in unbalancedstate. Such conditions usually arise during late-log and stationary phases inculture conditions.End-product inhibition of enzymes is a well established phenomenon to prevent theaccumulation of a particular metabolite. In case of reversible reactions,end-product accumulation can change the direction of the reaction. Similarly, Pi isknown to inhibit a number of phosphatases 32P]orthophosphoric acid in the presenceof co-expressed STPK (PknA or PknB) in E. coli lead to the specificphosphorylation of PstPc and PstPcD38G.Co-expression in pETDuet-1 has previously been utilized extensively to assess theinteraction of mycobacterial STPKs with their cognate substrates in the surrogatehost E. colic andPstPcD38G revealed the higher activity ofPknA-phosphorylated phosphatase as compared to the PknB-phosphorylated protein.Prominent variations in the activity of phosphorylated and unphosphorylatedPstPcD38G were observed with phosphorylated protein beingproficient to hydrolyze pNPP to a greater extent (∼15-foldhigher) in contrast to the unphosphorylated protein. The difference in theactivities of phosphorylated and unphosphorylated PstPc was not asprominent as that of PstPcD38G (∼2–3 fold higher).These differences may be attributed to the fact that PstPc may getauto-dephosphorylated to a greater extent than PstPcD38Gduring expression and purification procedures. Higher activity of phosphorylatedphosphatase is suggestive of reverse regulation of signaling cascade emanating fromthe kinases. In the constitutively active state, STPKs perform their regularfunctions and phosphorylate the target substrates following the stimulus. This mayultimately lead to the phosphorylation of PstP. The resulting increase in theactivity of phosphatase may itself act as a control mechanism for kinases,eventually impeding the continued effect of that particular stimulus. The overallprocess has to be dynamic due to auto-dephosphorylation of PstP, eventually ceasingthe effect of signaling cascade. In the conditions of high Zn2+ orhigh Pi content of the cell, PstP may not be active and will allow the kinase towork at its maximal activity. The proposed phosphorylation of PstP in suchconditions may act as a mechanism to overcome the inhibition of PstP, hencebalancing the cellular signaling pathways.Metabolic labeling by [Figure S1pNPP-assay. To confirm theauthenticity of pNPP assay, increasing amounts of alkaline phosphatase(0-100 ng) was used a positive control and PknBc (0–5µg) was used as a negative control. The assay was performed for 30mins at 37°C and the activity is calculated as µmoles ofpNPP hydrolyzed per min at a given amount of enzymeused. As clearly evident, alkaline phosphatase showed very high activitywhile no such activity was detected in PknBc.(TIF)Click here for additional data file.Figure S2c.Effect of mutations on the activity of PstP To showthat the loss in activity of PstPc was specifically due tomutations of Arg20, Asp38 and Asp229,PstPc was mutagenized on irrelevant residues Thr5and Thr141 to Ala and Glu, respectively and pNPPhydrolysis was performed for 30 mins at 37°C. Activity ofPstPc was taken as 100% and relative activity wascalculated. As evident from the bar graph, there were no significant changesin the activity of the mutants PstPcT5A andPstPcT141E as compared to PstPc.(TIF)Click here for additional data file.Figure S3Time-dependentpNPP-assay.pNPP-hydrolysis was performed in a time-dependent mannerfor 30 mins using PstPc, PstPcR20G,PstPcD38G and PstPcD229Gvariants (2 µg each) at 37°C. Alkaline phosphatase (2 ng) was useda positive control and PknBc (5 µg) was used as a negativecontrol. Activity was calculated as nmoles of pNPPhydrolyzed per µg of enzyme used at a given time and depicted inlogarithmic scale. Nevertheless, the results are essentially similar as thatof time-dependent dephosphorylation of PknBc (of PknBc .(TIF)Click here for additional data file.Figure S4In vitrocD38G. dephosphorylation activity ofpETDuet-1 purified PstPAutophosphorylated PknAc was incubated with unphosphorylated andphosphorylated PstPcD38G. As shown in theautoradiogram, the PknA-phosphorylated PstPcD38Gdephosphorylated the kinase to a greater extent in comparison to theunphosphorylated PstPcD38G. The image was alsoanalyzed by ImageGauge software and corresponding values are depicted bybar-graph (ar-graph .(TIF)Click here for additional data file.Figure S5c.Phosphorylation of PstP Autoradiogram showing thephosphorylation of PstPc (1 µg) by His6-taggedSTPKs PknAc (upper panel) and PknBc (lower panel) inpresence of 0.2 mM Zn2+ and 0.5 mM Pi. Due to overlappingmolecular weights of PknAc and PknBc withPstPc, the bands were not separated properly. Still, thephosphotransfer on PstPc was evident in presence ofZn2+ and Pi by both the kinases. The reaction was alsoperformed with GST-tagged STPKs to clearly depict the reaction (reaction .(TIF)Click here for additional data file.File S1Detailed protocol of sample processing for identification ofphosphorylation sites.(DOC)Click here for additional data file.


