This mutation does not alter basal CaV1.2 current properties or regulation of CaV1.2 current by PKA and the beta-adrenergic receptor, but abolishes CaV1.2 phosphorylation by PKA. Here, we test the contribution of the corresponding PKA phosphorylation site of the human α11.2 subunit S1898, to the regulation of catecholamine secretion in bovine chromaffin cells. Chromaffin cells were infected with a Semliki-Forest viral vector containing either the human wt or a mutated S1898A α11.2 subunit. Both subunits harbor a T1036Y mutation conferring nifedipine insensitivity. Secretion evoked by depolarization in the presence of nifedipine was monitored by amperometry. Depolarization-triggered secretion in cells infected with either the wt α11.2 or α11.2/S1898A mutated subunit was elevated to a similar extent by forskolin. Forskolin, known to directly activate adenylyl-cyclase, increased the rate of secretion in a manner that is largely independent of the presence of S1898. Our results are consistent with the involvement of additional PKA regulatory site(s) at the C-tail of α11.2, the pore forming subunit of CaV1.2.A PKA consensus phosphorylation site S1928 at the  V1.2, is modulated by protein kinase A (PKA) and protein kinase C (PKC) [β-adrenergic receptor activates cAMP-dependent phosphorylation by PKA, which increases the amplitude of voltage-dependent Ca2+ currents. It is now well established that the β-adrenergic signaling enhances inward Ca2+ currents via CaV1.2 phosphorylation in cardiac myocytes [The voltage-gated calcium channels (VGCCs) are regulated by kinases that are activated by second messenger pathways –4. The dmyocytes , adrenalmyocytes , 8, rat myocytes  and in smyocytes .Po) of the channel [β-adrenergic signaling pathways increases calcium currents ~4-fold and is attributed to the phosphorylation of CaV1.2 [PKA phosphorylation of the L-type channel leads to an increase in the open probability ( channel  and an i channel . In the f CaV1.2 .α11.2 subunit is phosphorylated by PKA on serine 1928 (S1928) [β-adrenergic regulation of CaV1.2 [The L-type channel assembles with Gs, adenylyl cyclase, PKA, and phosphatase PP2A into a signaling complex that enables the rapid modulation of the channel by the adrenergic receptor . Biochem (S1928) –17. Phosf CaV1.2 , 17–21.α11.2 pore forming subunit of rabbit CaV1.2 that is phosphorylated both in vitro and in vivo, was suggested to be responsible for the increase in Ca2+ currents [The phosphorylation of S1928, the PKA consensus phosphorylation site at the β-adrenergic regulation of cardiac L-type Ca2+ channels [β-subunit of the channel [α11.2 subunit to increase channel activity [Additional phosphorylation sites that can also participate in the channels  include  channel , 23. Sinactivity .V1.2 by the β-adrenergic pathway in mouse heart was tested in vivo using knock-in mouse with a targeted mutation of S1928 to alanine [β-adrenergic regulation of the L-type current. The conclusion led to the suggestion that PKA phosphorylation of S1928 in cardiomyocytes is functionally not involved in the β-adrenergic stimulation of CaV1.2-mediated calcium influx [More recently, the regulation of Ca alanine . This mum influx .2+-bound channel were suggested to transmit the signal from the channel to the exocytotic machinery and trigger fast release of vesicles assembled with the channel [2+ channel [In neuroendocrine cells such as bovine chromaffin and PC12 cells, membrane depolarization triggers catecholamine (CA) secretion primarily by the opening of the L-type channels –28. In t channel , 33–35.  channel . Based oα11.2 (Hu/wt) or α11.2/S1898A (Hu/S1898A) mutant subunit. The S1898 site corresponds to S1928 of rabbit cardiac cells. The frequency of the secretory events mediated by Hu/S1898A and Hu/wt-infected cells was accelerated by forskolin to a similar extent, indicating that mutating serine 1898 to alanine did not affect the forskolin-induced increase of catecholamine secretion.We examined the modulation of L-type channel and its role in mediating evoked secretion using forskolin. Secretion was triggered in cells infected with the human wt Overall, our results suggest that additional PKA-sensitive phosphorylation sites contribute to the PKA modulation of catecholamine release.α11.2 subunit, accession number AJ224873, rabbit β2b accession number X64298, and rabbit α2/δ accession number NM_001082276, were kindly donated by Dr. M. Sanguinetti (University of Utah). A modified pSFV1 (Invitrogen) plasmid, where an internal ribosome entry site from poliovirus had been inserted followed by the gene for enhanced GFP, was provided by Dr. U. Ashery.The complete human cDNAs of α11.2/S1989A. The CaV1.2α1 subunit coding sequence (AJ224873) was used as a template. The S1898A mutation was introduced by mutating the codon TCC to GCC by using the Bio-X-Act Long DNA Polymerase kit  and Quick-change site-directed mutagenesis PCR was applied . The following primers were used: 5′-TGCCTCACTAGGTCGAAGGGCCGCCTTCCACCTGGAATGTCTGAA and the 3′-TTCAGACATTCCAGGTGGAAGGCGGCCCTTCGACCTAGTGAGGCA. For amperometry studies, plasmids α11.2/T1036Y and α11.2/S1898/T1036Y were inserted upstream of the internal ribosome entry site using BamHI and BssHII restriction sites. All constructs were verified by DNA sequencing and restriction site mapping.Standard methods of plasmid DNA preparation were used to prepare the human 4 cells/cm2on glass cover slips placed in 35 mm plates, in DMEM (Gibco) supplemented with ITS-X . Cells were incubated at 37°C in 5% CO2  and were used for amperometric recordings 1–4 days after preparation at 23°C.Bovine adrenal glands were obtained at a local slaughterhouse. The adrenal medulla cells were isolated as previously described in  and platμm thin carbon fiber electrodes  and a VA-10 amplifier  held at 800 mV as described previously [2, 2 mM CaCl2, 10 mM Glucose, and 10 mM HEPES, pH 7.3 at ~23°C (adjusted with NaOH)). Individual cells were stimulated to release by a 10 sec application of isoosmotic 60 mM KCl buffer from a ~3 μm tipped micropipette placed 5–7 μm from the cell in the bath. Amperometric currents were sampled at 10 kHz, using clampex 9.2 (Axon Instruments) and low-pass filter at 1 kHz.Amperometry recordings were carried out using 5 eviously . Cells wSecretion of catecholamines from fluorescent cells identified at 480 nm excitation was recorded 16–24 hrs after infection by amperometry.in vitro transcribed RNA from pSFV-GFP, or pSFV expressing GFP through an internal ribosome entry site (IRES) motif, pSFV-Nifedipine-insensitive α11.2-IRES-GFP, or pSFV-Nifedipine-insensitive α11.2/S1898A-IRES-GFP was coelectroporated into BHK-21 cells with pSFV-Helper-2 RNA. Virus stocks were harvested 24 hr later and activated with alpha-chymotrypsin before infection studies. Approximate titers were estimated by infection of known numbers of BHK-21 cells with serial dilutions of SFV stocks, and the GFP-positive cells were counted. Generally, titers in the range of 5 × 108 infectious particles/mL were obtained.Recombinant SFV particles were generated as described previously in . BrieflyμL/dish) were added to the cultured cells (2 × 105 cells per dish) 5–48 h after plating. Infected cells were identified by their GFP fluorescence.SFV particles  and were averaged to obtain a cell mean. The rate of secretion during 10–40 sec was determined from the slopes of the corresponding cumulative spike plots. Data was analyzed as described in the text and figure legends. Error bars give standard errors. Spikes exceeding three times the background noise (>10 pA) were analyzed. All peaks identified by the program IGOR PRO  were inspected visually and bad signals were excluded manually.Xenopus laevis oocytes were removed surgically from the ovaries of anesthetized animals and transferred to a Ca2+-free medium  containing 1 mg/mL collagenase (253 U/mg) (Wortington Bioche. Corp. USA). The follicular cell layer was removed by shaking the oocytes in this buffer for 1.5–2 hr at room temperature. After extensive wash, the oocytes were transferred to ND96 buffer  containing 2.5 mM pyruvate, 100 U/mL penicillin, and 10 μg/mL streptomycin. Oocytes were incubated in ND96 for 12–20 hr before cRNA injection.Stage V-VI In vitro transcribed capped cRNA of the channel subunits were injected into the defolliculated oocytes in a final volume of 40 nL using a Drummond 510 microinjector . Oocytes were maintained at 18°C for 5 days after injection [cRNAs were prepared using the T7 Fermentas transcription kit (Lituania), and the product was monitored by gel electrophoresis and optical density measurements. njection .μm thick were captured. Exposure time was 8 μsec/pixel.Single optical sections through the oocytes were obtained with an Olympus FV1000  equipped with a 40x oil objective (N.A. 1.3). Two excitation lasers were used sequentially: 488 nm GFP and narrow-band emission filters 505–525 nm. Sequential scanning was performed with a resolution set to 512 × 512 pixels (0.621 mm/pixel), and single optical sections ~0.5 α11.2 pore forming subunit corresponds to S1898 in the human α11.2, as shown in The sequence homology of the phosphorylation consensus site of S1928 at the distal C-terminal of the α11.2 subunit was mutated to Ala and inserted into a frog expression vector. The cRNAs encoding the GFP-tagged human α11.2 subunit of CaV1.2 or the GFP-α11.2/S1898A mutant were coinjected into oocytes along with the cRNAs encoding the auxiliary rabbit α2δ and β2b channel subunits . Averaged time courses of exocytosis in the absence and in the presence of 5 μM nifedipine are shown by the cumulative histograms (upper right). Spike frequencies were quantified for individual cells as initial rates, which correspond to the linear fits to the averaged cumulative histograms (bold lines) collected from cells (n = 49) during the 10 sec of 60 mM KCl (K60) stimulation and a 20 sec poststimulus period (range 10–40 sec) (lower right).At first, using a selective Ca channel . The evothe bath   inset.inset). The overall time course of secretion determined from the normalized waiting time distributions was 0.52 spike/sec in the GFP-infected cells (lower) and 0.53 spike/sec in noninfected cells (lower). No release was detected when the cells were stimulated in a Ca2+-free solution (left) or in the absence of K60 (right).We then tested the effect of Semliki Forest virus (SFV) infection on the rate of secretion in cells using SFV viral vector harboring GFP, as previously described . The infed cells , lower aed cells , lower. solution , left ore of K60 , right.α11.2 human channel subunit (Hu/wt). As a control we monitored the kinetics of secretion induced in cells that were infected with pSFV vector expressing GFP (see above). Secretion mediated by the wt α11.2(Hu/wt) pSFV vector was monitored in the presence of 5 μM nifedipine. The nifedipine insensitivity was used to distinguish between the endogenous cellular channels and our cloned channels that were made resistant to nifedipine (Nif) by a single-amino-acid mutation, T1036Y [Xenopus oocytes [V1.2. Representative amperometric spikes elicited by a 10 sec pulse of K60 from single GFP- and in Hu/wt-infected cells are shown in Q, gray area) as well as foot width, foot amplitude, and integrated foot , are presented schematically in To study secretion mediated by the human channel, we created a pSFV vector expressing wt , T1036Y . The T10 oocytes , 39. DifThe foot amplitude and the kinetics of foot width were not significantly different, calculated by analyzing the distribution of the data and depicted as cumulative probability in the GFP- and the Hu/wt-infected cells Figures .