Phosphorylation of the T cell receptor (TCR) by the kinase Lck is the first detectable signaling event upon antigen engagement. The distribution of Lck within the plasma membrane, its conformational state, kinase activity, and protein–protein interactions all contribute to determine how efficiently Lck phosphorylates the engaged TCR. Here, we used cross-correlation raster image correlation spectroscopy and photoactivated localization microscopy to identify two mechanisms of Lck clustering: an intrinsic mechanism of Lck clustering induced by locking Lck in its open conformation and an extrinsic mechanism of clustering controlled by the phosphorylation of tyrosine 192, which regulates the affinity of Lck SH2 domain. Both mechanisms of clustering were differently affected by the absence of the kinase Zap70 or the adaptor Lat. We further observed that the adaptor TSAd bound to and promoted the diffusion of Lck when it is phosphorylated on tyrosine 192. Our data suggest that while Lck open conformation drives aggregation and clustering, the spatial organization of Lck is further controlled by signaling events downstream of TCR phosphorylation. T lymphocytes participate in an immune response when they become activated through the T cell receptor (TCR). However, despite the identification of the major players and sequences of events involved in T cell signaling pathways, the question of “How does T cell receptor signaling begin?” remains poorly understood , 2. TCR The spatial organization of Lck is regulated by several different mechanisms. Lck can bind to and diffuse with the coreceptor CD4, which in turn binds to the pMHC complex on the antigen-presenting cell , 2. ThisThe SH2 and SH3 domains of Lck mediate intramolecular interactions and the binding to a great variety of signaling proteins, such as TCRζ, Zap70, Csk, and CD45, as well as adaptor proteins, such as LIME and TSAd . These iOur previous results suggest a relationship between the clustering of Lck and a local increase in signaling ability. Because T cells tightly regulate the strength and extent of TCR signaling, it is likely that the molecular processes following TCR activation impact on Lck distribution, thereby retroactively modulating Lck activity. Such a feedback mechanism has already been shown for the regulation of Zap70 clustering by SLP-76 . The SH2Two recent studies illustrate the role of Y192 in regulating Lck activity and interactions. First, Granum et al. showed that preventing phosphorylation of Y192 (Lck Y192F mutation) led to a greater extent of tyrosine phosphorylation, including CD3ζ. This study also identified various proteins, including the adaptor protein TSAd, which displayed a greater affinity for the SH2 domain of Lck when Y192 was phosphorylated . TCR actThe second study by Sjölin-Goodfellow et al. demonstrated that selective inhibition of the kinase Zap70 led to a pronounced decrease in Y192 phosphorylation on Lck in resting and activated cells. This coincided with an increased phosphorylation of the Lck-activating tyrosine 394 (Y394) . Lck andvia a fundamentally different mechanism. While clustering of open Lck was found to be intrinsic and only attenuated by the absence of Zap70 and Lat, clustering of Lck(Y192F) was not associated with self-aggregation and was dramatically modified in cells lacking Zap70 or Lat. These data suggest that while Lck open conformation drives aggregation and clustering, Lck spatial organization is further controlled by signaling events happening downstream of TCR ITAMs phosphorylation.Hence, we set out to quantify the contribution of the phosphorylation on Y192 Lck, Zap70, and Lat to the spatial organization of Lck in activated Jurkat T cells using cross-correlation raster image correlation spectroscopy (ccRICS) and photoactivated localization microscopy (PALM). We used an open mutant, Lck(Y505F), as well as a mutant that cannot be phosphorylated on Y192, Lck(Y192F), and measured their diffusion and propensity to aggregate and cluster in Lck-deficient Jurkat T cells reconstituted with Lck and in Jurkats lacking Zap70 or Lat. Our results show that similarly to locking Lck in an open conformation, preventing phosphorylation on Y192 promotes Lck clustering, albeit We used raster image correlation spectroscopy (RICS) to extend the data on Lck spatial organization obtained previously in fixed and live cells using PALM . The RIC2/s, respectively). These values reflected the fact that Lck10 only contained the membrane anchor that cannot interact with other proteins while full-length Lck is larger and has the potential for protein–protein interactions. These values are also within the same range than those previously measured by single particle tracking , and LckY192F), fused to either EGFP or mCherry in the Lck-deficient Jurkat T cell line JCaM1. Lck(Y505F) cannot be phosphorylated on the inhibitory Y505 and is therefore locked into an open conformation. When Lck cannot be phosphorylated on Y192, its SH2 domain is prevented from binding to many potential interactors, including the kinases Pyk2 and Itk, the phosphatase SHP-1, and the adaptor protein TSAd . We co-e92F, fuse2/s, respectively). Interestingly, the tendency of Lck(Y505F)–EGFP to diffuse with WT Lck–mCherry was similar to that measured for WT Lck, suggesting that open Lck only interacts with open Lck and does not recruit WT Lck into clusters of open Lck . In parallel to the ccRICS experiments, we expressed WT Lck, Lck(Y505F), and Lck(Y192F) fused to the photo-switchable fluorescent protein PS-CFP2 in JCaM1 cells and imaged them after 10 min of activation using PALM. As previously described  − r], assembling in clusters that were denser, larger, and less numerous than WT Lck  and 55% of LckY192F) were found to self-associate , although to a slightly lower extent than what we observed in the presence of Lat. Lck(Y192F) self-aggregation and diffusion coefficient were not affected by the absence of Lat  was more clustered than WT Lck in Lat-deficient cells, although to a much lesser extent to what we observed in cells expressing Lat Figures C,D. Morevia SH2 domain interacting partners. Additionally, Lck(Y505F) clustered less in absence of Lat and Zap70; however, the potentiating effects of Zap70 on the clustering of open Lck were far greater than those of Lat on open Lck clustering. This is in agreement with a TCR network topology, where Zap70 is more closely located to Lck than Lat.In light of these results, it appears that Lck(Y192F) was allowed to “cluster freely” even more in the absence of Lat, suggesting that the protein network organized by Lat contributes to restraining Lck clustering It was unexpected that preventing the TCR-dependent increase of the affinity of the Lck SH2 domain in the Lck(Y192F) mutant enhanced its clustering. It is logical to assume that the association of Lck with a protein network would rather immobilize Lck and promote cluster formation. In an attempt to understand this apparent contradiction, we focused on the adaptor protein TSAd, which was recently shown to associate with Lck upon Y192 phosphorylation . Lck–EGFWe have demonstrated previously that TCR activation leads to an increase in Lck clustering and that this clustering is driven by the open/active conformation of Lck . Here, wIn cells lacking Zap70, conformation-induced clustering of Lck was significantly attenuated compared to what we observed in JCaM1 cells. If we put the possible contribution of untagged open Lck in these cells aside, this suggests that Zap70 further promotes intrinsic clustering of open Lck. This could be achieved by favoring the confinement of Lck. Indeed, Zap70 kinase activity is essential to the assembly of the protein network downstream of Lat, which in turn is directly linked to actin regulation at the immunological synapse. Interactions both with the protein network installed by Lat and with the actin cytoskeleton could regulate Lck confinement. Zap70 can also have a kinase-independent scaffolding function , which cLat interacts with Lck  and thisWe further observed that upon TCR activation, Y192 phosphorylation contributes to