This suggests that the reciprocal regulation of MLC phosphorylation status by β-arrestins-1 and 2 causes turnover in the phosphorylation status of MLC that is required for cell contractility and subsequent chemotaxic motility.Over the last decade, it has been established that G-protein-coupled receptors (GPCRs) signal not only through canonical G-protein-mediated mechanisms, but also through the ubiquitous cellular scaffolds β-arrestin-1 and β-arrestin-2. Previous studies have implicated β-arrestins as regulators of actin reorganization in response to GPCR stimulation while also being required for membrane protrusion events that accompany cellular motility. One of the most critical events in the active movement of cells is the cyclic phosphorylation and activation of myosin light chain (MLC), which is required for cellular contraction and movement. We have identified the myosin light chain phosphatase Targeting Subunit (MYPT-1) as a binding partner of the β-arrestins and found that β-arrestins play a role in regulating the turnover of phosphorylated myosin light chain. In response to stimulation of the angiotensin Type 1a Receptor (AT1aR), MLC phosphorylation is induced quickly and potently. We have found that β-arrestin-2 facilitates dephosphorylation of MLC, while, in a reciprocal fashion, β-arrestin 1 limits dephosphorylation of MLC. Intriguingly, loss of either β-arrestin-1 or 2 blocks phospho-MLC turnover and causes a decrease in the contraction of cells as monitored by atomic force microscopy (AFM). Furthermore, by employing the β-arrestin biased ligand [Sar Cellular contraction, governed by actin-myosin-mediated motor activity, is central to many physiological processes in both muscle and non-muscle cells, including cell division, adhesion, chemotaxis and cytokinesis . DynamicSeveral G-protein-coupled receptors (GPCRs), such as the Angiotensin II type 1a receptor (AT1aR), are involved in the regulation of cellular contraction. The AT1aR agonist, Angiotensin II (Ang II), a potent vasoconstrictor, is well known to contribute to the development of cardiovascular diseases . ATa1R iMyosins (both smooth muscle and non-muscle) are regulated by the phosphorylation of MLC . PhosphoGodin et al. also reported recently the importance for β-arrestins-1 and 2 in the regulation of Rho-A-dependent membrane blebbing -Angiotensin II (SII) was purchased from the Cleveland Clinic Core Synthesis Facility. Q-serum was prepared as follows: 5ml Source 30Q beads  were washed two times in phosphate buffered saline (PBS) then incubated with 25 ml fetal bovine serum (FBS) (Sigma) for 30 min with agitation. The mixture was then centrifuged at 2000 rpm and the treated serum was recovered and filtered through 0.22 µm.Angiotensin II was purchased from Sigma. [SarAT1aR-stable HEK-293 (AT1aR-293) cells were maintained in minimum essential Eagle’s medium (EMEM)  plus 10% FBS, 0.6 IU/ml penicillin (Sigma) and 600 µg/ml streptomycin (Sigma) in the presence of 100 μg/ml zeocin (Invitrogen) as described previously . Rat vasEscherichia coli, the pAS2-1 β-arrestin-1 plasmid was transformed into yeast strain PJ69-4A and a yeast two-hybrid screen was carried out essentially according to the Clontech Matchmaker protocol using a GAL4 activation domain fusion library in pACT2 . Library clones containing putative β-arrestin-1 interacting proteins were identified by positive growth on Trp/Leu/His/Ade dropout plates. Plasmids were isolated from positive clones using standard methodologies and analyzed by DNA sequencing . One of these clones corresponded to amino acids 643 to 943 of human MYPT-1 (NM_001143886). To confirm this interaction and to map the β-arrestin-1 interacting domain, DNA fragments corresponding to amino acids 1-163 or 1-253 of rat β-arrestin-1 were PCR amplified and subcloned into pAS2-1 using EcoRI and BamH1 restriction sites. Plasmids corresponding to full length (1-418), 1-163, or 1-253 β-arrestin-1 were then co-transformed with pACT2 MYPT-1(643-943) into the PJ69-4A yeast strain and selected on Trp/Leu dropout plates. Yeasts containing the β-arrestin-1 and MYPT-1 plasmids were then analyzed for growth on Trp/Leu/Ade dropout plates.The full length cDNA of rat β-arrestin-1 was subcloned into the yeast expression vector pAS2-1 using EcoRI and BamHI restriction sites. Following amplification in 3 days before β-arrestin overexpression experiments, AT1aR-293 cells were transfected with either 1μg of plasmid pcDNA3-β-arrestin-1-FLAG or pcDNA3- β-arrestin-2-FLAG (encoding rat β-arrestins) ,19 usinguucuccgaacgugucacgudTdT-3, 5-agccuucugcgcggagaaudTdT-3 and 5-ccaaccucauugaauuugadTdT-3, respectively. The SMARTpool siRNA targeting human MYPT-1 consists of 4 different siRNA sequences . The custom made siRNA sequences targeting rat β-arrestin-1 and rat β-arrestin-2 were 5-AGC CUU CUG UGC UGA GAA C-3 and 5-CCA ACC TCA TTG AAT TCG A-3 respectively [3 days before β-arrestin and MYPT-1 siRNA-mediated knockdown experiments, 50% confluent human AT1aR-293 or rVSMC cells were transfected with siRNA using Gene Silencer (Gene Therapy Systems) as previously described ,19 or Prectively . The SMArVSMCs were seeded to 80% confluency in 6-well plates and cultured overnight. The following day, siRNA delivery system was prepared by mixing 10 µl of 40 µM siRNA diluted in water with 9 µl of 200 µM PTD-dRBD protein pre-diluted in 64.2 µl of PBS-10% Glycerol. While the siRNA delivery mix was incubating on ice for 15 minutes, cells were washed with serum free medium and incubated in 1 ml 10% Q-serum-medium. After incubation on ice, the PTD-dRBD:siRNA mix was diluted 1:5 in 10% Q-serum-medium. Media was removed from cells and replaced with 400 ul PTD-dRBD:siRNA complexes. After 4-6 hrs of incubation at 37°C in 5% CO2 incubator, the cells were washed twice with 10% FBS medium and kept under culture conditions until assays (motility or western blotting) were performed 96 hours later.Cells were solubilized in lysis buffer containing 50 mM HEPES (pH 7.5), 0.5% Nonidet P-40, 250 mM NaCl, 2 mM EDTA, 10% (v/v) glycerol, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, 5 μg/ml leupeptin, 5 μg/ml aprotinin, 1 μg/ml pepstatin A and 100 μM benzaminidine. For immunoprecipitation, 15 μl of protein A/G plus-agarose (SantaCruz) and 1 μg of specified antibody were added to whole-cell lysates. After 3 hour incubation at 4 °C on a rocker platform, the immunoprecipitates were collected by centrifugation at 2,500 rpm for 2 min. The supernatant was discarded, whereupon the pellet was washed 4 times and then dissolved in sample buffer. For direct detection of protein levels in lysates, whole-cell lysates were directly used for SDS-polyacrylamide gel