α11.2 (Hu/wt) or the α11.2/S1898A (Hu/S18989A) subunits that harbored a second mutation T1036Y that rendered the channel nifedipine insensitive were subcloned into a pSFV, which expresses GFP through an internal ribosome entry site (IRES) motif [μM nifedipine, 16–24 hrs after infection as shown by representative amperometric currents  motif . Both HuS) motif , 39. We  Figures . The amp Figures . For eve Figures .In addition, foot values that correspond to the pre-spike signal and depict the properties of the fusion pore  did not in vitro and in vivo, either directly by β-adrenergic agonists or indirectly by forskolin [It has previously been shown that S1928 is phosphorylated by PKA, both orskolin , 15.V1.2, we applied forskolin to cells infected with Hu/wt and Hu/S1898A.To gain insight into the specific contribution of the corresponding consensus PKA site S1898 to the regulation of catecholamine release mediated via CaμM forskolin when applied to GFP-infected cells increased the frequency of the amperometric events. The analysis of cumulative spikes showed an increase from 21 ± 3 to 33 ± 3.1 spikes/cell (left). The increase in the frequency of the amperometric events induced by forskolin also led to a 1.6-fold increase in total catecholamine secretion, which was calculated as the total mean charge of spike area within the range of 10–40 sec during and after stimulation . A smaller increase (1.3-fold compared to 1.5-fold) was observed in Hu/S1898A-infected cells.In spite of the increase in the frequency of secretion by forskolin, no effect on the kinetics of the individual amperometric spikes and prespikes (foot) was observed, as presented in Table SI of the supplementary material available online at doi:10.1155/2011/746482.V1.2 like other VGCC is further modulated by phosphorylation [In bovine chromaffin cells membrane depolarization triggers catecholamine release primarily via the activation of the dihydropyridine-sensitive VGCC, known as the L-type channel. This was previously shown using selective DHP L-type channel blockers , 28. Therylation .V1.2 by protein Kinase A (PKA) and protein kinase C (PKC) further modulates the kinetics of the channel [α11.2 subunit is phosphorylated by PKA on serine 1928 (S1928) [V1.2, and here we have tested whether this site at the human pore forming α11.2 subunit contributes to the regulation of catecholamine release in bovine chromaffin cells.Phosphorylation of the L-type channel or Ca channel . Biochem (S1928) –17. ThisAt first we confirmed that bovine chromaffin cells infected with pSFV vector containing GFP retained their sensitivity to nifedipine. Secretion of CA that was monitored using carbon fiber amperometry was reduced by more than 85%.α11.2 (Hu/wt) and the α11.2/S1898A (Hu/S1898A) subunits, which are equally expressed and targeted to the cell membrane, were introduced into chromaffin cells via Semliki forest viral infection. The two channel subunits each harbored an additional mutation that rendered them nifedipine insensitive. Secretion in the infected cells was carried out in the presence of nifedipine, which enabled the functional discrimination of L-type-mediated secretion contributed by exogenously expressed human channels from that of native bovine Ca2+ channels. The frequency of secretion and the total catecholamine released that was mediated by the endogenous bovine channel was similar to that of the human α11.2 (Hu/wt) channel. Also the spike parameters and the prespike (foot) parameters were indistinguishable.Both the human wt α11.2 [β-adrenergic receptor modulation of channel activity.Protein microsequencing of phospho-peptide mapping confirmed that phosphorylation occurs at a PKA site, at serine 1928 near the C-terminus of cardiac α11.2 . Based oα1 subunits of the L-type channel thereby, upregulating its activity. It was previously shown that forskolin elevation of cAMP could be fully accounted for by the activation of the β-adrenergic receptor [α11.2 subunit to secretion by mutating Ser1898 to Ala, the site that corresponds to the S1928 in rabbit cardiac α11.2 subunit.Forskolin is known to activate adenylyl cyclase, which by producing cAMP and activating PKA, leads to the phosphorylation of the receptor , 42, 43.The modulation of secretion by the PKA activation pathway was tested by forskolin that was applied to chromaffin cells infected with the Hu/wt or Hu/S1898A subunits.α1 subunit was most likely phosphorylated by PKA to the same extent as the endogenous α1 subunit.The direct activation of adenylyl-cyclase by forskolin in GFP-infected and in the Hu/wt-infected cells led to a similar fold increase in the rate of depolarization-evoked secretion and total catecholamine release. These results suggest that the human On the other hand, the positive impact of forskolin on the rate of secretion was slightly lower in cells infected with the Hu/S1898A subunit. These results suggest that forskolin upregulates evoked secretion only marginally via S1898.β-adrenergic regulation of calcium currents in cardiomyocytes [V1.2S1928-129B6F2 mice, no detectable differences in heart rate were detected compared to control animals during isoproterenol infusion [α11.2 subunit or in other channel subunits [These results are in agreement with others  who havemyocytes . Furtherinfusion . It is psubunits , 44 and subunits .2+ channels are phosphorylated in basal state by high levels of PKA phosphorylation, while dephosphorylation reduces their activity [In our studies, both the Hu/wt and the Hu/S1898A channel subunits mediate faster rate of secretion by forskolin. Since forskolin modified secretion largely independently of the presence of Ser or Ala at position 1898, we concluded that the cAMP-dependent PKA has contributed to the modulation of secretion via other selective sites . Other sactivity , 45.In summary, our results show that mutating S1898 hardly affected the forskolin-induced increase in depolarization-evoked secretion in the neuroendocrine cells. Given that a similar forskolin-mediated increase in the rate of secretion was observed in cells infected with Hu/wt and Hu/S1898A mutant, our results indicate that other phosphorylation sites either at the L-type channel auxiliary subunits or other proteins of the secretory apparatus are involved in the upregulation of depolarization-evoked secretion in chromaffin cells.α111.2 subunits of Hu/wt and Hu/S1898A. The cells were stimulated by a 10 sec puff of 60mM KCl .Supporting material: The kinetic parameters of the spikes and foot of elicited in bovine chromaffin cells infected by pSFV vectors of the Click here for additional data file.


Susceptibility-weighted imaging (SWI) has been proven to be superior to T2*-weighted imaging and also other existing magnetic resonance imaging (MRI) techniques for the detection of iron content and hemorrhage in the brain. The purpose of this study was to compare SWI with T1WI, T2WI and T2*WI in detecting splenic siderotic lesions.P<0.05). The average size of the nodules detected by SWI was larger than that of those detected by T1WI, T2WI or T2*WI . SWI provided superior contrast and visibility for splenic siderotic nodules compared to any other sequence .Twenty-two patients with splenic siderotic nodule were imaged with non-contrast MRI T1WI, T2WI, T2*WI and SWI at 3.0 Tesla. Imaging data were independently analyzed by two experienced radiologists. The number of splenic siderotic nodules was counted, and the size (largest diameter) was measured. The conspicuity was calculated as the nodule to background parenchyma intensity ratio. We found that SWI detected a larger average number of splenic siderotic nodules than T1WI, T2WI, or T2*WI (all SWI may be a better detection scheme for splenic siderotic nodules than T1WI, T2WI and T2*WI. Iron is a critical element for human life. Abnormalities of iron metabolism are associated with the formation of siderotic nodules in the spleen, liver and bone marrow, contributing to numerous diseases such as hepatic cirrhosis, thalassemia, hemochromatosis, sickle cell disease, and aplastic anemia Because of their clinical importance, many techniques have been developed to detect hepatic and splenic siderotic nodules. Ultrasound lacks the ability to discriminate iron contents. It is notorious for user dependents and can be particularly difficult with obese patients. In comparison, computed tomography (CT) produces high-quality images, especially with the enhancement multi-detector CT and contrast agents. However, CT also has limited sensitivity for the detection of iron and has several disadvantages including radiation exposure, anaphylactic shock and nephrotoxic impairment By adding phase information, susceptibility-weighted imaging (SWI) achieves even greater sensitivity for detecting focal iron than T2* contrast imaging. It has been well documented that SWI is superior to T2*WI and to other existing magnetic resonance imaging (MRI) techniques for the detection of iron content and hemorrhage in the brain All research procedures were approved by the Institutional Review Board of our institution and were conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained for all patients prior to the study.From March 2011 to December 2012, twenty-six consecutive patients participated in this study at our hospital. Patient inclusion criteria were as follows: 1) Patient were able to sustain a 20-second breath hold; 2) Patient age was between 18 years and 80 years; 3) Patient had at least one visible splenic siderotic nodule previously detected by MR; and 4) no other lesions in the spleen were found by previously MR. The diagnosis of siderotic nodules was based on hypointense on MRI compared to background parenchyma excluding cross sections of blood vessels in the spleen 2, the matrix was 384 × 250, and slice thickness was 5 mm with a 1 mm gap. Two to three breath holds of 15- to 20-s duration were used for each sequence. The recently developed multislice 2D GRE sequence with SWI reconstruction  was used to perform abdominal SWI. SWI post-processing was done online using the following procedure: 1) Original images from each channel were passed through a 32 × 32 high pass filter to remove background artifacts; 2) Modulus-weighted magnitude and phase images were combined into a complex image; 3) High pass filter corrected phase images were created from the complex images; and 4) a normalized phase mask was calculated from each corrected phased image and multiplied with the magnitude image to produce the final SWI and phase image For all subjects, MR imaging of the upper abdomen, without intravenous or oral contrast enhancement, was performed on a 3.0-T whole body system  equipped with a standard 12-channel matrix coil. The imaging sequence included transverse T1-weighted 2D gradient echo (GRE) , transverse T2-weighted 2D fast spin echo , transverse T2*-weighted 2D GRE  and transverse abdominal 2D SWI . For all sequences, field of view (FOV) was 380 × 285 mmTwo radiologists, with 10 and 12 years of experience in abdominal imaging, independently analyzed the images. They were blinded to previous clinical MR interpretations, clinical history and pathologic results. Based on a previous study, spots that were hypointense relative to background spleen parenchyma on T1-, T2-, T2*- weighted, and SWI images were considered siderotic nodules after excluding cross sections of blood vessels in the spleen t test. Cohen’s kappa method was used to assess inter-reader agreement.All statistics analyses were performed using SPSS statistical software . P values of 0.05 were considered statistically significant. The number, size, and conspicuity of splenic siderotic nodules identified by SWI and by T1WI, T2WI and T2*WI were compared by paired The average number of splenic siderotic nodules detected by SWI 38.2±19.7) was greater than that by T1WI, T2WI or T2*WI . The average size of splenic siderotic nodules on SWI (2.83±1.35) was higher than that on T1WI, T2WI or T2*WI,  , 2, 3. T±19.7 wasRegarding conspicuity, SWI performed significantly better than T1WI, T2WI and T2*WI (P<0.001) , 3. LesiInter-reader correlation, based on results provided by each individual reader and determined using the K coefficient, was estimated at 0.82–0.89.To our knowledge, this is the first study of SWI applied to splenic siderotic nodule detection. We found that the average number and size of splenic siderotic nodules detected by SWI were greater than those detected by T1WI, T2WI or T2*WI. There was no case where SWI showed a smaller number or size than the other imaging techniques. With regards to nodule conspicuity, a statistically significant superiority of SWI over T1WI, T2WI and T2*WI was found. Splenic siderotic nodules detected on SWI had superior contrast and visibility than on any other MRI sequence. These findings suggest that SWI is powerful, efficient and the best imaging method for the identification and evaluation of spleen iron detection. In past studies, T2*WI has been used reliably to quantify myocardial and hepatic iron Detection of siderotic nodules has broad medical applicability. With approximately 60,000 new cases each year, thalassemia is the most common genetic disorder worldwide. Iron overload in patients with thalassemia results from both excessive iron absorption and transfusion. Precise iron overload detection can prevent iron toxicity and avoid excessive administration of chelator This study has several recognized limitations. First, this is a retrospective study with a small sample size, and only a single representative image per imaging technique was chosen for evaluation. Correspondingly, there is potential for bias. Second, not all splenic siderotic nodules shown on SWI could be verified by pathological examination. Third, because abdomen SWI is sensitive to motion artifact from respiratory movement, our use of three consecutive breath-hold acquisitions may not be feasible in all cirrhotic patients, especially those with pulmonary compromise from hepatopulmonary syndrome or ascites.In conclusion, we demonstrated the value of SWI in improving the detection of splenic siderotic nodules. SWI may be a better detection scheme for splenic siderotic nodules than T1WI, T2WI and T2*WI. SWI should be a useful addition to imaging techniques currently in clinical practice.