promoting Lck association with TSAd, an adaptor protein lacking enzymatic activity –23. TherIt has been previously suggested that binding to TSAd would promote the open conformation of Lck by breaking the SH2–pY505 intramolecular interaction . HoweverFinally, Couture et al. reported that when phosphorylated on Y192, Lck bound to much less proteins, although these proteins were not identified . Hence, In conclusion, when bearing in mind the inhibitory effect of Y912 phosphorylation, we could speculate that the “declustering” of Lck when Y192 is phosphorylated is a way of downregulating Lck signaling, similarly to what has been described for Zap70 clusters . It coulvia a short 4 amino acid (GGGG) linker. Lck–EGFP–mCherry was made by cloning the mCherry coding sequence into pm-Lck–EGFP-N1 using AgeI.Mammalian expression constructs encoding full-length wild-type human Lck and the constitutively open Lck(Y505F) mutant were a gift from T. Harder. PS-CFP2 expression backbone was obtained from Evrogen. The Y192F single point-substitution mutants of Lck were made by site-directed mutagenesis. The 10- and 15-amino acid N-terminus regions of Lck and Src were fused to EGFP and mCherry, respectively, For the knocking out of Lat, Jurkat cells were transfected with two gRNAs (guide RNA) that were specifically designed to target genomic Lat DNA, together with cas9 expression plasmid. Twenty-four hours post-transfection, transfected single cells were FACS sorted and seeded into 96-well plates. Cell clones were screened by using western blotting with a Lat antibody  and clones lacking Lat eventually grown to an appropriate population for around 20 days.E6.1, JCaM1, P116, and Lat KO cells were cultured in RPMI media (Gibco) supplemented with 10% fetal calf serum (FCS) and transfected by electroporation  to express WT and mutant Lck, Sr15, Lck10, and TSAd fused to EGFP, mCherry, or PS-CFP2. For ccRICS experiments, cells were activated on anti-CD3ϵ  and anti-CD28  antibody-coated coverglass by allowing the cells to settle upon the activating surface for 10 min at 37°C prior to imaging. For PALM experiment, cells were activated for 10 min and subsequently fixed in 4% paraformaldehyde for 13 min. Antibody was adsorbed onto surfaces by incubating clean glass coverslips with 10 μg/ml antibody for at least 1 h at 37°C.www.lfd.uci.edu) as described in the previously published papers . Lck–GFP was excited with the 488-nm emission line of an Argon laser. Lck–mCherry was excited with the 561 nm emission line of a diode pump solid state (DPSS) laser. Lck–GFP and Lck–mCherry were measured simultaneously with GaAsP detectors using the 493–556-nm and 613–696-nm collection ranges, respectively. For each channel, the pinhole was set to 1 AU. For each ccRICS experiment, we acquired a stack of 100 frames in a selected field next to the cell edges. The pixel frame size of the image field was set to 256 × 256 and collected at an electronic zoom that resulted in a pixel size of 50 nm. The pixel dwell time was set to 12.61 μs/pixel, which resulted in a line time of 7.56 ms and frame time of 1.15 s. The acquired ccRICS data were processed and analyzed by the SimFCS software developed at the Laboratory for Fluorescence Dynamics  values and diffusion coefficients were derived from the fits recorded. The cross RICS function was then calculated between the two channels fit to a one-component diffusion model and the cross G(0) value and diffusion coefficient derived from the fit recorded. The fraction of molecules bound was then derived by taking the ratio of G(0)CROSS/G(0)CH1 if G(0)CH1 < G(0)CH2 or G(0)CROSS/G(0)CH2 if G(0)CH2 < G(0)CH2.Briefly, for each two-color experiment, the RICS function was calculated in channels 1 and 2 for the entire image stack, with a moving average applied to remove slow cell movements. The resulting 3D RICS profile was then fit to a one-component diffusion model in each channel and the Photoactivated localization microscopy images were acquired on a TIRF microscope  with a 100×, NA = 1.46 oil-immersion objective. For PS-CFP2, photoconversion was performed with 8 μW of 405-nm laser radiation and imaging of green-converted PS-CFP2 with15–30 mW of 488-nm light. For PALM, 15,000–20,000 images were acquired per sample using a cooled, electron-multiplying CCD (EMCCD) camera  with an exposure time of 18 ms. Recorded images were analyzed using Zeiss ZEN software. Drifting of the sample during acquisition was corrected relative to the position of surface-immobilized 100 nm colloidal gold beads  that were placed on each sample.SMLM data were analyzed using custom software written in MATLAB (MathWorks) for detection of clusters and extraction of clustering parameters. Typically, for each cell, one to five non-overlapping representative regions of 4 μm × 4 μm were selected for analysis.K function as previously described . The DBSCAN routine is implemented in MATLAB and subsequently coded in C++ and compiled in a Matlab executable (MEX) file to improve the speed of processing.To retrieve information on individual clusters, we used density-based spatial clustering application with noise (DBSCAN) analysis  to identt-test with Welch’s correction for the ccRICS data sets, which displayed normal distributions, and with a Mann–Whitney test for the PALM data sets, which did not all have normal distributions.Statistical significance of the means of two data sets was assessed with unpaired NK-K, FK, EC, and YY did and analyzed ccRICS and PALM experiments; EH conceived and analyzed ccRICS experiments; ZY and JL contributed to molecular biology; TT designed the analysis of PALM data; KG and SP contributed to the manuscript preparation; JR was responsible for conceptualization and the manuscript preparation.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.


S1101 and IRS2S1149 are Pim substrates. In xenograft tumor experiments and in a human phase I clinical trial, a pan-Pim inhibitor administered in vivo to animals or humans decreased IRS1S1101 phosphorylation in tumor tissues. This phosphorylation was shown to have effects on the half-life of the IRS family of proteins, suggesting a role in insulin or IGF signaling. These results demonstrate that IRS1S1101 is a novel substrate for the Pim kinases and provide a novel marker for evaluation of Pim inhibitor therapy.The Pim family of serine/threonine protein kinases  contribute to cellular transformation by regulating glucose metabolism, protein synthesis, and mitochondrial oxidative phosphorylation. Drugs targeting the Pim protein kinases are being tested in phase I/II clinical trials for the treatment of hematopoietic malignancies. The goal of these studies was to identify Pim substrate(s) that could help define the pathway regulated by these enzymes and potentially serve as a biomarker of Pim activity. To identify novel substrates, bioinformatics analysis was carried out to identify proteins containing a consensus Pim phosphorylation site. This analysis identified the insulin receptor substrate 1 and 2 (IRS1/2) as potential Pim substrates. Experiments were carried out in tissue culture, animals, and human samples from phase I trials to validate this observation and define the biologic readout of this phosphorylation. Our study demonstrates in both malignant and normal cells using either genetic or pharmacological inhibition of the Pim kinases or overexpression of this family of enzymes that human IRS1 The oncogenic activity of Pim kinases has been well described in Eμ-myc driven mouse lymphomagenesis, where expression of Pim accelerated Myc driven tumorigenesis . Deletio pathway . Given t pathway , 7. For  pathway  entered  pathway , 9 and pconsensus K/RXRHXpS/pT could help in identifying potential substrates of Pim protein kinase.Overexpression of Pim kinases in cells exerts a pleiotropic effect on various signal transduction pathways, including cell cycle progression, cell proliferation and apoptosis. To understand how Pim is regulating this diverse set of pathways, it is essential to define the substrates for this protein kinase. Several proteins have been suggested to be regulated by Pim