electrophoresis. SDS-PAGE were conducted on 10% polyacrylamide tris-glycine gels (Invitrogen) and transferred to nitrocellulose membranes for immunoblotting according to standard protocols. Immunoblots were performed using the following antibodies: β-arrestin-1 polyclonal A1CT and β-arrestin-2 polyclonal A2CT , FLAG M22-independent EMEM media containing HEPES at 37°c were adapted to room temperature one hour prior to an AFM experiment. YFP fluorescence was used to select cells that incorporated siRNAs and contrast phase microscopy was used to manually position the cantilever tip over the apical region of the cell. With the help of a 15 µM piezo scanner (Physik Instumente) an individual cell was approached until a contact force of approximately 200pN was measured. Baseline measurements were typically recorded for 5 mins after which vehicle injection was performed as control. After an additional 5 min, agonists were injected. Simultaneously to AFM data recording, micrographs were taken every second in phase contrast microscopy using a 40X objective and an AxioCam MRm camera (Carl Zeiss). All AFM experiments were performed at room temperature to limit mechanical vibration and thermal drift introduced by a heating/cooling system. Cell mechanical responses are reported as variations in cell height measured by the cantilever deflection (nm) and cell contractions were reported as the maximal variation of the cell height with respect to the baseline. A custom-made AFM-based force measurement device was mounted on an inverted epifluorescent microscope (Carl Zeiss) as described previously . Silicon2. Cells were then removed from the upper chamber and the upper side of the membrane, leaving only those cells that migrated through the membrane and to the lower chamber. Membranes were then fixed with 4% paraformaldehyde and stained with 1% crystal violet before being dried, excised, and mounted on microscope slides. Membrane densities were quantified using the GeneTools image analysis software (Syngene). The chemotactic index was calculated by dividing values from membranes in the stimulated conditions by values from control membranes (Vehicle).Boyden-Chamber assays were performed in transwell chambers of 24-well inserts with 8-μm pore membranes coated with fibronectin (5µg/ml) for 1 hour. rVSMC were serum-starved for 2 hours in M199 then assays were conducted in serum-free M199 media as well. Agonists were placed in the lower chamber, and 100 000 cells were placed in the upper chamber and incubated for 5 h at 37°C and 5% COQCM chemotaxis cell migration assays  were performed in 96-well plates. Briefly, rVSMC were starved overnight in M199 and 25 000 cells per well were used using the protocol supplied by the manufacturer. The cell fluorescence was measured by a fluorescence plate reader using 480/520 nm filter set. The chemotactic index was calculated by dividing values from the different treatments by values from control conditions. In order to identify binding partners of β-arrestin-1, a yeast-II hybrid screen was performed using pAS2-1β-arrestin-1 as the bait and a human heart cDNA library as the prey. We isolated one clone corresponding to the c-terminus amino acids (643 to 943) of MYPT-1 NM_001143886). Amino acids 643 to 943 contain the regulatory and myosin binding domains of the MLCP complex, indicating that these domains confer the ability of MLCP to interact with β-arrestin-1. To determine the regions of β-arrestin-1 that interact with MYPT-1, β-arrestin-1 fragments were co-expressed with MYPT-1(643-943) in a yeast-II hybrid assay. The results demonstrate that β-arrestin-1 (1-418) and (1-253) interact with MYPT-1(643-943), while β-arrestin-1 (1-163) did not 1A. Thes86. Amino To confirm the interaction of β-arrestins with MYPT-1, co-immunoprecipitations were performed following Ang II (1 nM) stimulation of AT1aR-293 cells overexpressing β-arrestin-1 or β-arrestin-2. , upper left and 3C, solid circles) and then showing a slight decrease before leveling off as the turnover of phospho-MLC reaches an apparent equilibrium for at least 30 min. In cells knocked down for β-arrestin-1, Ang II-stimulated MLC phosphorylation is essentially ablated  suggesting that β-arrestin1 is required for Ang II driven MLC phosphorylation. Interestingly, Ang II stimulation of β-arrestin-2 knockdown cells leads to a dramatic increase in MLC phosphorylation, , suggesting that β-arrestin-2 normally functions to suppress phosphorylation of MLC. MYPT-1 knockdown leads to a significant increase in both basal and Ang II stimulated MLC phosphorylation as would be predicted for cells lacking MLCP activity . Since the loss of β-arrestin-2 or MYPT-1 leads to a similarly high level of MLC phosphorylation, our results suggest that β-arrestin-2 is a positive regulator of MYPT-1 and, consequently, MLCP activity. Taken together, these data point towards opposing roles for β-arrestin-1 and β-arrestin-2 on MLC phosphorylation where β-arrestin-1 promotes MLC phosphorylation while β-arrestin-2 acts as a repressor of MLC phosphorylation by promoting the activity of MYPT-1 and the MLCP complex. These experiments identify the β-arrestins as important regulators of MLC phosphorylation and suggest that they may in turn affect myosin-dependent cell contraction and motility. Work from our group and others has revealed a critical role for both β-arrestins in cytoskeletal dynamics downstream of the AT1aR ,15. SincSince phosphorylation of MLC is associated with cell contractility and motility, we next determined if these β-arrestin-mediated regulations of MLC phosphorylation could also affect cellular contraction. In order to examine this question, we conducted AFM-based measurements of single cell contractility in response to AT1aR activation triggered by the full agonist Ang II (1nM) or the β-arrestin biased agonist SII (10 µM) 4. AFM eTo examine the