Branched structures arise in the intra-cellular signaling network when a molecule is involved in multiple enzyme-substrate reaction cascades. Such branched motifs are involved in key biological processes, e.g., immune response activated by T-cell and B-cell receptors. In this paper, we demonstrate long-range communication through retrograde propagation between branches of signaling pathways whose molecules do not directly interact. Our numerical simulations and experiments on a system comprising branches with JNK and p38MAPK as terminal molecules respectively that share a common MAP3K enzyme MEKK3/4 show that perturbing an enzyme in one branch can result in a series of changes in the activity levels of molecules “upstream” to the enzyme that eventually reaches the branch-point and affects other branches. In the absence of any evidence for explicit feedback regulation between the functionally distinct JNK and p38MAPK pathways, the experimentally observed modulation of phosphorylation amplitudes in the two pathways when a terminal kinase is inhibited implies the existence of long-range coordination through retrograde information propagation previously demonstrated in single linear reaction pathways. An important aspect of retrograde propagation in branched pathways that is distinct from previous work on retroactivity focusing exclusively on single chains is that varying the type of perturbation, e.g., between pharmaceutical agent mediated inhibition of phosphorylation or suppression of protein expression, can result in opposing responses in the other branches. This can have potential significance in designing drugs targeting key molecules which regulate multiple pathways implicated in systems-level diseases such as cancer and diabetes. Such modules, often associated with specific functions, are distinguished by a relative level of insulation from the activity of other molecules The intra-cellular signaling machinery is an extremely large and complex network that is best understood in terms of interactions between branched pathways. For example, the MAP3K MEKK-1/2/3 are known to be capable of activating both JNK and ERK pathways However, such linear or chain-like reaction schemes imply a rigid relation between stimulus and response, precluding the possibility of the system switching to a different response for the same signal under altered circumstances. This adaptive ability is essential for survival of a cell in a noisy and dynamic environment. In fact, many linear cascades are actually part of In this paper we show that, in addition to having more flexibility compared to linear cascades, branched pathways allow complex long-range coordination of activity even in the absence of any direct long-range connections  between molecules. This allows complex dynamical control in conjunction with economy of wiring, measured by the total number of different possible chemical reactions, in the intra-cellular signaling network. Restricting the total number of links is desirable in a complex system, as high connectivity in a network can reduce the specificity of the response as many different signals can elicit the same activity The possibility of reverse communication between components of the intracellular signaling network implies that branched pathways cannot be considered as simple open-loop circuits. Instead, retrograde propagation effectively implement closed-loop or feedback circuits, where perturbing one of the “output” elements can result in changes at the branch-point (at the “input”-end of the module), and thus, eventually to all other outputs of the module. Although the existence of implicit feedbacks in activation-deactivation reaction pathways have been shown earlier in the case of a simple linear MAPK cascade A,B-MAPKA,B cascade where the MAP3K, upon activation by an input signal (stimulus) S, phosphorylates two different types of MAP2K (designated MAP2KA and MAP2KB respectively). The doubly phosphorylated MAP2Ks in turn act as enzymes for the phosphorylation of the respective MAPK molecules designated as MAPKA and MAPKB respectively  using an ATP inhibitory agent targeting the ATP binding site, and (ii) using siRNA to block the expression for the gene coding for MAPKA, with both interventions having less than 100% efficiency.The perturbation we have discussed above corresponds to complete blocking of MAPKA  in [MAPKA] leads to increase (decrease) in [MAP2KB**] and [MAPKB**] respectively (A), viz., the siRNA-type inhibitor which inhibits a fraction of total concentration of MAPKA and the pharmaceutical inhibitor which blocks the ATP binding site of a fraction of total MAPKA. The simulations reveal that while concentration depletion of MAPKA results in depletion of [MAP2KB**] and [MAPKB**]  for the common kinase enzyme . We have investigated the contribution of such competition in producing retrograde propagation by comparing the system with an artificial cascade model that allows only single phosphorylation of MAPKA,B and MAP2KA,B so that competitive inhibition is absent. We observe that the magnitude of retrograde propagation (and hence the amplification of kinase activation in the unperturbed branch) for the system with singly phosphorylated MAP2K and MAPK is significantly reduced  for the same enzyme (MAP3K*), both contribute to the magnitude of retrograde propagation seen in a branched network. If these are the only factors affecting the degree of amplification of kinase activity in the unperturbed branch then the results of the original two-branch system should be reproducible in a hypothetical four-branch model network with doubly phosphorylated MAPK but only singly phosphorylated MAP2K (A), the effect on the unperturbed branches is relatively less compared to the original system where the MAP2K molecules are doubly phosphorylated  the competition between branches for a common enzyme at the branch-point, (ii) sequestration of a kinase through binding in an ESK complex In a biological setting, the concentrations of kinases and phosphatases are usually of comparable magnitude and the systems are exposed to a wide range of signal strengths Our demonstration that local intervention in a signaling network can have remarkable non-local consequences has implications for understanding how the intricate machinery of information processing functions in the cell. In particular, reciprocal inhibition between parallel pathways can occur without the involved agents directly reacting with each other. This allows a high level of adaptability in control without a concomitant increase in the wiring complexity in the network, leading to a more efficient system design. Long-range effects also assume importance in light of the current experimental paradigm in systems biology where the observation of up- or down-regulation of activity for a molecule as a result of perturbing another molecule is assumed to indicate the existence of a direct interaction between them The results reported here have potential significance for designing drugs against systems-level diseases such as cancer that proliferate through complex orchestration of multiple signaling molecules We can, for example, consider the proteins JNK and p38MAPK, both of which are now established to be intimately involved in the proliferation of cancer and its cure. However, the respective functions of these two molecules in cancer development are not well-understood and their contribution to genesis and propagation of cancer may sometimes appear to be contradictory Complexity of a signaling network We have shown here that different types of perturbations having the same objective , while having similar consequences in a linear signaling cascade, can give rise to very different results in a branched network. Such distinct responses to apparently similar perturbations may be crucial when dealing with co-regulated diseases. For example, JNK signaling is enhanced and p38MAPK signaling is abrogated in different cancers Our results may also be used to understand the evolutionary advantage of intracellular pathogens which only target molecules in one branch of parallel MAPK pathways but which can nevertheless modulate the cellular response to its own advantage. These pathogens often target the MAPK module as the upstream signaling intermediates converge to MAPK for final integration of the signal deciding the cellular responses. As a result, the pathogens do not need to devise extremely complicated interception strategies targeting many types of molecules in order to survive within the host. As the signal strength cannot be adjusted beyond the MAPK module, the pathogen strategy would be a winning one. The retrograde propagation capability of a branched motif described here can explain how host cell signaling can still be adjusted to maintain the MAPK-dependent cellular functions.The time-evolution of the different molecular concentrations in the branched motif has been described using a set of coupled ordinary differential equations (ODEs) where each enzyme (E)-substrate kinase (SK) reaction has two steps: (i) a reversible enzyme-substrate kinase complex (ESK) formation step . The robustness of the system response is measured as a function of the degree of variation in its parameter values quantified by the Total Parameter Variation (TPV) i denotes the value of the ith parameter in a given realization and pA** and MAP2KB** on blocking MAPKA activation for signal strengths [S] = 10−12 M (A–B), 10−10 M (C–D), and 10−8 M (E–F) as a function of TPV. We observe that even relatively large deviations from the HF parameter values produce results qualitatively similar to that reported in our paper.Robustness analysis for the results reported here with respect to variation in the parameters have been carried out by performing Monte Carlo simulations over BALB/c mice from Jackson Laboratories  have been bred in our (NCCS) experimental facility. All animal experiments have been performed according to the animal use protocol approved by the Instiutional animal care and use committee . P388D1, a macrophage-like cell line Thioglycolate-elicited macrophages have been treated with an agonistic anti-CD40 antibody, as indicated in In a 6-well tissue culture plate, 2×105 cells/well have been cultured in antibiotic-free RPMI-1640 supplemented with 10% FCS. The experiments have been performed three times and the data from a representative experiment has been shown.Pharmaceutical inhibitors have been used for blocking the phosphorylation of JNK and p38MAPK. For blocking p38MAPK phosphorylation the agent SB203580 has been used and JNK phosphorylation has been blocked using SP600125. For the kinetic studies with inhibitors the cells have been pre-incubated with specific inhibitor type (SB203580 or SP600125) for 1 hour and subsequently the cells have been stimulated for the desired time.Western blot data have been subjected to densitometry analysis and phosphorylation ratio (PR) =  for individual kinase has been calculated. For the kinetics of phosphorylation in the wild-type condition and in kinase inhibition (p38MAPK and JNK), relative PR values at different time points under inhibited conditions have been compared to their respective controls. For the inhibitor studies PR before inhibition for all kinases has been scaled to 1 and the respective changes (increase or decrease) in the PR after inhibition have been calculated accordingly. To avoid possible artifacts resulting from transcription mediated feedback such as the induction of MAPK phosphatase, which can become significant at longer durations Figure S1Role of competition between the two branches in binding to MAP3K*. Relative increase of response as a function of the ratio of the binding rates of MAP2KA (B (A activation and (B) MAPKB activation. Relative increase of response as a function of the ratio of the binding rates of MAP2KA (B (A activation and (D) MAPKA activation.