phosphorylation, including cdc25A, BAD, PTP-U2S, PAP-1, p21, NFATc1 and SOCS-1 . RecentlThis analysis led to the discovery that IRS1 contains a highly conserved Pim phosphorylation sequence at S1101. Given the role of Pim in regulating a signal transduction pathway related to metabolism , 14, 15,in vivo substrate for Pim protein kinases, MEF cells derived from wild type (WT) and triple knockout of Pim1, Pim2 and Pim3 (TKO) FVB mice were examined. Western blot analysis demonstrated that phosphorylated IRS1 protein expression was undetectable in TKO cells when protein was probed with anti-phospho S1101 IRS1 antibody  Pim1 [The Pim1 protein kinase plays an important role in the initiation and progression of human prostate cancer and is elevated in both epithelial and stromal tumor cells. To determine whether overexpression of Pim1 in prostate cancer increases IRS1 S1101 phosphorylation, human prostate cancer PC-3 cells and human prostate stromal cells, BHPrS1 and WPMY1, expressing tet-inducible Pim1 were studied. The latter cell line was stimulated with varying doses of doxycycline. After 48 hours stimulation, both Pim1 expression and IRS1 phosphorylation increased in parallel to the concentration of doxycycline applied Figure , suggest7M) Pim1 , 20. Incin vitro kinase assay, the immunoprecipitants were incubated with 0.1 μg of recombinant active Pim1 kinase in the presence of ATP and then subjected to Western blot analysis with anti-phospho S1101 IRS1 antibody. These results demonstrated that recombinant Pim1 could phosphorylate S1101  antibodies. Phosphorylation at IRS1 on tyrosine residues was increased after IGF1 stimulation but it was unchanged by GNE-652 treatment Figure . In contS1101 is reportedly phosphorylated by protein kinase C (PKC) [S1101 phosphorylation. In our experiments PMA appeared to increase phosphorylation by 20%, and this phosphorylation was inhibited by GNE-652 . These results indicate that the Pim protein kinases are the primary regulator of IRS1S1101 phosphorylation, and suggest that these enzymes might function downstream of PKC in the control of the phosphorylation of this site.IRS1 C (PKC) , which r C (PKC) , 27. Pho C (PKC) , 29 and  C (PKC) . The add2 Figure . In contS1101 phosphorylation, Pim inhibitors did not alter Cyclin D3 or c-Myc protein turnover. To further examine whether Pim expression alters IRS1 protein stability, cycloheximide chase was performed in TKO MEF cells expressing Pim2 only or empty vector. As Pim2 expression leads to phosphorylation of S1101, a half-life of IRS1 protein was significantly shortened  between the treated and control mice. After sacrifice, the cells were removed from the mouse bone marrow, and IRS1S1101 expression was measured. The cellular extracts from the treated mice demonstrated that AZD1208 reduced IRS1S1101 phosphorylation, while inhibiting leukemic cell growth in vivo.We have previously demonstrated that T-ALL cells show moderate inhibition of growth when treated with Pim inhibitors . Treatmen Figure . To exams Figure . The totin vivo measurable changes in IRS1 phosphorylation could be observed. Additional patient samples from solid and liquid tumors will be needed to examine in detail this protein phosphorylation site as a biomarker for Pim inhibitor activity.To test whether this decrease in IRS phosphorylation also occurred in leukemia patients, samples from a phase I trial of AZD1208 in humans were evaluated. These samples consisted of both human bone marrow and peripheral blood. Inhibition of S1101 after AZD1208 treatment was detected in bone marrow samples (PBM) isolated from patients 3002 Figure , while pin vitro, using immunoprecipitated proteins, and in vivo, with siRNA to the three Pim kinases and pan-Pim inhibitory drugs, demonstrated that Pim is capable of phosphorylating in humans IRS1S1101 and IRS2S1149. Phosphorylation of these sites was absent in MEFs from mice that do not contain any of the Pim kinase isoforms (TKO), validating the hypothesis that IRS1 and 2 are targets for the Pim kinases. In human prostate cancer cells, it was necessary to knockdown all three Pim kinases to inhibit phosphorylation of IRS proteins. Overexpression of a single Pim isoform in TKO was sufficient to induce IRS phosphorylation, suggesting that each of these enzyme can phosphorylate the IRS proteins and they have overlapping biologic activities. Likewise, each of the Pim kinases is capable of collaborating with Myc to enhance transformation [S406 [Using a bioinformatics approach, the IRS1 and 2 proteins have been identified as potential substrates for the Pim kinases. Experiments carried out both on [S406 , and thiS1101 expression. This observation is consistent with a previous report showing that S1101 phosphorylation was absent in TSC1/2-null cells where p70S6K and AKT kinases are highly activated [S1101 was previously suggested to be regulated by PKCθ [S1101 phosphorylation was observed after treatment of myoblasts with the PKC activator, PMA, and this increase was inhibited by the addition of a small molecule Pim inhibitor. It is possible that PMA increased Pim levels through its ability to regulate NF-κB, which then increased the phosphorylation of IRS1. Alternatively, PKC or a downstream kinase activated by PMA could be stimulating Pim activity [S1101 phosphorylating kinase.Previous experiments carried out by others , 32 suggctivated . The pho by PKCθ . In our activity . AdditioS1101, suggesting that the insulin stimulatory pathway regulates Pim activity. The Pim and Akt pathways appear to be regulated in parallel, as evidenced by their ability to modulate protein synthesis through the regulation of TSC2 and eIF4B [S1101 phosphorylation contributes to a decrease in the essential tyrosine phosphorylation needed for IRS1 function [In multiple cell types, structurally different Pim kinase inhibitors blocked the ability of insulin to stimulate the phosphorylation of IRS11nd eIF4B , 38, 39.nd eIF4B  suggestsfunction . HoweverS1101 phosphorylation. This data points to the possibility that IRS1 phosphorylation could be used as a biomarker of Pim activation or inhibition in clinical trials of anticancer agents targeting this protein kinase.Administration of Pim kinase inhibitor AZD1208 to mice bearing tumors blocked S1101 phosphorylation of IRS1 in tumors extracts. Additionally in 2 patient samples that we were able to obtain from patients treated with AZD1208 in a phase I trial for myeloid leukemia, Pim inhibition blocked IRS1The following antibodies were purchased from Cell Signaling Technology: anti-phospho-tyrosine (p-Tyr-100), anti-Pim1, anti-Pim2, anti-Pim3, anti-Cyclin D3, anti-phospho-eIF4B, anti-AKT, anti-phospho-AKT (S473), anti-phospho-AKT (T308), anti-p70S6K, anti-phospho-p70S6K (T389), anti-phospho-S6 (S240/244), anti-phospho-GSK3β (S9), and anti-c-Myc, and anti-phospho IRS1  antibodies which recognize the following residues in human (h) or mouse (m) IRS1; anti-phospho IRS1 hS1101 (m1097); mS318 (hS323); mS302 (hS307); mS307 (hS312); hS636/S639 (mS632/635); mS612 (hS616). This numbering refers to human IRS1 (P35568); and mouse IRS1 (P35569). Anti-IRS1 (R & D systems), anti-RPS6 (Santa Cruz Biotechnology), anti-His (GenScript), anti-HA and anti-β-actin (Sigma) antibodies were used in these studies. Horseradish peroxidase (HRP)-linked enhanced chemiluminescence (ECL), mouse IgG and rabbit IgG secondary antibodies were purchased from GE Healthcare Life Sciences. Various protein kinase inhibitors, GNE-652 (Genentech), LGB321 (Novartis), AZD1208, AZD5363 (AstraZeneca) and PP242  were used in these studies. All other chemicals including cycloheximide (CHX) and PMA were purchased from Sigma. Human recombinant insulin and insulin-like growth factor 1 (IGF1) were obtained from Peprotech.2 as reported previously. HeLa and C2C12 cells were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 2 mmol/L Glutamax and 10% fetal bovine serum at 37°C under 5% CO2. T-lineage acute lymphoblastic leukemia (T-ALL) cell lines including H-SB2, SUP-T1, DU.528, CUTLL1, KOPT-K1 and HPB-CLP were a gift of Dr. Jon C. Aster  and cultured in RPMI supplemented