contribution of the β-arrestins in Ang II-dependent cell contraction, we conducted similar AFM experiments in cells transfected with β-arrestin-1 or β-arrestin-2 siRNA. When compared to the contractile response observed with Ang II (1nM) stimulation (179±24 nm), the loss of β-arrestin-1 or 2 leads to smaller contractions of 82±18 nm and 97±24 nm, respectively . MoreoveTo determine if the SII-mediated effects on contractility seen in Since it appears that both β-arrestins play a role in the regulation of MLC phosphorylation, and both are required for AT1aR-induced cellular contractility, we wished to examine the involvement of β-arrestin-1, β-arrestin-2, and MYPT-1 in a physiologically relevant system that expresses endogenous levels of AT1aR receptors. Therefore, we used primary cultures of rat vascular smooth muscle cells (rVSMC) as they endogenously express the AT1aR and chemotax robustly in response to Ang II  or SII  7. The rThe activation of distinct GPCRs has been reported to initiate numerous physiological responses including contraction in muscle and non-muscle cell types. In addition to their G-protein-mediated signaling, these receptors also trigger the recruitment and activation of β-arrestins that are not only responsible for mediating receptor internalization and desensitization, but also for driving G-protein-independent, β-arrestin-mediated signaling. The AT1aR is well-known to induce contractile responses in muscle cells . MoreoveWe have shown here that the β-arrestin-mediated regulation of phosphorylated MLC is a significant component of AT1aR-induced mechanical response. Depletion of β-arrestin-1 from cells decreases levels of phospho-MLC in response to AT1aR stimulation 3 suggesAdditionally, we have shown that β-arrestin-2 is important for AT1aR-mediated dephosphorylation of MLC 3. AgoniIn 2008, Matsumura et al. proposed a model where high levels of phosphorylated MLC do not necessarily translate into increased cell contraction or migration. In their model, high MLC phosphorylation coupled with low MLCP activity result in low phospho-MLC turnover leading to increased cell adhesion and stress fiber formation. Conversely, high MLC phosphorylation coupled with high MLCP activity result in high phospho-MLC turnover leading to morphological changes and cell motility by accelerating focal adhesion turnover and limiting myosin-induced actin polymerization . Our resCycling of MLC phosphorylation correlates with cell contractility  and we s


Activated α2M (α2M*) binds to cancer cell surface GRP78 and triggers proliferative and antiapoptotic signaling. We have studied the role of α2M* in the regulation of mTORC1 and TORC2 signaling in the growth of human prostate cancer cells.Tetrameric αEmploying immunoprecipitation techniques and Western blotting as well as kinase assays, activation of the mTORC1 and mTORC2 complexes, as well as down stream targets were studied. RNAi was also employed to silence expression of Raptor, Rictor, or GRP78 in parallel studies.2M* promotes phosphorylation of mTOR, TSC2, S6-Kinase, 4EBP, AktT308, and AktS473 in a concentration and time-dependent manner. Rheb, Raptor, and Rictor also increased. α2M* treatment of cells elevated mTORC1 kinase activity as determined by kinase assays of mTOR or Raptor immunoprecipitates. mTORC1 activity was sensitive to LY294002 and rapamycin or transfection of cells with GRP78 dsRNA. Down regulation of Raptor expression by RNAi significantly reduced α2M*-induced S6-Kinase phosphorylation at T389 and kinase activity in Raptor immunoprecipitates. α2M*-treated cells demonstrate about a twofold increase in mTORC2 kinase activity as determined by kinase assay of AktS473 phosphorylation and levels of p-AktS473 in mTOR and Rictor immunoprecipitates. mTORC2 activity was sensitive to LY294002 and transfection of cells with GRP78 dsRNA, but insensitive to rapamycin. Down regulation of Rictor expression by RNAi significantly reduces α2M*-induced phosphorylation of AktS473 phosphorylation in Rictor immunoprecipitates.Stimulation of cells with α2M* to prostate cancer cell surface GRP78 upregulates mTORC1 and mTORC2 activation and promotes protein synthesis in the prostate cancer cells.Binding of α While the liver produces large amounts of the proteinase inhibitor α2-macroglobulin (α2M) it is produced by cancer cells and is linked with tumor growth 2M is also produced locally in tumor stromal tissue such as associated with prostate cancer 2M subunits, thiol esters rupture and the protein undergoes a very large conformational change exposing receptor recognition sites in each subunit 2M to small primary amines or ammonia, by direct attack on the thiol esters, also induces a large conformational change exposing these receptor recognition sites 2M*. Although GRP78 (glucose regulated protein of Mr ∼78000) is primarily known as a resident endoplasmic reticulum chaperone, it appears on the cell surface of many types of malignant cells 2M* to tumor cell surface GRP78 causes its autophosphorylation 2-terminal domain of GPR78 appear in the sera of prostate cancer and melanoma patients where they are a biomarker of aggressive behavior 2M* binds 2M* and anti-GRP78-NH2-terminal domain antibodies in cell culture and mice 2M functions like a growth factor and cell surface-associated GRP78 as a growth factor-like receptor The ability of cancer cells to thrive S473 in poorly differentiated prostate cancer Akt is a Ser/Thr kinase expressed as isoforms, Akt1, Akt2, and Akt3, encoded by three different genes Mammalian target of rapamycin (mTOR), an evolutionarily conserved Ser/Thr kinase, is a key regulator of Akt phosphorylation . Antibodies against mTOR, p-mTORS2481, GβL, p-TSC2T1462, Rheb, S6-Kinase, p-S6-KinaseT235/236, p-S6-KinaseT389, p-S6-KinaseT229, 4EBP1, p-4EBP1T37/46, Akt1, p-AktT308, p-AktS473 were purchased from Cell Signaling Technology . Antibodies against Raptor, Rictor, Protor were from Santa Cruz Biotechnology . Anti-mSIN1 antibody was from Bethyl Laboratories, Inc . Anti-actin antibody was from Sigma . PHAS-1 (4EBP1) was from Stratgene. GST-S6-Kinase construct was expressed and purified according to the protocol provided by Prof Blenis . Antibodies against the carboxyl-terminal domain of GRP78 were from Aventa Biopharmaceutical Corp . [3H]Leucine (specific activity 115.4 Ci/mmol) and [33P]-γ-ATP (specific activity 3000Ci/mmol) were from Perkin-Elmer Life Sciences. Rapamycin and LY294002 were from Biomol . All other materials were of analytical grade and were procured locally.Culture media were purchased from Invitrogen. Receptor-recognized α2 (5%) incubator. At 90% confluency the medium was aspirated. The monolayers washed with ice-cold HHBSS, a fresh volume of medium added and the cells used for the experiments described below.In an earlier report, we studied two prostate cancer lines 1-LN and DU-145, which express GRP78 on their cell surface and the PC-3 prostate cancer line, which expresses little GRP78 on its cell surface 3 cells/well) in 48 well plates were grown in RPMI-S medium in a humidified CO2 (5%) incubator at 37°C. At about 90% confluence, the medium was aspirated a volume of RPMI-S was added followed by the addition of either buffer or α2M* (50 pM). To each well was added [3H] leucine (2 µCi/ml) and cells were incubated overnight. The reactions were terminated by aspirating the medium and monolayers washed thrice twice with ice-cold 5% TCA, followed by three washings with ice-cold PBS. Cells were lysed in a volume of 1 NaOH (40°C/2 h), protein was estimated and the lysates were counted in a liquid scintillation counter. In experiments where modulation of α2M*-induced protein synthesis was studied, cells were pretreated with the PI 3-kinase inhibitor LY294002 (20 µM/20 min), mTOR inhibitor rapamycin (100 nM/15 min) or actinomycin D (5 µg/ml/20 min), before adding α2M* (50 pM/overnight). Other details of quantifying [3H] leucine incorporation into cellular proteins were as described 1-LN cells (300×103 cells/well) in RPMI-S medium in 6 well plates were incubated as above. At 90% confluency, the medium was aspirated, a volume of RPMI-S medium added to each well. In one set of experiments, cells were stimulated with varying concentrations of α2M* and incubated for 30 min. In another set of studies, cells were stimulated with 50 pM of α2M* and incubated for various times at 37°C in a humidified CO2 (5%) incubator. The reactions were terminated by aspirating the medium, a volume of lysis Buffer A containing 50 mM Tris•HCl (pH 7.5), 120 mM NaOH, 0.1% NP40, 25 mM sodium fluoride, 1 mM sodium pyrophosphate, 0.1 mM sodium orthovanadate, 1 mM PMSF, 1 mM benzamidine, and leupeptin (10 µg/ml) added and placed over ice for 15 min. The lysates were transferred into Eppendorf tubes and centrifuged at 1000 rpm for 5 min at 4°C to remove cell debris. The supernatants were removed to new tubes and their protein contents determined S2481, p-TSC2T1462, Raptor, Rictor, GβL, Rheb, p-S6-KinaseS235/236, p-4EBP1T37/46, p-AktT308 or p-AktS473 for 16 h at 4°C with rotation. The detection and quantification of immunoblots for the respective antigens were performed by ECF and Storm 860 phosphorimager. The respective membranes were reprobed for the protein loading controls, unphosphorylated target protein or actin, in this and the following studies. The specificity of antibodies employed here and in the experiments described below was determined by treating the cells with non-immune antibodies and cell lysates processed as above. Under the experimental conditions, no reactivity of these controls was observed 1-LN cells (300×106 per well in 6 well plates) incubated overnight in RPMI-S medium were washed twice with ice-cold HHBSS and a volume of RPMI-S medium added to each well. After temperature equilibration, cell in the wells were exposed to either buffer or α2M* (50 pM/25 min) and incubated as above. Reactions were stopped by aspirating the medium and a volume of CHAPS lysis buffer (Buffer B) containing 40 mM Hepe (pH 7.5), 120 mM NaCl, 1 mM EDTA, 10 mM sodium pyrophosphate, 10 mM β-glycerophosphate, 0.5 mM sodium orthovanadate, 0.3% CHAPS and a Roche Protease inhibitor cocktail tablet (1 tablet/10 ml) was added and cells lysed over ice for 15 min. The lysates were transferred to separate Eppendorf tubes, centrifuged (1000 rpm/5 min/4°C) and supernatants used for protein estimation and immunoprecipitation. Equal amounts of lysate protein (200–250 µg) in separate experiments were used for immunoprecipitation with Raptor antibodies , or Rictor antibodies  followed by the addition of 40 µl of protein A agarose and contents incubated with rotation overnight at 4°C. Raptor and Rictor immunoprecipitates were recovered by micro-centrifugation (2000 rpm/5 min/4°C) and washed twice with cold lysis buffer B. To Raptor and Rictor immunoprecipitates a volume of 4× sample buffer was added, tubes boiled for 5 min, centrifuged, electrophoresed , transferred to Hybond-P membranes and the membranes immunoblotted with antibodies specific for mTOR, Raptor, Rictor, GβL, mSIN1 and Protor. The protein bands on the membranes were quantified as above 1-LN cells (3×102M*-induced activation of mTORC1 kinase in prostate cancer cells was determined in mTOR and Raptor immunoprecipitates of cells by assaying the phosphorylation of S6-Kinase1 and 4EBP1 as described earlier 6 cells/per well in 6-well plates) incubated overnight in RPMI-S medium were washed twice with ice-cold HHBSS and a volume of RPMI-S medium added to each well. After temperature equilibration, cell in the wells were exposed to either buffer or α2M* (50 pM/25 min) and incubated as above. Reactions were stopped by aspirating the medium and a volume of CHAPS lysis buffer (Buffer B) was