(TIF)Click here for additional data file.Figure S2Role of asymmetry for reaction parameters in the two branches. Relative increase of response as a function of the signal on blocking MAPKA activation when (A) the product formation rates for branch A ((TIF)Click here for additional data file.Figure S3Robustness of the effect of retrograde propagation in branched motifs with respect to parameter variation. The variation in the relative increase in response, i.e., concentrations of MAPKB** and MAP2KB** on blocking the phosphorylation of MAPKA, measured in terms of Total Parameter Variation (TPV) on randomly varying the 38 parameters in the model. The dots in each figure indicate the individual values obtained from 104 realizations for three different signal strengths: (a–b) [S0] = 10−12 M, (c–d) [S0] = 10−10 M and (e–f) [S0] = 10−8 M. The 38 parameters, which include total concentrations and reaction rates for all kinases and phosphatases at a given branch  are randomly chosen from uniform distributions bounded between physiologically plausible minimum and maximum values for the parameters that are given in (TIF)Click here for additional data file.Table S1Parameter values for reactant total concentrations and reaction rates used in the model simulation. The same values for the corresponding parameters are used in the different branches.(XLS)Click here for additional data file.Text S1Equations describing the dynamics of the model system with two branches.(PDF)Click here for additional data file.


Consistently, EGLYA has the highest score of in silico prediction of p140Cap phosphorylation. To further investigate the p140Cap function, we performed site specific mutagenesis on tyrosines inserted in EGLYA and EPLYA, a second sequence with the same highest score of phosphorylation. The mutant protein, in which both EPLYA/EGLYA tyrosines were converted to phenylalanine, was no longer tyrosine phosphorylated, despite the presence of other tyrosine residues in p140Cap sequence. Moreover, this mutant lost its ability to bind the C-terminal Src kinase (Csk), previously shown to interact with p140Cap by Far Western analysis. In addition, we found that in vitro and in HEK-293 cells, the Abelson kinase is the major kinase involved in p140Cap tyrosine phosphorylation on the EPLYA and EGLYA sequences. Overall, these data represent an original attempt to in vivo characterise phosphorylated residues of p140Cap. Elucidating the function of p140Cap will provide novel insights into its biological activity not only in normal cells, but also in tumors.Protein phosphorylation tightly regulates specific binding of effector proteins that control many diverse biological functions of cells . p140Cap is an adaptor protein, specifically expressed in brain, testis and epithelial cells, that undergoes phosphorylation and tunes its interactions with other regulatory molecules via post-translation modification. In this work, using mass spectrometry, we found that p140Cap is  We have already shown that p140Cap is tyrosine phosphorylated in epithelial cells upon integrin-mediated adhesion and EGF treatment p140Cap contains several serine and tyrosine residues, which could undergo phosphorylation upon different biological stimuli. Using large-scale phosphoproteomic studies (Csk and the Csk-homologous kinase (Chk) are endogenous inhibitors constraining the activity of the Src-family kinases (SFKs) in cells. Both kinases suppress SFKs by selectively phosphorylating their consensus C-terminal regulatory tyrosine 3) using liquid chromatographic separation (LC) and hybrid linear ion trap (LTQ)-FT mass spectrometers 2 mode automatically triggered data-dependent MS3 fragmentation of the precursor ion. This results in high yield of peptide backbone fragments, determining a high confidence peptide identification and phosphorylation site assignment Mass spectrometry (MS)-based proteomics has been widely used for studies of protein phosphorylation 3-based analytical strategy targeted toward the detection and sequencing of in vivo phosphopeptides derived from p140Cap and we identified one phosphotyrosine and three phosphoserine residues. By site directed mutagenesis we defined the tyrosines contained in the sequences EGLYA and EPLYA as the major residues responsible for p140Cap tyrosine phosphorylation and functionally validated their essential role in binding to the Csk kinase. Moreover, we found that in vitro an in Human Embryonic Kidney (HEK)-293 cells, the Abelson (Abl) kinase is the major tyrosine kinase able to trigger p140Cap phosphorylation on these motifs.Here we applied pdMSHuman embryonic kidney HEK-293 and human breast cancer MCF7 cells were obtained from ATCC and cultured in DMEM with 10% FCS and Penicillin/Streptomycin. p140Cap monoclonal and polyclonal antibodies were produced in our laboratory as previously described in 2, and neutral loss-dependent MS3 acquisition. Survey full scan MS spectra were acquired between m/z 300 Th and 1600 Th by FTICR with resolution r 25000 at m/z 400. Then, the three most intense ions of each survey full scan MS spectrum were sequentially isolated for accurate mass measurements by a FTICR “selected ion monitoring” (SIM) scan. These ions were also fragmented in the linear ion trap using collision-induced dissociation. Data-dependent settings were enabled to trigger a MS3 scan when a neutral loss of 97.99, 48.99 or 32.66 Da  was detected among the 10 most intense fragment ions of the MS2 spectra.Nanoscale liquid chromatography mass spectrometry experiments were performed on an Agilent 1100 nanoflow system (Agilent Technologies) coupled a 7-tesla Finnigan LTQ-FT mass spectrometer  equipped with a nanoelectrospray ion source  as described previously n data were processed  using the program DTASuperCharge . The processed files were subsequently searched against the human sequence library in the International Protein Index (IPI) and UniProtKB/Swiss-Prot protein sequence databases using an in-house Mascot server . The databases searches were performed choosing trypsin as enzyme and one miss cleavage was allowed. Carbamidomethyl (Cys) was considered as the fixed modification. Oxidation (Met), N-acetylation (protein) and phosphorylation (STY) were chosen as variable modifications. The data were searched with a peptide mass tolerance of ±30 ppm and a fragment mass tolerance of ±0.6 Da.MSpEGFP-N1-p140Cap cDNA EPLY/FA F 5′GAAGGAGCCGTTGTTTGCTGCTTTTCCTGGCEPLY/FA R 5′GCCAGGAAAAGCAGCAAACAACGGCTCCTTCEGLY/FA F 5′GGGCGAGGGCCTCTTTGCCGATCCCTACGGGEGLY/FA R 5′CCCGTAGGGATCGGCAAAGAGGCCCTCGCCCFY/FELE F 5′GCTCGCAATGTCTTCTTCGAGCTGGAAGACGTCFY/FELE R 5′GACGTCTTCCAGCTCGAAGAAGACATTGCGAGCDPY/FG F 5′TGCCGATCCCTTCGGGCTGCTGCACGAGGDPY/FG R 5′CCTCGTGCAGCAGCCCGAAGGGATCGGCA.pcDNA3-BCR-Abl coding for the active form of Abl Csk full length was cloned into pQE vector by amplification of I.M.A.G.E. full Length cDNA clone IRAVp968G01111D, using specific primers. The Csk SH3 (37–182 bp from ATG) and SH2 (239–513 bp from ATG) domains were amplified by Csk full length using specific primers, and the amplification products were cloned into pGEX 4T3 vector using BamHI and EcoRI restriction enzymes. Csk Delta SH2 was originated by cloning in the pQE vector two fragments, one amplified from 1 to 239 bp with BamHI and XbaI restriction sites at the end, and the other amplified from 513 to the stop codon with XbaI and HindIII restriction sites at the end. Recombinant fusion proteins were produced in E. coli as described Far-Western analysis was performed as described in 2, 1 mM EDTA, 1% Triton X-100, and 10% glycerol) containing protease and phosphatase inhibitors . In some experiments cells were treated for 5 minutes with 100 micromolar of Pervanadate Stock solution  or starved overnight and stimulated with 20% Fetal Bovine Serum (FBS) for 30 min. In specific experiments cells were transfected with p140Cap and its mutant together with the pcDNA-3-BCR-Abl construct. Cell extracts were centrifuged at 13,000×g for 10 minutes, and the supernatants were collected and assayed for protein concentration using the Bio-Rad protein assay method (Bio-Rad). For immunoprecipitation, Dynabeads (Invitrogen) were cross linked with the specific p140Cap monoclonal antibody, following manufacture protocol. Cell extracts were incubated with the Dynabeads-conjugated antibody overnight at 4°C. The immunoprecipitates were washed three times with 1 ml of lysis buffer, resolved on SDS-PAGE, transferred onto nitrocellulose and reacted with specific antibodies. As input, fifty micrograms of cell extracts were analysed by SDS-PAGE and Western Blotting.To analyze phosphorylation of p140Cap  and of its mutants, HEK-293 cells were transiently transfected with the corresponding cDNAs by conventional calcium phosphate precipitation protocols. 