with 2 mmol/L Glutamax and 10% fetal bovine serum. Wild-type (WT), triple-knockout (TKO), Pim1−/−, Pim2−/−, and Pim3−/− mouse embryonic fibroblasts (MEFs) and TKO MEFs expressing Pim1, Pim2 or Pim3 have been described elsewhere [PC-3, LNCaP, A549, Calu-1, HEK-293T, HeLa and C2C12 cells were purchased from the American Type Culture Collection  and were not passaged or maintained for more than 6 months. PC3-LN4 cells were described before  and huma® Lightning Site-Directed Mutagenesis Kit according to the manufacturer's protocol. The HA-Pim1 construct was obtained by subcloning human Pim1 cDNA into pLEX vector (Open Biosystems), as described elsewhere [The his-tagged IRS1  and HA-tlsewhere . These pReverse transcriptase PCR and quantitative real time PCR (qPCR) assay was performed as previously described . The folmouse IRS1-forward, 5′-CTC AGT CCC AAC CAT AAC CAG-3′; mouse IRS1-reverse, 5′-TCC AAA GGG CAC CGT ATT G-3′; mouse β-Actin-forward, 5′-GAC ATG GAG AAG ATC TGG CA-3′; mouse β-Actin-reverse, 5′-GGT CTC AAA CAT GAT CTG GGT-3′.Cells were harvested in lysis buffer consisting of 50 mmol/L Tris, pH 7.4, 150 mmol/L NaCl, 1% NP-40, 5 mmol/L EDTA and 1% protease/phosphatase inhibitor cocktail. Following 15 min of incubation in lysis buffer at 4°C, lysates were cleared by centrifugation at 15,000 rpm for 15 min at 4°C, and then protein concentrations were determined by the DC protein assay (Bio-Rad Laboratories). His-tagged IRS1 or HA-tagged IRS2 was immunoprecipitated in a lysis buffer with anti-His or anti-HA antibodies and protein A/G-agarose (Pierce). Densitometry was determined with Image Studio Lite Ver 5.0 (LI-COR Biosciences) with normalization to the corresponding controls.2, and 2 mmol/L dithiothreitol) and then incubated with 0.1 μg of recombinant active Pim1 kinase [His-IRS1, its mutant (S1101A), or HA-IRS2 transfectants were immunoprecipitated with anti-His or anti-HA antibodies. Immune complexes were washed three times in lysis buffer, then washed twice in 1 × kinase buffer  was utilized to uncover Pim kinase substrates. NetworKIN is a database of predicted kinase-substrate relationships and the underlying computational approach combines predictions of kinase families expected to phosphorylate particular motifs with background information on substrates and kinases to forecast which kinases mediate phosphorylation at specific sites. The method integrates four sources of evidence, a genomic context, experimental evidence (protein interactions and co-expression), pathway databases and literature mining. A context score is provided to help eliminate false positive results based on consensus motifs alone [The NetworKIN resource Version_2_0  were injected via tail vein into irradiated (2.5 Gy) 4–6 week old NOD/SCID male mice (Charles River Laboratories). Starting on day 3, the mice were treated with AZD1208  or control vehicle  daily for ~17 days. Tumor growth was followed by bioluminescence imaging at the time-points indicated.All animal studies were performed in compliance with institutional guidelines under an IACUC approved protocol. To analyze IRS1 S1101 (S1097 in mouse) phosphorylation levels in various tissues including kidney, liver, abdominal fat and muscle tissues were obtained from WT and TKO FVB mice. Tissue extracts were prepared by homogenizing the tissues in RIPA lysis buffer, as described previously . To examLysates of bone marrow and peripheral blood mononuclear cells of AZD1208 phase I clinical trial patients were kindly provided by AstraZeneca and used as reported previously .


We show that Ser239 of murine CGI-58 is a substrate for PKA using phosphoamino acid analysis, MS, and immunoblotting approaches to study phosphorylation of recombinant CGI-58 and endogenous CGI-58 of adipose tissue. Phosphorylation of CGI-58 neither increased nor impaired coactivation of ATGL in vitro. Moreover, Ser239 was not required for CGI-58 function to increase triacylglycerol turnover in human neutral lipid storage disorder fibroblasts that lack endogenous CGI-58. Both CGI-58 and S239A/S240A-mutated CGI-58 localized to perilipin 1A-coated lipid droplets in cells. When PKA was activated, WT CGI-58 dispersed into the cytoplasm, whereas substantial S239A/S240A-mutated CGI-58 remained on lipid droplets. Perilipin phosphorylation also contributed to CGI-58 dispersion. PKA-mediated phosphorylation of CGI-58 is required for dispersion of CGI-58 from perilipin 1A-coated lipid droplets, thereby increasing CGI-58 availability for ATGL coactivation.CGI-58/ABHD5 coactivates adipose triglyceride lipase (ATGL). In adipocytes, CGI-58 binds to perilipin 1A on lipid droplets under basal conditions, preventing interaction with ATGL. Upon activation of protein kinase A (PKA), perilipin 1A is phosphorylated and CGI-58 rapidly disperses into the cytoplasm, enabling lipase coactivation. Because the amino acid sequence of murine CGI-58 has a predicted PKA consensus sequence of RKYS The control of lipolysis in adipocytes of vertebrates is a carefully orchestrated process. Lipolysis, or the hydrolysis of stored triacylglycerols, releases fatty acids, monoacylglycerols, and diacylglycerols that serve as substrates for energy production and the synthesis of phospholipids used for membrane synthesis and repair. The enzymes that catalyze triacylglycerol hydrolysis in adipocytes include adipose triglyceride lipase (ATGL), hormone-sensitive lipase, and monoacylglycerol lipase, to cleave the first, second, and third ester bonds, respectively –4. AdipoThe best-characterized signaling pathway that initiates adipose lipolysis occurs when catecholamines bind to β-adrenergic receptors on adipocyte plasma membranes. Hormone binding triggers a G protein-mediated cascade that activates adenylyl cyclase, increasing levels of cAMP, in turn, activating protein kinase A (PKA). The subsequent PKA-mediated phosphorylation of multiple proteins enables lipolysis. These proteins include, but are not limited to, perilipin 1A and hormone-sensitive lipase , 5, 6.Perilipin 1A at the surfaces of lipid droplets is a master regulator of adipose lipolysis. Under basal (fed) conditions, perilipin 1A provides a protective barrier against lipolysis of stored triacylglycerols  and bindCGI-58 was identified as an important factor in cellular triacylglycerol homeostasis when mutations in CGI-58 were established as the cause of Chanarin Dorfman syndrome , a neutrAll chemicals were reagent grade. Growth media for cultured cells were obtained from Mediatech, Inc.  or Sigma. FBS, isobutylmethylxanthine (IBMX), forskolin, and protease and phosphatase inhibitor cocktails were purchased from Sigma. The DNA purification kit and nickel-nitrilotriacetic acid agarose matrix were purchased from Qiagen. Coomassie Plus protein assay reagent was purchased from Thermo Scientific/Pierce; Bradford and DC protein assays were purchased from Bio-Rad. PfuUltra high-fidelity DNA polymerase was purchased from Stratagene, Inc. . BODIPY 439/503, Alexa Fluor 546 goat-anti rabbit IgG, Alexa Fluor 546 donkey-anti goat IgG, and Alexa Fluor 488 donkey-anti rabbit IgG were purchased from Molecular Probes. Radioactive compounds were purchased from Perkin Elmer Biosciences. Oligonucleotide primers were purchased from Operon BioTechnologies, Inc. . Phospholipids were purchased from Avanti Polar Lipids, Inc.  and Sigma. Bovine heart PKA catalytic subunit and modified trypsin were purchased from Promega. Peptides were synthesized and purified by Bio-Synthesis, Inc. . P81 phosphocellulose paper was from Whatman International Ltd. Hybond-P polyvinylidene difluoride (PVDF) membranes were purchased from Amersham Biosciences. Protein A-Sepharose was from GE Healthcare. All remaining chemicals, scintillation counting supplies, and solvents were purchased from Fisher Scientific or Sigma.Escherichia coli cell extracts with nickel-nitrilotriacetic acid agarose, as described (Preparation of mouse CGI-58 cDNA subcloned into the pET-28a(+) bacterial expression vector was described previously ; recombiescribed . All ste® site-directed mutagenesis kit (Stratagene) with forward (5′-CCTGATTTCAAGCGGAAGTACGACTCTATGTTTGAAGATGACACG-3′) and