added and cells lysed over ice for 15 min. The lysates were transferred to separate Eppendorf tubes, micro-centrifuged (1000 rpm/5 min/4°C) and supernatants used for protein estimation and immunoprecipitation. In separate experiments, equal amounts of lysate protein (200–250 µg) were used for immunoprecipitation with mTOR antibodies (1∶50) or Raptor antibodies (1∶50), followed by the addition of 40 µl of protein A agarose and contents incubated with rotation overnight at 4°C. mTOR and Raptor immunoprecipitates were recovered by centrifugation (2000 rpm/5 min/4°C). In experiments where anti-mTOR and -Raptor immunoprecipitates were assessed for S6-Kinase1 phosphorylation, the respective immunoprecipitates were first washed once with lysis buffer containing 10 mM Tris•HCl, pH 7.2, 0.5% sodium deoxycholate, 0.1% NP-40, 100 mM NaCl, 1 mM EDTA, 1 mM sodium orthovanadate, 2 mM DTT, 10 µg/ml leupeptin and 5 µg pepstatin followed by washing with above buffer without NP 40 and containing 1 M NaCl and then with buffer containing 50 mM Tris•HCl pH 7.2, 5 mM Tris•base and 100 mM NaCl. The immunoprecipitates after each wash were recovered by micro-centrifugation (2500 rpm/5 min/4°C). To these immunoprecipitates, 40 µl of reaction buffer containing 20 mM Hepes pH 7.2, 10 mM MgCl2, 50 uM ATP and 3 µg substrate peptide was added. The reactions were started by adding 5 µCi of [33P] γ-ATP. The contents were incubated for 15 min at 30°C in a shaking water bath. The reactions were stopped by adding a volume of 4× sample buffer and tubes boiled for 5 min, centrifuged, electrophoresed in 12.5% gel transferred to membranes and membrane autoradiographed. Phosphorylated S6-Kinase substrate peptide bands were visualized and quantified as above. In experiments where the sensitivity of mTORC1 kinase in mTOR and Raptor immunoprecipitates towards PI 3-kinase inhibitor LY294002 (20 µM/25 min) and mTORC1 inhibitor rapamycin (100 nM/15 min) was determined, the cells were pretreated with these inhibitors before stimulation with α2M* (50 pM/25 min/37°C) and cell lysates processed as above. Other details for autoradiographic visualization of phosphorylation of S6-Kinase substrate peptide were as above. We also determined α2M*-induced activation of mTORC1 kinase by assaying the phosphorylation of S6-Kinase1 in vivo in mTOR and Raptor immunoprecipitates by Western blotting. Experimentation details were identical to those described above except that to mTOR and Raptor immunoprecipitates isolated from cells treated as above, was added a volume of 4× sample buffer, tubes boiled, centrifuged, electrophoresed (12.5% gel), transferred to membrane, immunoblotted with p-S6-KinaseT235/236 antibody and protein bands quantitified as above. In experiments where the effect of pretreatment of cells with antibodies directed against the carboxyl-terminal domain of GRP78 on α2M-induced phosphorylation of S6-Kinase was determined, the antibody (3 µg/ml/60 min) was added 60 min before α2M* addition. Other details were as above.α2M*-induced phosphorylation of 4EBP1 in prostate cancer cells was determined in mTOR and Raptor immunoprecipitates of 1-LN, DU-145, or PC-3 cells 6 cells/per well in 6 well plates) were incubated in RPMI-S medium overnight, washed twice with ice-cold HHBSS and a volume of RPMI-S medium added to each well. After temperature equilibration, cell in the wells were exposed to either buffer or α2M* (50 pM/25 min) and incubated as above. Reactions were stopped by aspirating the medium and a volume of CHAPS lysis buffer (Buffer B) was added and cells lysed over ice for 15 min. The lysates were transferred to separate Eppendorf tubes, micro-centrifuged (1000 rpm/5 min/4°C) and supernatants used for protein estimation and immunoprecipitation. In separate experiments, equal amounts of lysate protein (200–250 µg) were immunoprecipitated with mTOR antibodies (1∶50) or Raptor antibodies (1∶50), followed by the addition of 40 µl of protein G agarose and contents incubated with rotation overnight at 4°C. mTOR and Raptor immunoprecipitates were recovered by centrifugation (2000 rpm/5min/4°C) and washed twice with lysis buffer B and micro-centrifuged (2500 rpm/5 min/4°C). Respective immunoprecipitates were suspended in 40 µl of kinase buffer containing 10 mM Hepes pH 7.4, 50 mM NaCl, 50 mM β-glycerophosphate, 0.1 mM EDTA, 1 mM DTT, 20 mM MnCl2, 200 µM ATP and 4 µg PHAS. The reactions were initiated by adding 5 µCi of [33P]-γ-ATP, tubes incubated in a shaking water bath for 15 min at 30°C. The reactions were stopped by adding a volume of 4× sample buffer, tubes boiled for 5 min, and supernatants electrophoresed on 12.5% acrylamide gels. The protein bands were transferred to membranes and membranes autoradiographed. Phosphorylated PHAS peptide bands were visualized and quantified as above. In experiments where the sensitivity of mTORC1 kinase in mTOR and Raptor immunoprecipitates towards PI 3-kinase inhibitor LY294002 (20 µM/25 min) and mTORC1 inhibitor rapamycin (100 nM/15 min) was determined, the cells were pretreated with these inhibitors before stimulation with α2M* (50 pM/25 min/37°C) and cell lysates processed as above. Other details are as described above. We also determined α2M*-induced activation of mTORC1 kinase by assaying the phosphorylation of 4EBP1 in vivo in mTOR and Raptor immunoprecipitates by Western blotting. Experimentation details were identical to those described above except that to mTOR and Raptor immunoprecipitates isolated from cells treated as above was added a volume of 4× sample buffer added, tubes boiled, centrifuged, electrophoresed (12.5% gel), transferred to membranes, immunoblotted with p-4EBP1T37/46 antibody, and protein bands quantified as above α6 cells/per well in 6 well plates) incubated overnight in RPMI-S medium were washed twice with ice-cold HHBSS and a volume of RPMI-S medium added to each