48 hrs after transfection, cells were extracted with lysis buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, 1.5 mM MgClThis assay was performed by ProQinase . The phosphorylation profile of two biotinylated sample peptides, SIIKIYRKEPLYAAFPGSHLTNGDL and LAGKAGGMVLVKGEGLYADPYGLLH, synthesized by JPT Peptide Technologies  was determined at three concentrations each  in triplicate in a radiometric activity assay based on streptavidin-coated FlashPlateTM Plus, using three recombinant protein kinases, Abl, Alk and Src, respectively.2, 3 mM MnCl2, 3 micromolar Na3VO4, 1.2 mM DTT, 50 microgram/ml PEG20000, 1 micromolar ATP/[gamma P33]-ATP (8.0×1005 cpm per well), protein kinase and sample peptide (1/0.5/0.25 micromolar). The assay plates were incubated at 30°C for 60 minutes. Subsequently, the reaction cocktails were stopped with 20 microliters of 4.7 M NaCl/35 mM EDTA. The reaction cocktails were transferred into 96-well streptavidin-coated FlashPlateTM Plus , followed by 30 min incubation at room temperature on a shaker. Subsequently the plates were aspirated and washed three times with 250 microliters of 0.9% NaCl. Kinase activity dependant transfer of P33 (“counting of cpm”) was determined with a microplate scintillation counter .The reaction cocktails were pipetted into 96 well, V-shaped polypropylene microtiter plates (“assay plates”) in the following order: 10 microliters of kinase solution, 40 microliters of buffer/ATP/test sample mixture. The reaction cocktails contained 60 mM HEPES-NaOH, pH 7.5, 3 mM MgCl3 approach to detect the in vivo phosphorylation of p140Cap in human breast cancer cells. p140Cap was purified from extracts of MCF7 cells by immunoprecipitation with specific monoclonal antibodies against p140Cap, and subjected to tryptic digestion. The MS and computational analyses confirmed that the protein recovered from the SDS-PAGE gel band contained only the p140Cap protein. They also revealed four singly phosphorylated peptides with masses 1208.5540, 1580.7613, 1593.7919 and 1825.9329 Da : 913.9091 Th, charges (z): 2+] showed an extensive peptide backbone fragmentation, allowing a high-confidence phosphorylation assignment  did not contain a phosphoryl group and ruled out the possibility of phosphotyrosine-400. Therefore, the in vivo mapping revealed the presence of a tyrosine phosphorylated residue, inserted in the sequence 392-GEGLpYADPYGLLHEGR-407 (briefly indicated as EGLYA)  . Interes2 spectrum the peptide with mass 1208.5540 Da,  lost 48.9975 Th generating an ion  which was subjected to MS3 analysis , is shown in 7 ion (indicated as b*7) and of y8, which lost phosphoric acid (yDelta8) suggested the presence of the phosphorylated serine-1003. The b2, b4, b6 ions are not modified, excluding the presence of a phosphoryl group on threonine-997 or serine-1001. Overall, three peptides contained phosphorylated serine residues (http://www.uniprot.org/uniprot/Q9QWI6), underlying the relevance of phosphorylation of these sites across the species.In MSanalysis . The fraentation , indicatentation . The MS2residues , namely in vivo phosphorylation on multiple sites, specifically identified in one tyrosine and three serine residues. Since we have shown that integrin-mediated adhesion or serum treatment induce p140Cap tyrosine phosphorylation In human breast cancer cells p140Cap undergoes Since we have already demonstrated that p140Cap directly binds to Csk We closely examined our liquid chromatography data in order to find evidence of the in vivo Y264 phosphorylation in MCF7 cells. The non-phosphorylated form of the peptide 261-EPLYAAFPGSHLTNGDLR-278 was automatically subjected to MS/MS fragmentation. This peptide (mass: 1956.96 Da) was detected as 3+ (m/z: 653.33), at retention time (RT) 30.17 min (data not shown). Thanking into account these features, we calculated a possible mass of the 261–278 phosphorylated form (2036.93 Da). In a chromatogram at RT 30.46 min, there was peak at m/z 679.99, (z: 3+), indicating a mass of 2036.93. This could correspond to the phosphorylated 261–278 peptide, but it was not selected for MS/MS fragmentation. In other runs we did not find the 679.99 peak suggesting that the tyrosine 264 residue (EPLYA) can be phosphorylated in a very dynamic way whose kinetics is different from the tyrosine 396 (EGLYA) in human breast cancer MCF7 cells (data not shown).in vivo data, we used the NetPhos program to predict the score of putative phosphorylation by tyrosine kinases Taking into account our Y/FA or EGLY/FA, p140Cap tyrosine phosphorylation was decreased compared to WT, indicating that both tyrosine residues contribute to p140Cap phosphorylation. Strikingly, the double mutant  was completely defective in phosphorylation (Y/FELE or ADPY/FG (not shown) and the double mutant  were still phosphorylated on tyrosine at a similar extent of the WT (Wild type p140Cap (WT) and the mutant cDNAs were transiently transfected in Human Embryonic Kidney 293 (HEK-293) cells. Within 48 hours upon transfection, cells were treated with 100 micromolar pervanadate solution for 5 minutes to significantly inhibit phosphatase action and enhance tyrosine phosphorylation. p140Cap and its mutants were then immunoprecipitated and analysed for tyrosine phosphorylation by blotting with antibodies to phosphorylated tyrosines. In cells expressing the single tyrosine mutant EPLrylation . In contf the WT . TherefoOur previous results showed that p140Cap can directly interact with the Csk protein by Far western analysis Y/FA, EGLY/FA), indicating that these two phosphorylated tyrosines mediate Csk binding.Tyrosine phosphorylation at the EPIYA-like motif allows the protein CagA and Pragmin/Sgk223 to interact with the SH2 domain of Csk Y/FA, EGLY/FA double mutant, upon FBS treatment, p140Cap tyrosine phosphorylation did not increase over the basal level that was already much lower than that of p140Cap WT. In these conditions Csk was not detectable in p140Cap EPLY/FA, EGLY/FA double mutant immunoprecipitates , we took advantage of the KinaseFinder service package  to find out whether the EPLYA and EGLYA peptides could function as substrates for these three recombinant protein tyrosine kinases in an in vitro system. As shown in EPLYAAFPGSHLTNGDL including the EPLYA sequence, were incubated with recombinant purified kinases, only the Abl kinase was able to significantly trigger peptide phosphorylation at both 0,5 and 1 micromolar substrate concentrations. On the other hand, both Abl and Alk kinases were able to phosphorylate the synthetic peptide LAGKAGGMVLVKGEGLYADPYGLLH, including the EGLYA sequence, although with low efficiency, at the high substrate concentration (1 micromolar) (in vitro assays and the ALK kinase is not expressed in HEK-293 cells (data not shown), we focused our analysis on the ability of Abl to trigger p140Cap tyrosine phosphorylation in these cells.In order to identify the tyrosine kinases involved in phosphorylation of the EPLYA and EGLYA sequences, we first performed romolar) . Since Sin vitro kinase assays, the use of Src inhibitor SU6656 did not affect p140Cap tyrosine phosphorylation  together with the constitutive active form of Abl, namely BCR-Abl Y/FA, EGLY/FA) was poorly phosphorylated by active BCR-Abl , based on the use of the NetPhos algorithm in silico data match accurately with the in vivo analysis. Moreover, this tyrosine residue has also been identified as phosphorylated by a global phosphoproteomic analysis of murine brain in silico prediction (0.980), leading us to analyse the relevance of both residues by site specific mutagenesis. The observation that p140Cap mutated in EPLYA or EGLYA is still phosphorylated on tyrosine even though at a low level, suggests that each of these two tyrosines can be independently phosphorylated. Moreover, the fact that the double mutant in EPLYA and EGLYA is no longer phosphorylated, demonstrates that these two tyrosines are the most relevant in determining p140Cap tyrosine phosphorylation. Regarding the dynamics of the EPLYA modification in human breast cancer MCF7 cells, liquid chromatography data indicated that the tyrosine 264 residue (EPLYA) could be in-vivo phosphorylated in a very dynamic way, with a kinetics different from the tyrosine 396 (EGLYA).The tyrosine embedded in the EGLYA sequence (GEGLpYADPYGLLHEGR) is phosphorylated in vivo phosphorylated GEGLpYADPYGLLHEGR - do not account for p140Cap phosphorylation. Indeed, while both EGLYA and EPLYA have been both found phosphorylated in human breast cancer cells (http://www.phosphosite.org/), FYELE and ADPYG were not found phosphorylated in human samples. Overall, these results represent the first characterisation of the most significant p140Cap phosphotyrosines in the human setting.Notably, the tyrosine residues in the sequence FYELE and in the sequence ADPYG - which is very close to the EGLYA and included in the Csk kinase is a potent negative regulator of Src, due to its ability to phosphorylate the negative regulatory tyrosine 527 on the C-terminal domain of Src in silico analysis coupled to in vitro kinase assays with recombinant tyrosine kinases allowed the identification of Abl as the major kinase involved in tyrosine phosphorylation of EPLYA and EGLYA synthetic peptides. Consistently, in HEK-293 cells, the specific Abl inhibitor Imatinib strongly reduces p140Cap tyrosine phosphorylation. Moreover, in the same cellular system, tyrosine phosphorylation triggered by the constitutive active BCR-Abl kinase was heavily reduced on the double p140Cap mutant . ABL specificity for a given substrate is conferred by both target sequence and domain-guided protein-protein interactions. Notably, the EPLYA and EGLYA sequences fit well with the Abl target site consensus sequence We have shown here that the EPLYA and the EGLYA sequences are relevant for both vanadate- and serum-dependent phosphorylation, indicating that these tyrosine residues might be substrate of several tyrosine kinases. Our data provide evidence that p140Cap is the substrate for the Abl kinase. Abl is an ubiquitous tyrosine kinase that controls actin remodelling, cell motility and adhesion, and is involved in cell differentiation processes through association with specific substrates, most of which mediate signal transduction and cytoskeleton dynamics In conclusion, we identified serine and tyrosine phosphorylated residues on the human p140Cap adaptor in breast cancer cells. Moreover we validated two tyrosine residues inserted in the EGLYA and EPLYA sequences as the major phosphorylated sites responsible for p140Cap tyrosine phosphorylation and Csk kinase binding. We also found that p140Cap phosphorylation on the EPLYA and EGLYA sequences in HEK-293 cells is dependent on the Abl tyrosine kinase activity. Overall, elucidating the role of the EGLYA and ELPYA sequences in p140Cap may give further insights into the mechanisms underlying p140Cap biological activity in both physiological and pathological conditions.Figure S1Expression of p140Cap EPLYA/EGLYA double mutant partially rescues Src activation. cDNAs encoding GFP, GFP-p140Cap WT and GFP-p140Cap EPLYA/EGLYA double mutant were used to transfect HEK-293 cells. After 48 hours, cells were starved overnight and treated with 20% FBS for the indicated times. Cell extracts were western blotted using polyclonal antibodies to p140Cap, active Src (pY416) and total Src. The results are representative of two independent experiments. Numbers express the quantification of the ratio between active Src and total Src, with 100 as arbitrary value assigned to the levels of Src phosphorylation in GFP-transfected cells.(PSD)Click here for additional data file.Figure S2Abl silencing in HEK-293 cells causes down-regulation of p140Cap tyrosine phosphorylation. cDNA encoding GFP-p140Cap WT was used to transfect HEK-293 cells together with Abl shRNA construct G10. After 48 hours, cell extracts were immunoprecipitated with a specific antibody to p140Cap and analysed by western blotting using monoclonal antibodies to phosphotyrosine PY99 and p140Cap. Abl silencing was evaluated on cell extracts. The results are representative of two independent experiments.(PSD)Click here for additional data file.