reverse (5′-CGTGTCATCTTCAAACATAGAGTCGTACTTCCGCTTGAAATCAGG-3′) mutagenic primers for S239D CGI-58 and forward (5′-CCTGATTTCAAGCGGAAGTACGAGTCTATGTTTGAAGATGACACG-3′) and reverse (5′-CGTGTCATCTTCAAACATAGACTCGTACTTCCGCTTGAAATCAGG-3′) mutagenic primers for S239E. The mouse ATGL cDNA encoding amino acids 1–288 was subcloned into His-pSumo with 5′ TEV site using forward (5′-GCTATGGATCCATGTTCCCGAGGG-3′) and the reverse (5′-GGCGCTCGAGTCATTTTTCGAACTGCGGGTGGCTCCAATCCTCCTCTCCAGC-3′) oligonucleotide primers including endonuclease cleavage sites and a stop codon following the nucleotide sequence for D288, followed by the sequence encoding a Strep-tag® . Mutations were confirmed by DNA sequencing .Additionally, the coding sequences of mouse CGI-58 and mouse ATGL cDNAs were amplified as described  for subcE.coli (Agilent Technologies). Expression of proteins was induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside when cells reached an optical density of 0.6 at 600 nm. Induced His-pSumo-CGI-58 cells were grown for 9–12 h, while His-pSumo-ATGL 1-288 and His-pSumo cells were grown for 4 h at 30°C. Expression of the proteins was confirmed with SDS-PAGE.His-pSumo-CGI-58, His-pSumo-ATGL 1-288, and the His-pSumo vector encoding 6-His-smt protein were transformed into BL21(DE3) RIPL CodonPlus E. coli expressing recombinant CGI-58 in His-pSumo were disrupted by probe sonication in buffer-1  supplemented with protease inhibitor cocktail , 750 U benzonase nuclease HC (Novagen), and 1 mg/ml lysozyme. Cell debris was removed by centrifugation at 30,000 g for 40 min and soluble recombinant CGI-58 was purified with affinity chromatography using a 5 ml His-Trap FF column . The column was washed extensively with buffer-1 and eluted with a linear gradient of 10 column volumes with buffer-2 . Cleavage with tobacco etch virus protease was performed at room temperature for 4 h and the cleaved CGI-58 was further purified on a Superdex 200 (Sigma-Aldrich) column equilibrated in buffer-3 . Residual 6-His-smt was removed by reverse affinity chromatography with a 5 ml His-Trap FF column equilibrated in buffer-3. Recombinant CGI-58 was then dialyzed into the PKA assay buffer.E. coli expressing recombinant ATGL 1-288 and 6-His-smt protein were disrupted by sonication in buffer-4  supplemented with 0.1 mM phenylmethanesulfonylfluoride and protease inhibitor cocktail. Cell debris was removed by centrifugation and soluble protein was loaded onto a 1 ml His-Trap FF column. The column was washed extensively with buffer-4 and the proteins were eluted with a gradient of 12 column volumes of buffer-5 . Subsequently, ATGL 1-288 was dialyzed into buffer-6  and used directly in the triacylglycerol hydrolase activity assay.32P]ATP  in 10 mM MgCl2, 60 mM DTT, 50 mM Tris-HCl (pH 8.0); reactions were incubated for 10 min at 30°C. Reactions were stopped by the addition of 4× Laemmli’s sample buffer  were incubated with the indicated concentrations of PKA and 50 μM , proteins were eluted using hot SDS-PAGE XT (Bio-Rad) sample buffer for 10 min at 90°C. Proteins were separated by electrophoresis on 4–20% gradient gels for SDS-PAGE (Bio-Rad) and transferred to nitrocellulose membranes. Membranes were immunoblotted with anti-CGI-58   (Bio-Rad) by a gradual increase in voltage from 200 V to 8,000 V at the rate of 4 V/min . The second dimensional separation was performed using 4–12% gradient SDS-PAGE gels (Bio-Rad). Antibodies used for immunoblotting were rabbit polyclonal anti-mouse CGI-58 antiserum . ProteinP < 0.05.Kinetics data were analyzed according to the Michaelis-Menten equation using the GraphPad Prism kinetic model-fitting program. Data are reported as mean ± standard deviation or standard error and were analyzed using two-way ANOVA and Tukey’s post hoc test with SPSS software. Differences between samples were considered significant at Caenorhabditis elegans or Drosophila melanogaster , were expressed and purified from E. coli lysates, incubated with PKA and ATP, reduced, alkylated and digested with trypsin, chymotrypsin, or AspN. Digests were then analyzed by LC-MS/MS using different fragmentation and scanning techniques. A search of the mammalian SwissProt database unambiguously identified Ser239 as the major phosphorylation site in CGI-58 peptides derived from three different digests , and continued to decrease over 48 h  or S239A/S240A-mutated CGI-58 in cultured human NLSD fibroblasts that lack functional CGI-58 , and havver 48 h . The ratver 48 h . In summInitial experiments to test CGI-58 function in NLSD fibroblasts were conducted in the absence of compounds to activate PKA. We next investigated whether the activation of PKA would alter the rate of triacylglycerol turnover in NLSD cells expressing ectopic CGI-58. Adenoviruses driving the expression of either intact (WT) or S239A/S240A-mutated CGI-58 were added to NLSD cells 6 h prior to the addition of forskolin and IBMX. Measurement of the triacylglycerol content of cell lysates over the next 2 h revealed no increase in the rate of lipolysis due to the activation of PKA, and no difference in the rate of triacylglycerol turnover when comparing data from cells expressing WT CGI-58 to those from cells expressing S239A/S240A-mutated CGI-58 . Thus, PWe next asked whether the PKA-mediated phosphorylation of CGI-58 affects subcellular localization of the protein. PKA controls lipolysis in adipocytes in part by modulating the subcellular localization of CGI-58 , 37. UndWe conducted a similar experiment using cells expressing S239D CGI-58 and perilipin 1A. Under basal conditions, S239D CGI-58 localized efficiently to perilipin 1A-coated lipid droplets , suggesting that the addition of negative charge to amino acid 239 of CGI-58 is insufficient to cause dispersion of CGI-58 from perilipin 1A-coated lipid droplets without the activation of PKA. When cells were stimulated with forskolin and IBMX, S239D CGI-58 efficiently dispersed into the cytoplasm . These data suggest that negative charge at position 239 is important for CGI-58 dispersion when PKA has been activated.In this study, we employed a variety of approaches to demonstrate that CGI-58 is phosphorylated on Ser239 by PKA. The finding is supported by data from the phosphorylation of recombinant CGI-58 in vitro, immunoblotting of immunoprecipitated proteins from mammalian tissue and cells in which phosphorylation occurred in intact cells, and MS. All of our studies were conducted using mouse CGI-58; however, human CGI-58 is likely phosphorylated, given the complete conservation of the PKA consensus sequence in chordates.In adipocytes, the PKA-mediated phosphorylation of key mediators of lipolysis is a crucial event in the initiation of lipolysis. Phosphorylation of hormone-sensitive lipase is required to trigger movement of the lipase from a diffuse cytoplasmic localization to the surfaces of lipid droplets –42, and Although the PKA-mediated phosphorylation of CGI-58 is an important step in the activation of lipolysis for cells in which perilipin 1A is the major lipid droplet-associated protein, it is likely less important for cells in which lipid droplets are coated with other members of the perilipin family. Perilipin 1A is an abundant lipid droplet protein in adipocytes from white and brown adipose tissue , is lessAlthough the phosphorylation of CGI-58 increases the availability of CGI-58 to bind to ATGL in adipocytes, it does not affect the coactivation mechanism itself. In vitro lipolysis assays suggest that phosphorylated CGI-58 activates ATGL as efficiently as nonphosphorylated protein. The mechanism by which CGI-58 increases ATGL-mediated hydrolysis of triacylglycerols is as yet unknown. Control of ATGL activity is complex; triacylglycerol hydrolase activity is inhibited by a small protein named G0S2 , 55, yetWe have shown that Ser239 of CGI-58 is phosphorylated by PKA in vitro, as well as in tissue samples and intact cultured cells. Interestingly, 2D electrophoresis revealed three to four isoforms of CGI-58, suggesting additional posttranslational modifications. Additional studies are needed to address this hypothesis.