well. After temperature equilibration, cell in the respective wells were exposed to either buffer or α2M* (50 pM/25 min) and incubated as above. Reactions were stopped by aspirating the medium and a volume of CHAPS lysis buffer (Buffer B) was added and cells lysed over ice for 15 min. The lysates were transferred to separate Eppendorf tubes, centrifuged (1000 rpm/5 min/4°C) and supernatants used for protein estimation and immunoprecipitation. In separate experiments, equal amounts of lysate protein (200–250 µg) were immunoprecipitated with anti-mTOR antibodies (1∶50) or Rictor antibodies (1∶50), followed by the addition of 40 µl of protein A agarose, and the contents incubated with rotation overnight at 4°C. mTOR and Rictor immunoprecipitates were washed with lysis Buffer B containing 0.5 M NaCl, and Tris•HCl (pH 7.4) supplemented with 1 mM DTT, 1 mM PMSF, and 1 mM benzamidine. The tubes were centrifugated at 2500 rpm for 5 min at 4°C. To the immunoprecipitates, 40 µl of kinase buffer containing 50 mM Tris•HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 1 mM PMSF, 1 mM benzamidine and 20 µg/ml leupeptin was added followed by the addition of 30 µM of AktS473 kinase substrate (NH2-RRPHFPQ-FSYSA-COOH) to the respective tubes. The peptide (NH2-GGEEEEYFEL VKKKKCOOH (ZAK 3 tide) served as the control. The reaction was initiated by adding 50 µM ATP and 5 µCi of [33P]-γ-ATP in each tube and the tubes incubated for 30 min at 30°C in a shaking water bath. The reaction was stopped by the addition of 5 µl of 0.5 M EDTA in each tube, the tubes centrifuged at 3000 rpm for 3 min, 40 µl of each supernatant applied on p81 phosphocellulose paper  allowed to dry and papers washed four times each time by immersing them in a liter of 1 M phosphoric acid for 3 min. The papers were rinsed with acetone and their radioactivity was counted in a liquid scintillation counter. In preliminary experiments the kinase activities of AktS473 kinase towards control Zak 3 peptide were always 50–60% of buffer control. Hence, control peptide activities are not being shown. Experiments were performed to study the sensitivity of mTORC2 kinase activities in mTOR or Rictor immunoprecipitates with respect to the PI 3-kinase inhibitor LY294002 (20 µM/25 min) and the mTORC1 inhibitor rapamycin (100 nM/15 min). These cells were pretreated with these inhibitors before stimulation with α2M* (50 pM/25 min/37°C) and cell lysates processed as above. Other details of quantifying AktS473 kinase activation were identical to those described above. We also determined α2M*-induced activation of mTORC2 in mTORs and Rictor immunoprecipitates by Western blotting. Cells incubated overnight in RPMI-S medium were treated as above S473 and p-AktS473 bands detected by ECF and quantified in a Storm860 phosphorimager. Phosphorylation of Akt at Thr308 in mTOR and Rictor immunoprecipitates was also determined as above for AktS473 except that AktThr308 kinase substrate peptide (NH2-KTFCGTPEYLAPEVRR-COOH) was used. In experiments where we determined the effect of antibodies directed against the carboxyl-terminal domain of GRP78 on α2M*-induced AktS473 phosphorylation, the antibody (3 µg/ml) was added 60 min before α2M* addition. Other details of assaying and quantifying AktS473 phosphorylation were as above.1-LN, DU-145, and PC-3 prostate cancer cells  were incubated as above and transfected with 75 nM annealed GRP78 dsRNA and control cells were transfected with lipofectamine as described previously 2M* (50 pM/25 min). Cells transfected with scrambled dsRNA (75 nM/48 h) and treated with α2M* were used as the control. The reaction was terminated by aspirating the medium, and cells lysed in lysis buffer B. Equal amounts of lysate protein was immunoprecipitated with anti-Raptor antibodies. mTORC1 kinase activities in washed Raptor immunoprecipitates were determined by phosphorylation of S6-Kinase and 4EBP1. The effect of silencing GRP78 gene expression on mTORC2 kinase activity in mTOR immunoprecipitates and Rictor immunoprecipitates was also determined by assaying AktS473 phosphorylation.To determine the requirement of cell surface GRP78 for α2M-induced phosphorylation of Akt at S473 is brought by mTORC2, we silenced Rictor gene expression by RNAi to disrupt the assembly of mTORC2 complex and thus suppress mTORC2 kinase activation. Annealed and validated small interfering RNA of the gene was purchased from Ambion  of the sense sequence (5′-3′) GGG UUA GUU UAC AAU CAG Ctt and antisense (5′-3′) GCU GAU UGU AAA CUA ACCtt. The cancer cells were transfected with 100 nM of annealed Rictor dsRNA and control cells were transfected with lipofectamine as described previously. In preliminary experiments we found that transfection of cells with Rictor dsRNA was maximally effective at 75–100 nM and this knocked down Rictor by about 60–65% as measured by Rictor mRNA (data not shown) and protein levels. Forty eight h after transfection, the control cells were stimulated with either buffer α2M* (50 pM/25 min/37°C) or insulin (200 nM/15 min/37°C). Cells for the negative control were transfected with scrambled dsRNA  and then stimulated with either buffer, α2M* or insulin as above. The reactions were terminated by aspirating the medium and cells were lysed in a volume of Buffer B as described above. Equal amounts of lysate proteins was employed for immunoprecipitating Rictor with anti-Rictor antibodies. Rictor immunoprecipitates were used for measuring phosphorylation of Akt at S473. Equal amounts of lysate protein were also used for determining p-AktS473 by Western blotting.To further demonstrate that α2M*-induced the phosphorylation of S6-Kinase, we silenced the expression of Raptor by RNAi, which would disrupt the assembly of mTORC1 complex and thus suppress the phosphorylation of its downstream target S6-Kinase. Insulin has been widely used in studies on mTORC1 and