Kip1 and TRF1 in vitro. The inhibitory activity of these small molecules on ubiquitin conjugation has been traced to their inhibition of the ubiquitin E1 enzyme. To further dissect the mechanism of E1 inhibition, we analyzed the effects of these inhibitors on each of the two steps of E1 activation. We show that Largazole and its derivatives specifically inhibit the adenylation step of the E1 reaction while having no effect on thioester bond formation between ubiquitin and E1. E1 inhibition appears to be specific to human E1 as Largazole ketone fails to inhibit the activation of Uba1p, a homolog of E1 in Schizosaccharomyces pombe. Moreover, Largazole analogs do not significantly inhibit SUMO E1. Thus, Largazole and select analogs are a novel class of ubiquitin E1 inhibitors and valuable tools for studying ubiquitination in vitro. This class of compounds could be further developed and potentially be a useful tool in cells.Protein ubiquitination plays an important role in the regulation of almost every aspect of eukaryotic cellular function; therefore, its destabilization is often observed in most human diseases and cancers. Consequently, developing inhibitors of the ubiquitination system for the treatment of cancer has been a recent area of interest. Currently, only a few classes of compounds have been discovered to inhibit the ubiquitin-activating enzyme (E1) and only one class is relatively selective in E1 inhibition in cells. We now report that Largazole and its ester and ketone analogs selectively inhibit ubiquitin conjugation to p27 In humans, protein ubiquitination is a dynamic process, depending on a tightly regulated balance between the activity of two ubiquitin-activating enzymes (E1s), approximately 40 ubiquitin-conjugating enzymes (E2s), and hundreds of ubiquitin ligases (E3s). Protein ubiquitination and subsequent degradation regulates almost every aspect of eukaryotic cellular function including cell cycle regulation, endocytosis, signal transduction, apoptosis, DNA damage repair, transcriptional regulation, and many others Skp2 E3 ubiquitin ligase and subsequent proteasomal degradation Skp2 is a cullin-RING ligase (CRL), which is comprised of RING-box protein I (Rbx1), scaffold protein Cul1, linker protein Skp1, and F-box protein Skp2 In many human cancers, the ubiquitination system is often destabilized. For example, the cyclin-dependent kinase inhibitor p27 is mainly regulated at the protein level and is excessively degraded in approximately 50% of all human cancers Given that ubiquitination influences many cellular functions, malfunctions in the pathway play a role in the pathogenesis of human neurodegenerative disorders such as Parkinson's, Alzheimer's and Huntington's diseases, as well as cancer. Inhibiting components of the ubiquitination system seems to be an avenue of therapeutic development with clinical applications Symploca genus. They examined the compound for cytoxicity against cancer cells and observed remarkable antiproliferative activity in transformed mammary epithelial cells. In addition, they showed that Largazole preferentially targets cancer cells over normal cells, which makes this marine substance an important synthetic target as well as a potentially valuable cancer chemotherapeutic. Remarkably, the structure consists of several unusual features, such as a 16-membered macrocycle containing a 4-methylthiazoline fused to a thiazole ring and an octanoic thioester side chain, a unit rarely found in natural products. in vitro mechanistic studies that reveal a potential role of Largazole as an antagonist of the ubiquitin-activating enzyme E1. In contrast to HDAC inhibition, ketone and ester analogs of Largazole can actively block the ligation of ubiquitin onto E1, indicating a differential mode of inhibitory activity since the formation of a thiol metabolite is indispensible for E1 inhibition. More explicitly, Largazole's presence negatively affected the formation of ubiquitin adenylate, which we monitored through nucleotide exchange assay.In January 2008, the Leusch group at the University of Florida identified a natural product they named Largazole, which was isolated from cyanobacteria of the Mink lung epithelial cells expressing GFP-p27 were generated by retroviral-mediated gene transfer. pBabe-GFP-p27 amphotropic virus was made by cotransfecting pBabe-GFP-p27-Puro with pCL-Ampho in 293T cells. Viral supernatant was collected and used to infect mink lung epithelial cell line Mv 1 Lu (CCL-64) from ATCC in the presence of 8 µg/ml polybrene. Puromycin was added at 5 µg/ml and stable clones were selected. Each clone was subcultured and tested for GFP-p27 expression in the presence or absence of 10 µM MG132  for 24 hours. Clones expressing high levels of GFP in the presence of MG132 but low or unndetectable GFP in its absence were expanded. Immunoblotting using an anti-p27 antibody  was used to confirm the expression of the GFP-p27 fusion protein and stabilization of GFP-p27 upon MG132 treatment. One of the clones used for all subsequent studies was named Kip16.http://pubs.acs.org. Kip16 cells were seeded into 96-well flat clear-bottomed plates at 40,000 cells/well in 100 µl medium and incubated overnight at 37°C in a humidified 5% CO2 atmosphere. Largazole was then added to final concentrations ranging from 1 µM to 1 nM in 300 µl of fresh medium. 0.3% DMSO and 1 µM of MG132 were used as negative and positive controls, respectively. After 24 hours of incubation, the medium was removed, the cells were washed twice with phosphate-buffered saline (PBS), and the cells were fixed with 4% paraformaldehyde in PBS for 15 minutes and stored at 4°C for microscopy evaluation. Cells were visualized with a GFP filter set using a 10× objective on an Eclipse TE2000-S  equipped with a Photometrics camera .Total synthesis of Largazole and Largazole analogs is described in Eschericia coli. His-cdc34 was purified by Ni-NTA chromatography followed by ion exchange and size-exclusion chromatography. The purity and concentration of His-cdc34 were determined by SDS-PAGE analysis.Human ubiquitin E1 (UBA1) was expressed with an N-terminal GST tag fusion by means of recombinant baculovirus expression in Hi5 insect cells using the pFastBacHTA vector . The cells were lysed by sonication in the presence of protease inhibitors in a buffer containing 200 mM NaCl, 50 mM Tris-HCl pH 7.5, 1% NP40, 1 mM DTT, and 1 mM EDTA. Cleared lysate was incubated with glutathione beads  for one hour at 4°C. After three washes with lysis buffer, untagged E1 was produced by thrombin cleavage. The protein solution was passed through a S200 gel filtration column , and UBA1 concentration and purity was evaluated by SDS-PAGE and Coomassie Blue gel staining. The purity was generally greater than 90% and purified UBA1 was aliquoted and stored at −80°C after quick freezing in liquid nitrogen. N-terminal hexahistidine (His)-tagged human Cdc34 was cloned into the pQE-30 vector  and expressed in in vitro in a reticulocyte lysate system  in the presence of [35S]-labeled methionine. p27 was phosphorylated by purified recombinant Cdk2-CyclinE as outlined by Ungermannova et al Skp2 E3 ligase complex, 50 nM Cks1, 10 µM ubiquitin , 10 µM methylated ubiquitin , 1 µl of energy regeneration system  , 1 µM ubiquitin aldehyde, and 1 µM MG132 in a total volume of 15 µl. The reaction was quenched after 30 minutes in a 30°C water bath by addition of 4× SDS sample buffer. The products of ubiquitination were resolved by SDS-PAGE, destained in a 45% methanol and 10% acetic acid solution in water, dried and exposed overnight to a phosphoimager screen, and scanned using a Typhoon scanner 9400 . In vitro Trf1 ubiquitination was carried out as described in Zeng et al 33P]-ATP by CDK1-CyclinB, was incubated with 0.5 µM UBA1, 5 µM UbcH5a, 1 µM SCFFbx4 E3 ligase complex, 5 µM ubiquitin, 100 µM methylated ubiquitin, 1 µM ubiquitin aldehyde, and 1 µl 20×ER for two hours at 30°C. Ubiquitinated Trf1 was analyzed by SDS-PAGE followed by autoradiography.Mouse p27, cloned into pCS2, was translated S. pombe E1  or 0.5 µM human SUMO E1  were pre-incubated with 20×ER at 30°C for 5 minutes in thioester reaction buffer . After 5 minutes, 1 µM fluorescein-ubiquitin  was added to initiate attachment of ubiquitin. All components were allowed to react for another 5 minutes in a total volume of 5 µl. The reaction was stopped with 10 µl of SDS-PAGE loading buffer, minus DTT, and the proteins were resolved using 12% gels that were run on ice to prevent the reduction of E1-ubiquitin due to the heat generated during electrophoresis. Thioester bond formation was visualized by scanning the gel using Typhoon scanner 9400  that was set to fluorescence mode (532 nM). When necessary 100 nM of E2 (Cdc34) was added after the E1 enzyme was pre-charged with ATP. Serially diluted Largazole and its analogs were incubated with the reagents as stated in the text. ImageJ was utilized to quantify the fluorescence signal, and the dose response curves were generated by nonlinear least regression analysis of data using Prism .40 nM ubiquitin E1 (UBA1) or 1 µM 2 (reaction assay buffer), 150 nM human ubiquitin E1 (UBA1), 100 µM ATP, 2 mM AMP, 1 µM [α-32P]- or [γ-32P]- ATP , 500 µM PPi . A total of 5 µM ubiquitin was added to the mixture to initiate the ATP:AMP exchange. After incubation at 30°C for 10 minutes, the reactions were quenched with EDTA, and 0.5 µl aliquots of the reaction mixtures were spotted on Baker-flex® thin layer chromatography (TLC) polyethylenimine-modified