Psoralea corylifolia and shows several biological activities. Although there have been studies on the biological effects of bakuchicin, its modulation potency of CYP activities has not been previously investigated. Here, we investigated the inhibitory effects of bakuchicin on the activities of CYP isoforms by using a cocktail of probe substrates in pooled human liver microsomes (HLMs) and human recombinant cDNA-expressed CYP. Bakuchicin strongly inhibited CYP1A-mediated phenacetin O-deethylation with an IC50 value of 0.43 μM in HLMs. It was confirmed by human recombinant cDNA-expressed CYP1A1 and CYP1A2 with a Ki value of 0.11 μM and 0.32 μM, respectively. A Lineweaver-Burk plot indicated that the inhibition mechanism of bakuchicin was competitive inhibition. Overall, this is the first study to investigate the potential CYP1A1 and CYP1A2 inhibition associated with bakuchicin and to report its competitive inhibitory effects on HLMs.Bakuchicin is a furanocoumarin isolated from Cytochrome P450 (CYP) is a group of heme-containing enzymes embedded essentially in the lipid bilayer of the endoplasmic reticulum, and it acts in the metabolism of many drugs, steroids, and carcinogens . They maThe CYP1A subfamily consisted of two members CYP1A1 and CYP1A2. CYP1A is one of the important CYP subfamilies in humans and plays a role in the metabolism of some clinically used drugs including theophylline, clozapine, tacrine, and foodborne procarcinogens . The CYP Psoralea corylifolia which have been used in oriental medicine . Po. Po P. cβ-NADPH, and 1.0 U/mL glucose-6-phosphate dehydrogenase. To investigate the IC50 of bakuchicin during CYP1A inhibition, bakuchicin  was incubated without preincubation. In addition, the mixture with bakuchicin was preincubated for 15 min without the probe substrate cocktail in the presence or absence of 1 mM β-NADPH. After the reaction was initiated by adding the NGS for 60 min, incubated samples were terminated by adding 100 μL acetonitrile containing 0.1% formic acid and 2.5 μL of internal standard solution (5 μM reserpine) in methanol. After mixing and centrifuging at 13,000 ×g for 12 min, a 5 μL aliquot was injected onto a C18 column for LC-MS/MS analysis.The inhibitory effects of bakuchicin on the metabolism of 8 different CYP-specific substrates are listed in μM in 0.1 M potassium phosphate buffer (pH 7.4) for 60 min at 37°C. Those were terminated by adding 100 μL acetonitrile containing 0.1% formic acid and 2.5 μL of internal standard solution (5 μM reserpine) in methanol. After mixing and centrifuging at 13,000 ×g for 12 min, a 5 μL aliquot was injected onto a C18 column for LC-MS/MS analysis. Phenacetin was used as a probe substrate at 20, 40, or 80 μM. The inhibition pattern was investigated by Michaelis-Menten and its secondary plotting.To characterize the inhibition mode on CYP1A by bakuchicin, 0.5 mg/mL of HLMs was incubated with bakuchicin at 0, 0.2, 0.4, and 0.8  cDNA-expressed CYP1A1 and CYP1A2 was incubated with bakuchicin at 0.1–50 μM and NGS for 60 min at 37°C after the addition of 80 μM phenacetin as a selective CYP1A substrate. Those were terminated by adding 100 μL acetonitrile containing 0.1% formic acid and 2.5 μL of internal standard solution (5 μM reserpine) in methanol. After mixing and centrifuging at 13,000 ×g for 12 min, a 5 μL aliquot was injected onto a C18 column for LC-MS/MS analysis. CYP1A1 and CYP1A2 activities were determined by phenacetin O-deethylation. The inhibition patterns of bakuchicin on CYP1A in human recombinant cDNA-expressed CYP1A1 and CYP1A2 were confirmed by Michaelis-Menten and secondary plot.To confirm the selective inhibition of CYP1A by bakuchicin, 10 pmol of human recombinant™ LC system coupled to a TSQ Vantage triple quadrupole mass spectrometer  equipped with a HESI-II Spray source. Electrospray ionization was performed in positive mode at a spray voltage of 3,500 V (except for the detection of 6-hydroxy chlorzoxazone). Nitrogen was used as a sheath and auxiliary gas at optimum values of 45 and 20 (arbitrary units), respectively. Vaporizer and capillary temperatures were 150 and 300°C, respectively. For LC analysis an Inertsil® ODS-2  column  was used. The mobile phase consisted of acetonitrile (mobile phase A) and water (mobile phase B), both of which contained 0.1% formic acid, at a flow rate of 0.22 mL/min at 30°C.All measurements were performed using LC-MS/MS in multiple reaction monitoring mode. LC-MS/MS was performed using an Accela50 values (the concentration of inhibitor causing a 50% inhibition in enzyme activity) were obtained using percent activity versus log[I] concentration plots. Kinetic parameters were estimated by curve fitting using SigmaPlot .All incubations were performed in duplicate, and data are presented as means. IC50 values of bakuchicin on the 8 CYP activities were determined with or without microsomal preincubation for 15 min at 37°C to predict the inhibitory mechanism of bakuchicin. CYP2B and CYP2D6 were inhibited, but CYP1A was most strongly inhibited in HLMs compared to other CYPs at the IC50 value of 0.43 μM  was calculated from a secondary plot for CYP1A  by ethanol extraction [ in vivo can depend on variability in the environment, the preparation method, or the patient. Therefore, it is important to identify the modulation effect of bakuchicin on CYP activities in HLMs. In conclusion, we were the first to investigate potential herb-drug interactions associated with bakuchicin by evaluating its inhibitory effects on CYP1A in HLMs. Our study showed that bakuchicin was a strong and selective competitive inhibitor of CYP1A1 and CYP1A2 in HLMs.elmintic . Bakuchitraction . The con


O-GlcNAcylation, a generally undermined atypical protein glycosylation process, is involved in a dynamic and highly regulated interplay with phosphorylation. Akin to phosphorylation, O-GlcNAcylation is also involved in the physiopathology of several acquired diseases, such as muscle insulin resistance or muscle atrophy. Recent data underline that the interplay between phosphorylation and O-GlcNAcylation acts as a modulator of skeletal muscle contractile activity. In particular, the O-GlcNAcylation level of the phosphoprotein myosin light chain 2 seems to be crucial in the modulation of the calcium activation properties, and should be responsible for changes in calcium properties observed in functional atrophy. Moreover, since several key structural proteins are O-GlcNAc-modified, and because of the localization of the enzymes involved in the O-GlcNAcylation/de-O-GlcNAcylation process to the nodal Z disk, a role of O-GlcNAcylation in the modulation of the sarcomeric structure should be considered. Nowadays, it is well admitted that the phosphorylation does not act alone in the fine modulation of numerous cellular processes, but rather, presents a dynamic and highly regulated interplay with an atypical glycosylation, the O-linked N-acetyl-glucosaminylation (termed O-GlcNAcylation), occurring on nuclear, cytoplasmic and mitochondrial proteins  onto the hydroxyl group of serine and threonine residues of target proteins by the uridine diphospho-N-acetyl glucosaminyl transferase (O-GlcNAc transferase or OGT). The β-N-acetylglucosaminidase (O-GlcNAcase or OGA) catalyzes the removal of O-GlcNAc residues from proteins . MLC2 phosphorylation is not essential for skeletal muscle contraction but is an important regulatory mechanism since it can produce changes in thick filament structure and enhance crossbridge attachment . This atrophy results from a reduction in fiber diameter, protein content and is associated with slow to fast phenotype transitions accompanied by functional changes such as loss in force and increased fatigability  concomittantly to soleus muscle atrophy was correlated with an increase of the global level of MLC2 phosphorylation in rat hindlimb unloading model (Bozzo et al., Thus, the slow-to-fast MLC2 transition was associated to an increase of phosphorylation level of the two isoforms regardless of whether hypertrophy or atrophy develops. Moreover, although numerous data argue that MLC2 phosphorylation lead to higher calcium sensitivity in skeletal muscle, an increase in MLC2 phosphorylation was measured after muscle atrophy whereas the calcium sensitivity decreases (Bozzo et al., To support this hypothesis, the variation of O-GlcNAcylation level on MLC2, as well as its interplay with phosphorylation, was investigated in atrophied soleus muscle (Cieniewski-Bernard et al., This interplay between phosphorylation and O-GlcNAcylation in the fine modulation of MLC2 activity was also observed in humans (Stevens et al., Taken together, all these data clearly corroborated the interplay between phosphorylation and O-GlcNAcylation on MLC2, in animal and in human models of functional atrophy. Indeed, the decrease in calcium sensitivity as well as calcium affinity measured in skinned fibers of atrophied muscle soleus cannot be obviously explained by the increase in MLC2 phosphorylation state, as it was previously described (Bozzo et al., However, further functional experiments need to be performed to demonstrate unambiguously that the phosphorylation/O-GlcNAcylation balance of MLC2 might be directly involved in the decrease in calcium sensitivity and affinity observed in atrophied muscles. While recent reports focused on MLC2, other regulatory contractile proteins have been demonstrated to be O-GlcNAcylated such as Tropomyosin, TnT and TnI (Cieniewski-Bernard et al., From the data described above, it seems very likely that O-GlcNAcylation plays a major role in modulating the contractile activity in skeletal muscle. However, the role of the O-GlcNAcylation might also concern other mechanisms involved in the sarcomeric structure. The preferential localization of OGT and OGA in the sarcomere, more particularly at the Z disk region (Cieniewski-Bernard et al., 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.