mTORC2 signaling in various contexts, therefore, we have used insulin as a positive control in these studies. Annealed small interfering RNA of the gene was purchased from Sigma of the sense sequence (5′→3′) CUC AAC AAA UCU UUG CAG At (ID#SAS1_Hs01_00048387) and antisense (5′→3′) UCU GCA AAG AUU UGU UGA Gets (ID #SAS1_Hs)1_ 00048387_AS. The cancer cells were transfected with 100 nM of annealed Raptor and control cancer cells were transfected with lipofectamine as described previously. In preliminary experiments we found that transfection of cells with Raptor dsRNA (100 nM/48 h) knocked down α2M-stimulated Raptor pool by about 60–70% as judged by its mRNA (data how shown) and protein levels. Forty eight h after transfection the control cells were stimulated with either buffer, α2M (50 pM/20 min/37°C) or insulin (200 nM/15 min/37°C). Cells for the negative control were transfected with scrambled dsRNA  and then stimulated with either buffer, α2M (50 pM/20 min/37°C) or insulin  or Rictor dsRNA (100 nM/48 h) and cells stimulated with either α2M* (50 pM/25 min) or insulin (200 nM/15 min) as above. Cell lysates were assayed AktT308 and AktS473 phosphorylation by kinase assay as above. Levels of p-AktT308 and p-AktS473 were determined by immunoblotting as above.To further ascertain the specificity of mTORC1 in αStatistical significance of the data was determined by Students “t” test. P Values of 0.05 are considered statistically significant.2M* and its sensitivity to the mTORC1 inhibitor rapamycin and PI 3-kinase inhibitors, wortmannin and LY294002  2M* triggers pro-proliferation and pro-survival cellular responses 2M is about 2–5 µM and its proteinase activated form may comprise approximately 200–500 nM concentration of this pool 2M to α2M* 2M* is available to bind to cell surface GRP78 thus triggering mitogenic signaling and promoting cellular proliferation. In our earlier reports we observed inhibition of α2M*-induced elevated macromolecule synthesis by use of the mTOR inhibitor rapamycin and several PI 3-kinase inhibitors. α2M*-stimulated cells also showed increased phosphorylated S6-Kinase and Akt473 which is abrogated by pretreatment of cells with antibodies against the carboxyl-terminal domain of GRP78 The molecular chaperon GRP78 is constitutively expressed, but its synthesis is induced by stressful conditions that perturb protein folding and assembly within the ER 2M*- stimulated prostate cancer cells (6–10) involves cell surface GRP78 and activation of mTORC1 and mTORC2 signaling in these cells. We thus elucidated mTORC1 and mTORC2 signaling in 1-LN and DU-145 prostate cancer cells. The salient points of this study are: (1) α2M* treatment of cells causes about a twofold increase in protein synthesis which is sensitive to rapamycin and PI 3-kinase inhibitors; (2) a concentration and time-dependent increase in p-mTOR, Raptor, Rictor, p-TSC2, Rheb, p-S6-Kinase, p-4EBP1, p-AktT308 and p-AktS473 is observed in α2M*-stimulated cells; (3) stimulation of prostate cancer cells with α2M* and insulin elevates mTORC1-dependent phosphorylation of S6-Kinase and 4EBP1 as determined by kinase assays and phosphorylated protein levels in mTOR and Raptor immunoprecipitates; (4) α2M*-induces increase in S6-Kinase and 4EBP1 phosphorylation in mTOR and Raptor immunoprecipitates is greatly suppressed by pretreatment with LY294002, rapamycin, or transfection of cells with GRP78 dsRNA and Raptor dsRNA; (5) treatment of prostate cancer cells with α2M* and insulin significantly elevates mTORC2 activity as measured by phosphorylation of Akt at S473 by kinase assays and phosphorylation levels in mTOR and Rictor immunoprecipitates; (6) α2M*-induced increase in mTORC2 activity is insensitive to rapamycin, but is significantly reduced by LY294002 and transfection of cells with GRP78 dsRNA and Rictor dsRNA in mTOR and Rictor immunoprecipitates.In the present study, we hypothesized that cellular proliferation observed in α2M* is shown in Akt is activated by extracellular and intracellular stimuli to regulate survival, proliferation, differentiation, and metabolism. Full activation of Akt requires its phosphorylation at T308 in the catalytic loop by PDK1 and S473 in the hydrophobic domain by mTORC2 2M* promotes cell proliferation and cell survival in prostate cancer cells and possibly other malignancies. The availability of circulating or tissue-derived α2M regulates its conversion to receptor-recognized forms (α2M*) by complexing with proteinases, such as PSA. α2M* binds to cell surface GRP78, the availability of which is increased several fold under ER stress conditions which are prevalent in cancer tissue. Binding of α2M* to cell surface GPR78 causes its autophosphorylation triggering downstream signaling cascades which include RAS/MAPK, and PI 3-kinase/Akt/mTOR, which causes cellular proliferation and promotes survival. As a result, the TSC1-TSC2 complex is phosphorylated inhibiting its GAP activity towards Rheb, thus freeing it for mTORC1 activation. Likewise phosphorylation of PRAS40 and Deptor by Akt causes their dissociation from mTOR and promotes mTORC1 activation. α2M*-induced phosphorylation of PIP2 by PI 3-kinases promotes membrane recruitment of PDK1 and Akt1, where PDK1 phosphorylates Akt at T308 and mTORC2 phosphorylates Akt at S473 causing its full activation. This is the first demonstration of the involvement of prostate cancer cell-surface GRP78 in upregulating mTOR signaling thereby causing cancer cell proliferation and survival. These studies should be considered in the context of newer chemotherapeutic approaches for treating castration resistant prostate cancer with rapamycin analogues which target mTORC1 only 2M* in the progression of such tumors 2M*, appear in patients with prostate cancer Based on our earlier observations and our current data, we propose the following mechanism(s) by which α