cellulose plates  and developed in filtered 0.34 M potassium phosphate pH 7.0 for 20 minutes in a glass jar. The TLC plates were allowed to air dry for 10 minutes, covered in plastic wrap, and then exposed to a phosphoimager plate for 5–10 minutes. The separation of radiolabeled nucleotides was visualized using a Typhoon scanner 9400 .The reaction mixture contained, in a final volume of 10 µl, 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM MgClSkp2-mediated ubiquitination. Hence, stabilization of p27 degradation represents a rational approach in cancer therapeutics. To identify small molecule inhibitors that can stabilize p27Kip1, we performed a screen of ∼3000 compounds from NCI DTP diversity set along with several natural products in our collection. For the cell-based screen, we generated a mink lung epithelial cell line (Kip16) stably expressing p27 that was cloned in frame with green fluorescent protein (GFP). The resulting N-terminal GFP-p27 fusion, detectable by fluorescence microscopy, was used to determine the levels of p27 expression upon treatment of cells with the compound libraries in 96-well format. Much to our surprise, the most potent hit that emerged from this screen was the natural compound Largazole (L)  , which wzole (L) . As expein vitroin vitro, we added Largazole to a p27 ubiquitin ligation reaction. As shown in seco-Largazole (S) were tested in an in vitro p27 ubiquitination assay (Seco-Largazole (S) did not affect p27 ubiquitination, indicating the importance of the topology of the inhibitory compound. Furthermore, the result also suggests that the thioester moiety of Largazole is not required for inhibition, because the ketone and ester analogs were equally potent in blocking p27 ubiquitination. In addition, E1 inhibition is unrelated to HDAC inhibitor activity of Largazole as both ketone and ester fail to inhibit HDAC. Prior to ubiquitination, p27 is phosphorylated by the Cdk2-CyclinE complex. We carried out an in vitro p27 phosphorylation assay  failed to inhibit E1 . If E1 activity is inhibited, we expect to see that defects in E1 activation should impair the attachment of ubiquitin onto Cdc34 (E2). To further validate E1 inhibition, we included Cdc34, the E2 enzyme required for p27 ubiquitinaton, in the E1 reaction mixture. As shown in 50 of E1 inhibition. These results suggest that Largazole and Largazole ester exhibit selectivity towards ubiquitin E1. Also, this result suggests that either compound fails to promote the hydrolysis of ubiquitin thioesters on precharged E1.The decreased ubiquitin transfer could be attributed to either E1 or E2 inhibition; therefore, we produced E1 precharged with ubiquitin by incubating ATP and fluorescent ubiquitin for 15 minutes at room temperature followed by the addition of Cdc34, which was preincubated with either Largazole or Largazole ester. If either compound inhibits the transfer of ubiquitin from E1 to E2, then we would observe a significant decrease in Cdc34 fluorescence regardless of the order we added the compounds. Interestingly, Largazole, preincubated with Cdc34, fails to inhibit the transfer of ubiquitin from precharged E1 at concentrations <1 mM . Further32P]-AMP:[ α-32P]-ATP exchange assay guarantees that a thioester bond is formed between E1 and ubiquitin, while the release of PPi, measured by the [32P]-PPi:[γ-32P]-ATP exchange assay, signals the formation of ubiquitin adenylate. To further dissect the mechanism of Largazole inhibition, two nucleotide exchange assays were carried out in the presence of Largazole derivatives. For these experiments we used Largazole ketone, which is similar to Largazole and Largazole ester. From the results shown in 32P]-PPi signal suggests that the adenylation step did not occur; consequently, ubiquitin could not be transferred to the active site cysteine to trigger the release of AMP. Both steps of the E1-catalyzed reactions can be measured by the AMP:ATP exchange assay. The lack of an [α-32P]-AMP signal further suggests that the adenylation step is inhibited by Largazole ketone. Thus Largazole or Largazole derivatives act on the first step of ubiquitin activation pathway by blocking the formation of ubiquitin-adenylate.E1 forms an ubiquitin–adenylate intermediate during the course of its catalytic cycle 50 is approximately 450 µM as opposed to 30 µM for ubiquitin E1 (data not shown). Thus Largazole is relatively selective in perturbing ubiquitin E1 activation.In addition to ubiquitin, there exist several ubiquitin-like proteins that covalently modify other proteins. All of the ubiquitin-like proteins have activation pathways similar to ubiquitin S. pombe E1 (ptr3/Uba1p) at the amino acid sequence level. To test whether Largazole ketone inhibits the S. pombe E1, we carried out a thioester assay using Largazole ketone and the ubiquitin E1 homologue in S. pombe, Uba1p. The results in Ubiquitin and the ubiquitin E1 enzyme are highly conserved among eukaryotes in vitro. Also, we demonstrated that the inhibitory activity of Largazole is independent of its inhibitory activity towards the histone deacetylase enzymes. Structure-activity relationship analysis shows that the thioester bond is not required for inhibition but the macrocycle core and aliphatic tail are indispensible. Largazole blocks ubiquitin activation at the adenylation step and without perturbing ubiquitin transfer from E1 to E2. Finally we show that Largazole inhibition of E1 is highly selective as it does not inhibit a highly related ubiquitin E1 enzyme from S. pombe and is almost twenty fold less effective in inhibiting the activation of SUMO E1. Taken together, our results reveal that Largazole represents a new class of ubiquitin E1 inhibitors.In this study, we showed that Largazole and its analogs selectively inhibit ubiquitin E1 enzyme activity in vitro ubiquitination assay, we were able to delineate the inhibitory point where Largazole acts on in the ubiquitination pathway, namely the E1 enzyme. However, there is a disconnect between the potency of E1 inhibition in vitro and GFP-p27 stabilization in cells. The EC50 of Largazole for GFP-p27 stabilization is in the low nM range, yet E1 inhibition is at ∼30 µM. This results suggests that the stabilization of GFP-p27 is unlikely caused by E1 inhibition, but is most likely a result of HDAC activity, which is known to block cell cycle progression and cause cell growth arrest. Consistent with this hypothesis, Largazole ketone and ester, two Largazole analogs that do not inhibit HDACs, do not increase GFP-p27 levels when Kip16 cells were treated (data not shown). However, other interpretations may account for the failure of Largazole ketone or ester to raise GFP-p27 by inhibiting E1 in cells. For example, we do not know if or how these compounds penetrate cells and how stable they are once they enter the cells. These investigations have to be undertaken before these analogs can be further developed for in vivo applications.We identified that Largazole caused a robust increase in GFP-p27 expression in Kip16 cells. This observation led us to further investigate the mechanism of GFP-p27 stabilization by Largazole. Using an Panus rudis, and Himeic acid A, derived from the marine fungus Aspergillus, are the first and second discovered inhibitors of the ubiquitin-activating enzyme E1, respectively in vitro using recombinant E1; however, the cellular activity and mechanism were not determined 50 of PYR-41 is around 5 µM, thus more potent than the compounds described here. However, the exact mechanism of PYR-41 inhibition is not known. Ub-AMSN represents a distinct class of protein based inhibitors of ubiquitin E1. Ub-AMSN contains a sulfamide group attached to the carboxyl terminus of ubiquitin as a nonhydrolyzable mimic of the phosphate group in the cognate Ub/Ubl-AMP adenylate intermediate. Thus, like Largazole analogs, it blocks the first step of E1 reaction Panepophenanthrin, a natural compound derived from the mushroom strain in vitro and possible clinical success of the NAE inhibitor MLN4924 further supports the concept that E1 inhibitors are potential promising cancer therapeutics.One of the most important questions to be answered is whether or not ubiquitin or ubiquitin-like E1 inhibitors are therapeutically relevant. Since only one ubiquitin E1 enzyme is responsible for a majority of protein ubiquitination in humans, inhibiting E1 will influence the degradation of proteins across several pathways and may lead to toxicity and, consequently, poor therapeutic efficacy. Bortezomib is the first FDA-approved proteasome inhibitor for the treatment of relapsed/refractory myeloma and mantle cell lymphoma. Our preliminary structure activity relationship studies suggest that the pro-drug form of Largazole including both the hydrocarbon tail and the macrocycle are essential for E1 inhibition. For Largazole analogs to be developed as potential antitumor drugs, additional analogs are needed to be synthesized in order to improve its potency toward ubiquitin E1. The most promising aspect of Largazole analogs as ubiquitin E1 inhibitors is the selectivity and specificity of Largazole. Largazole analogs not only display discrimination over related SUMO E1 enzyme but also remarkable selectivity in targeting human ubiquitin E1. Future structural studies would be helpful to understand how Largazole analogs inhibit E1, and insights gained from such studies may help to develop more specific inhibitors of E1. Experiments to test these hypotheses are currently underway.