Collectively, our studies reveal a novel function of Src in priming Pyk2 (but not FAK) phosphorylation and subsequent activation downstream of integrins, and shed light on the signaling events that regulate the function of Pyk2.Proline-rich tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase (FAK) family of non-receptor tyrosine kinases and plays an important role in diverse cellular events downstream of the integrin-family of receptors, including cell migration, proliferation and survival. Here, we have identified a novel role for Src kinase in priming Pyk2 phosphorylation and subsequent activation upon cell attachment on the integrin-ligand fibronectin. By using complementary methods, we show that Src activity is indispensable for the initial Pyk2 phosphorylation on the Y402 site observed in response to cell attachment. In contrast, the initial fibronectin-induced autophosphorylation of FAK in the homologous Y397 site occurs in a Src-independent manner. We demonstrate that the SH2-domain of Src is required for Src binding to Pyk2 and for Pyk2 phosphorylation at sites Y402 and Y579. Moreover, Y402 phosphorylation is a prerequisite for the subsequent Y579 phosphorylation. While this initial phosphorylation of Pyk2 by Src is independent of Pyk2 kinase activity, subsequent autophosphorylation of Pyk2  Upon cell adhesion to extracellular matrix, the integrin-family of transmembrane receptors are clustered at sites termed focal adhesions and activate various intracellular signaling pathways. Focal adhesion kinase (FAK) family members are important downstream mediators of integrin signaling in events such as cell proliferation, survival, motility and invasion, and are therefore considered as plausible drug targets in various disease processes, such as inflammation and cancer ,2.-/- mouse embryonic fibroblasts (MEFs) and Pyk2 can compensate for some but not all FAK functions in these cells [Proline-rich tyrosine kinase 2 (Pyk2), also known as RAFTK and CAK-β, is a non-receptor tyrosine kinase that shares related structure and sequence similarity with FAK ,3,4. Botse cells ,10. Indese cells ,12.The underlying mechanism of FAK activation has been extensively studied. It has been shown that when FAK is inactive, the FAK FERM domain associates with and inhibits the FAK kinase domain, and deletion of the FERM domain promotes FAK activity . Upon rein trans in a manner that appears to be independent of Src [et al. have reported that oligomerization of Pyk2 is important for Pyk2 activity and that the FERM domain plays an important role in this process [et al. have demonstrated that Ca2+/calmodulin binds to the Pyk2 FERM-domain, which results in Pyk2 activation through homodimer formation and transphosphorylation [Given the similarity in amino acid sequence and structure between FAK and Pyk2, and given that Pyk2 contains sites Y402 and Y579/580 that are considered equivalent to sites Y397 and Y576/577 in FAK, it was expected that Pyk2 would be similarly regulated. Several differences have been observed, however. For example, while the FERM domain of Pyk2 inhibits Pyk2 activity, it appears to exert its effects without interacting with the Pyk2 kinase domain . Also, Pt of Src . Further process . In supprylation .in trans is required for full Pyk2 phosphorylation and activation. Collectively, our studies uncover a novel role for Src in priming Pyk2 phosphorylation and activation, and shed light on the signaling events that regulate the function of Pyk2.In this study, we show that Src activity is indispensable for the initial Pyk2 phosphorylation at site Y402 upon cell attachment. In contrast, and as shown before by others, fibronectin-induced autophosphorylation of FAK at the corresponding Y397 site occurs in a Src-independent manner. We further demonstrate that the Src SH2 domain is required for Src-Pyk2 interaction, and thus plays key role in Src phosphorylation of Pyk2. While this initial phosphorylation of Pyk2 by Src is independent of Pyk2 kinase activity, our results suggest that subsequent autophosphorylation of Pyk2 Anti-phospho-Pyk2 (Y402), anti-Pyk2, anti-Src and anti-α-tubulin antibodies were from Cell Signaling Technologies ; anti-phospho-Pyk2 (Y579) was from EMD Millipore ; anti-phospho-FAK (Y397) and anti-FAK monoclonal antibodies used in immunoblot analysis were from Transduction Laboratories ; anti-phospho-FAK (Y576) was purchased from Invitrogen ; and anti-β-actin antibody was from Sigma . Fibronectin was from Invitrogen . Src inhibitor SU11333  was provMyc-tagged wild-type and mutant Pyk2 plasmids were from Dr. Wencheng Xiong (Georgia Regents University), and were subcloned to pEGFP-C1 (Invitrogen) to create GFP-Pyk2 constructs. Wild-type Src and kinase-dead Src (K295N) constructs were obtained from Dr. Sara Courtneidge, and Src mutants Y416F, ΔSH2 and ΔSH3 were provided by Dr. Matthias Hentze (EMBL). Src SH2 and SH3 defective mutants R175L and W118K and the double mutant R175L/W118K were created by using the Quick Change Mutagenesis Kit . Pyk2-WT (amino acids 1–408) and Pyk2-Y402F (amino acids 1–408) were subcloned to pGEX-4T-1 as BamHI-XhoI fragments  to generate GST-Pyk2 constructs.++ MEF cells (SYF cells with Src-expression reconstituted) [HeLa cells, 293T cells, SYF fibroblasts derived from mouse embryos lacking three Src-family kinases  and Srctituted)  were froCell monolayers were serum-starved overnight, trypsinized and treated as described in the figure legends. Cells were then lysed in modified RIPA lysis buffer  containing a protease inhibitor cocktail , and clarified by centrifugation. Protein concentrations of each sample were determined by Bradford assay. Equal amounts of protein were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, blocked with 3% dry milk  in TBS-Tween-20 and exposed to specific primary antibodies as described for each experiment. Antibody binding was detected using horseradish peroxidase (HRP)-conjugated goat anti-rabbit or anti-mouse secondary antibodies (Sigma) and enhanced chemiluminescence . Tubulin or β-actin served as internal loading controls.SYF cells transfected as described in the figure legends were lysed in modified RIPA buffer and the lysates were clarified by centrifugation. After pre-cleared with protein A beads , the supernatants were incubated in anti-Src antibody  with gentle rocking at 4°C, followed by capture of the immunocomplexes with protein A beads for 1 h. The immunoprecipitates were thoroughly washed with lysis buffer to remove nonspecifically bound proteins, and then analyzed by immunoblotting as described above.E. coli strain and purified by using GST Spin Trap column  following manufacturer’s instructions. Purified GST-Pyk2 WT or Y402F were incubated with recombinant Src kinase , and phosphorylated Pyk2 was examined by immunoblotting using anti-Pyk2-pY402 antibody.GST fusion proteins of Pyk2-WT and Pyk-Y402F were expressed from pGEX4T plasmids in BL21 DE3 Previously, Src has been shown to interact with and phosphorylate FAK and Pyk2 upon integrin ligation by extracellular matrix proteins ,27. HereTo examine the role of Src in adhesion-mediated phosphorylation of Pyk2 and FAK, two Src kinase inhibitors, SU11333 and PP2, were used. We observed that fibronectin-mediated Pyk2 phosphorylation was significantly reduced at both the Y402 and Y579 sites by these inhibitors . In contIn support of these findings, we observed that transient over-expression of wild-type Src (Src-WT) significantly promoted fibronectin-induced Pyk2-Y402 phosphorylation, but it had no effect on FAK-Y397 phosphorylation . A kinas++)  , fibrone++) . As expeYF cells . All theIn addition