Background: This multi-site study compares resting state fMRI amplitude of low frequency fluctuations (ALFF) and fractional ALFF (fALFF) between patients with schizophrenia (SZ) and healthy controls (HC).Methods: Eyes-closed resting fMRI scans  were collected from 6 Siemens 3T scanners and one GE 3T scanner. Imaging data were pre-processed using an SPM pipeline. Power in the low frequency band (0.01–0.08 Hz) was calculated both for the original pre-processed data as well as for the pre-processed data after regressing out the six rigid-body motion parameters, mean white matter (WM) and cerebral spinal fluid (CSF) signals. Both original and regressed ALFF and fALFF measures were modeled with site, diagnosis, age, and diagnosis × age interactions.Results: Regressing out motion and non-gray matter signals significantly decreased fALFF throughout the brain as well as ALFF in the cortical edge, but significantly increased ALFF in subcortical regions. Regression had little effect on site, age, and diagnosis effects on ALFF, other than to reduce diagnosis effects in subcortical regions. There were significant effects of site across the brain in all the analyses, largely due to vendor differences. HC showed greater ALFF in the occipital, posterior parietal, and superior temporal lobe, while SZ showed smaller clusters of greater ALFF in the frontal and temporal/insular regions as well as in the caudate, putamen, and hippocampus. HC showed greater fALFF compared with SZ in all regions, though subcortical differences were only significant for original fALFF.Conclusions: SZ show greater eyes-closed resting state low frequency power in frontal cortex, and less power in posterior lobes than do HC; fALFF, however, is lower in SZ than HC throughout the cortex. These effects are robust to multi-site variability. Regressing out physiological noise signals significantly affects both total and fALFF measures, but does not affect the pattern of case/control differences. Resting state fMRI has numerous advantages over other neuroimaging techniques to elucidate the pathopysiology of disease states. Compared to other neuroimaging methods resting state fMRI is non-invasive and does not expose subjects to radiation , has higher spatial resolution (compared to EEG), and is easily applied in almost all clinical populations since it does not require participation in a cognitive task . The low-frequency fluctuations within resting state fMRI signals are considered to reflect spontaneous neuronal activity to some degree . WithinBesides temporal correlations, we can also analyze the within-voxel time course and consider its power across the range of spatial frequencies  and chronic, stable SZ patients have been shown to be highly repeatable  developed methods for multi-site fMRI studies in clinical populations, notably SZ  and 176 healthy volunteers  matched as much as possible for age, sex, handedness, and race distributions, recruited from seven sites, who participated in the study. Inclusion criteria for the patients were a SZ diagnosis based on the Structured Clinical Interview for DSM-IV-TR Axis I Disorders (SCID-I/P) , and diffusion tensor imaging (DTI) scans. We report here only on the resting state fMRI scans, which were collected for all subjects approximately halfway through the scanning session, following the structural and ASL scans, and half of the memory task scans.*-weighted AC-PC aligned echo planar imaging sequence . For the resting scan, subjects were instructed to lie still with eyes closed.The seven sites included six 3T Siemens TIM Trio scanners and one 3T MR750 GE scanner . The imaging protocol for the resting state scans at all sites was a T2dataQuality matlab package . The SFNR for each subject was plotted against maximal absolute motion and average frame to frame motion; subjects with SFNR less than 150 tended also to have greater average frame to frame motion. Thus we excluded subjects who had a SFNR less than 150, or who moved more than 4 mm total.The three translation and three rotational head movement parameters for each subject were checked for maximal overall movement relative to the first image. Subjects who moved more than 4 mm were excluded from the analysis. We additionally used a data driven criterion to select subjects for the full analysis. We computed signal fluctuation to noise ratio (SFNR)  as follows: Motion correction to the first image using INRIalign; slice timing corrected to the middle slice; and normalization to MNI space, including reslicing to 3 × 3 × 3 mm voxels. These normalized images were the input to the original ALFF analysis. For the regressed analyses prior to ALFF or fALFF calculations and smoothing, we orthogonalized each within-brain voxel's time series with respect to the mean time series from the subject's WM, CSF signals, and the six head motion parameters corresponding to the subject as well as linear and quadratic trends,. To compute WM and CSF masks, we obtained WM and CSF segmentations from each subject's high resolution structural scans using SPM's VBM framework. Average WM and CSF segmentations across all subjects were computed in MNI space. These mean segmentation maps were thresholded at a very conservative level (T > 0.99) to reduce cross-contamination from other tissue types, and binarized. These binarized maps were resampled to EPI resolution using nearest neighbor interpolation. Average timeseries from these WM and CSF ROIs have been shown to correspond to physiological noise related to breathing and cardiac pulsations   was used in all analyses.The effect of the motion and physiological noise regression on the ALFF values was assessed using a paired p < 0.05 FDR-corrected and an extent threshold of 10 voxels. Results were localized by transforming the MNI coordinates to Talairach using mni2tal , then using the Talairach Client .ALFF and fALFF with and without regression were predicted with diagnosis and site as factors, age as a covariate, and the diagnosis by age interaction using a general linear model (GLM) in SPM5. Preliminary analyses had shown no effects of sex or site by diagnosis interactions so these terms were eliminated in the final analysis. All significance thresholds were set to The analyses described above analyzed all the data together, as a “mega-analysis.” We also performed SZ > HC and HC > SZ contrasts for each site separately, in order to assess the replication of the effects from the entire dataset within smaller subsets. This allowed us to perform an immediate meta-analysis. Each site's results as statistical t-maps were converted to Cohen's d effect size maps Cohen, ; the weip > 0.83) or within any site. The maximum translation did not differ with diagnosis (p > 0.86), nor did average root mean square translation (p > 0.09). SZ showed slightly greater mean framewise displacement (absolute sum of scan to scan movement) only in two sites, represented here as sites 2 (p > 0.024) and 5 (p > 0.01). Six subjects were excluded because they withdrew from the study; of the 70 subjects whose data were not suitable for inclusion, 44 were patients and 22 were controls, 47 (71%) were male, and their mean age was 41.5 years old. The two sites with the highest number of rejected data sets were both Siemens sites, with 21 subjects and 10 subjects rejected; both these sites began recruitment early and continued late attempting to collect more subjects, and recruited a number of patients who were unable to lie still in the scanner. The GE and two other Siemens sites each had 9 datasets rejected, while the two sites with the fewest rejected datasets had 5 and 3 total.A total of 306 subjects  had resting state fMRI data that passed quality assurance criteria. There was no significant effect of diagnosis on SFNR in the subjects in the overall, retained sample (p > 0.05). A Two-Way Analysis of Variance (ANOVA) with site and diagnostic category as factors showed the mean ages did not significantly differ between SZ and HC, but site 3 had significantly older subjects overall than did the other sites . The HC group tended to be college graduates (SES code 2), whereas the SZ group had only “some college (>1 year)” (SES code 3), except for sites 2 and 3 where the median response for the SZ group was “high school graduate.”Patients and controls in the final sample were demographically similar across sites Table  and patit-test comparing the original ALFF and regressed ALFF across all subjects showed the regression strongly increased ALFF values  throughout the interior of the cortex and brainstem, while it decreased the ALFF values around the external edges, in the frontal and occipital/cerebellar extremes, the inferior temporal cortices and thalamus  = 25.77, p < 0.0001 FDR corrected].The F = 34.97, p < 0.0001], and the bilateral gyrus rectus . These effects were largely due to the 3T GE (first column on the x-axis) being significantly different from other sites, with the other sites having smaller effects . For the HC > SZ contrast, the maximal result at 27, −84, 33 in the right precuneus had an effect size of 0.76 in the mega-analysis and 0.77 at the same location in the meta-analysis.The original ALFF data from each site were separately analyzed with a GLM including diagnosis as a factor, age as a covariate, and the age × diagnosis interaction. The SZ > HC and HC > SZ contrasts were calculated for each, and a weighted Cohen's d image to correct for differences in between-site samples sizes was calculated over all seven sites for each contrast. This weighted Cohen's d image can be compared directly to the Cohen's d image from the original, mega-analysis which included site as an additional factor  = 11.6, p < 0.00001 FDR corrected], and again was due to the GE site being the most different from the other sites, as shown in Figure F = 10.6, p < 0.00001 FDR corrected], with a similar pattern of effects across the sites. The secondary analysis on the Siemens sites' data only, in both the fALFF analyses, showed no significant site effects. However, the effects of diagnosis as reported below were again not affected other than through a reduction in the maximal t-values.In both the original and regressed fALFF measures, there were significant effects of both site and diagnosis, but no significant age × diagnosis interactions. In the original fALFF analysis, the effect of site covered almost the entire brain; the largest contiguous cluster was over 70,000 voxels. The strongest effect of site in the original fALFF analysis was in the left precentral gyrus at −48, −6, 27 [p < 0.05 is shown in red for the original fALFF analysis, green for the regressed fALFF analysis, and yellow where the two overlap. The correlation between the t-values in these original and regressed fALFF contrasts was ρ = 0.96, with the original fALFF t-values in the HC > SZ contrast being higher on average across all voxels by 0.5. Table t-value did not translate to a change in the significant effects.The areas where HC > SZ reached significance were throughout cortical gray matter in both analyses, though the effects were more wide spread in the original fALFF measures, particularly in the frontal and midline cortex. The maximal voxels and clusters for HC > SZ in the original fALFF results are listed in Table The original fALFF data from each site were separately analyzed with a GLM including diagnosis as a factor, age as a covariate, and the age × diagnosis interaction. The HC > SZ contrasts were calculated for each, and a weighted Cohen's d image was calculated over all seven sites as in the ALFF analysis. This weighted Cohen's d image can be compared directly to the Cohen's d image from the original, mega-analysis which included site as an additional factor  were in the frontal inferior orbital areas, as well as the more medial gyrus rectus, well into the areas of B0 disortion. Given this pattern, together with the standardization of imaging protocols, slice selection profiles, and the combination algorithm for multiple coils, we conclude that the primary source of the remaining GE/Siemens differences is likely to be regional B0 distortion differences.While the fMRI protocols were developed to be as similar as possible across the scanners, with slice-selection parameters and multi-coil combination techniques the same across site  using fALFF rather than ALFF and (b) using a consistent make and model of scanner in a multi-site study, the fact of the matter is that the important differences between patients with SZ and HC were not affected by the inclusion of an outlier site. This supports the notion that both fALFF and ALFF effects are robust to make and model differences, and we continue to recommend including a site factor in any analysis model, as recommended by many others (Fennema-Notestine et al., The meta-analysis of diagnosis effects, in which the analysis was performed for each site separately and then combined in a weighted Cohen's d, consistently gave a slight but consistently larger effect size than in the mega-analysis. Aggregating resting state datasets across multiple scanners in a mega-analysis carries with it a small penalty, reducing the differences between subject groups in this case, even when the scanner effects are included in the model. The implications for large-scale aggregated data analyses such as those needed for imaging genetics or clinical trials are supportive of including approaches such as that used by ENIGMA (Stein et al., In the analysis of patients with SZ and HC, the additional step of regressing out head movement parameters along with WM and CSF mean signals has no effect on the pattern of differences in ALFF and fALFF between diagnostic groups; thus the choice to do this regression or not should be based on other considerations, such as ease of pre-processing or what else the resting state measures are being used for. In combining multi-site resting state fMRI datasets across multiple scanners of the same make and model, which have already been standardized to the greatest extent possible, variance normalization of the timeseries data as is performed implicitly in fALFF can greatly reduce inter-site variation. However, it does not remove the site effects of different scanner manufacturers. And without that normalization, as in ALFF analyses, the site effects can be quite prominent. In a multi-site dataset analysis, a comparison of the mega-analysis results with the meta-analysis can help identify the extent to which site effects are being captured by including site as a covariate in the mega-analysis.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.