to the tyrosine kinase catalytic domain, Src possesses N-terminal regulatory regions termed SH2- and SH3-domain. These modular domains mediate protein-protein interactions that are important in signal transduction ,30,31. TAs could be predicted, FAK-Y397 phosphorylation was not affected by expression of any of these Src constructs in SYF cells upon cell attachment on fibronectin . In contin trans [Conceivably, Src may mediate Pyk2 phosphorylation upon cell adhesion by two distinct mechanisms. That is, Src may directly phosphorylate Pyk2 on Y402, or Src may activate Pyk2, resulting in Pyk2 autophosphorylation. To discern between these two possibilities, we decided to employ 293T cells as an additional model system. It has been reported previously that these cells do not express detectable levels of endogenous Pyk2 , and we in trans .in vitro results show that Src can directly phosphorylate Pyk2 on Y402  demonstrated low, although detectable levels of phosphorylation at sites Y402 and Y579 upon cell attachment on fibronectin . We surm on Y402 . Taken tWe also examined the role of the Y402 site in Pyk2 phosphorylation by Src. When Pyk2-Y402F mutant was expressed in 293T cells with or without Src, this form of Pyk2 was not phosphorylated at the Y402 site, as expected due to the mutation . ImportaSimilar results were observed in the Src/Yes/Fyn-deficient SYF cells. In these cells, we expressed a GFP-tagged form of Pyk2 (with a relative molecular weight of ~145 kDa) so as to differentiate it from the endogenous Pyk2 (with a relative molecular weight of ~116 kDa) in immunoblots. When expressed in SYF cells, phosphorylation of the GFP-tagged Pyk2-WT (faint band on overexposure) or Pyk2-KD could not be detected upon cell attachment on fibronectin . Howeverin trans at the site Y402, and that this transphosphorylation is independent of Src kinase activity[et al. were used here next. When overexpressed in SYF cells in the presence of serum, a phosphorylated form of Myc-tagged Pyk2-WT, but not of Pyk2-Y402F or Pyk2-KD mutants, could be detected  phosphorylation and subsequent activation, and shed light on the signaling events that regulate the function of Pyk2. These insights may enable further pharmaceutical development of compounds that selectively modulate FAK and Pyk2 kinases in various therapeutic applications.S1 FigSYF cells were transfected with Pyk2 constructs in the presence or absence of Src-WT. The expression and phosphorylation of Pyk2 was examined by immunoblot.(TIF)Click here for additional data file.


Senescence can be triggered by many stimuli, including telomere attrition, DNA damage, oxidative stress, activation of oncogenes, and inactivation of tumor suppressor genes, and plays important roles in tumor suppression, organism aging, tissue repair, and embryonic development [Cellular senescence is a form of irreversible growth arrest historically associated with the exhaustion of replicative potential of elopment ,2.One key feature of senescence is irreversibility. Once become arrested, senescent cells cannot be stimulated to proliferate by any known physiological stimuli, despite the availability of space, nutrients and growth factors. This is in contrast to quiescence resulted from growth factor deprivation, which is reversible upon replenishment of the missing growth factors. Another type of quiescence is contact inhibition, in which cells become growth arrested when they contact each other at high density. Similarly contact inhibition is also reversible, as cells re-enter cell cycle and start proliferating when split and re-plated at low density. It had been a mystery what signaling pathways differentiate irreversible senescence from reversible quiescence induced by growth factor deprivation or contact inhibition. Now a recent paper published by Blagosklonny's group demonstrates that the conversion of reversible cell cycle arrest to irreversible senescence, a process termed geroconversion, is governed by the AKT-mammalian target of rapamycin (mTOR) pathway .The AKT/mTOR pathway is an important intracellular growth promoting pathway. Upon activation by receptor tyrosine kinases or G-protein coupled receptors, AKT activates mTOR, which in turn stimulates protein translation through inhibitory phosphorylation of eukaryotic translational initiation factor eIF4E-binding protein 1 (4E-BP1) that inhibits eIF4E, and activating phosphorylation of p70S6 kinase (S6K) that phosphorylates ribosomal S6 protein. A previous study suggests that the status of the mTOR differs between quiescence and senescence [This new study published by the Blagosklonny group focuses on the difference between senescence and quiescence induced by contact inhibition . The autThe authors then performed a reciprocal experiment by asking whether contact inhibition-mediated suppression of mTOR could convert senescence into reversible quiescence. To this end, a cancer cell line HT-p21 was used which ectopically expresses a key senescence effector p21 in an isopropyl-thio-galactosidase (IPTG)-dependent fashion. Addition of IPTG induces p21, leading to senescence in these cells. As HT-p21 is a cancer cell line that do not undergo contact inhibition, the authors cocultured HT-p21 cells with confluent RPE cells, thus creating an artificial contact-inhibition condition, which effectively inhibited mTOR activity, as compared to the HT-p21 cells cultured alone. Importantly, HT-p21 cells induced to overexpress p21 by IPTG in the co-culture with confluent RPE cells did not display senescence morphology, and retained proliferative potential in that they resumed proliferation and formed colonies upon IPTG removal and replating at low density. In contrast, IPTG-treated HT-p21 cells cultured alone became senescent and failed to proliferate or form colonies after replating. Taken together, these findings demonstrate that suppression of the mTOR pathway during contact-inhibition is responsible for the maintenance of the reversible growth arrest state and prevents the induction of irreversible senescence. Supporting the key role of mTOR in differentiating the quiescence and senescence states, the authors showed that other conditions that inhibit mTOR, such as treatment with rapamycin and exhaustion of nutrients in the medium by a high-density culture, also suppress the induction of senescence.The finding that contact-inhibited cells do not undergo senescence may have important physiological implications. Most cells in an organism are contact-inhibited and thus should retain the ability to resume proliferation under conditions such as tissue damage, where the tissue integrity needs to be restored. In addition, suppression of senescence in a densely packed tumor may explain why tumor cells are sometimes resistant to anti-cancer drugs. This study also raises several intriguing questions that need to be answered by further experiments. For example, what is the mechanism underlying mTOR suppression in contact inhibition? What act downstream of mTOR to promote quiescence-to-senescence conversion? Does the mTOR status differentiate senescence and quiescence under physiological or pathological conditions in vivo?


Prospective inhibition comes into play prior to the presentation of the stop signal and reactive inhibition follows the presentation of a signal to stop a particular action. The aim of this study is to investigate the neural activity evoked by prospective and reactive inhibition in adolescents with and without ADHD.Deficient response inhibition in situations involving a trade-off between response execution and response stopping is a hallmark of attention deficit hyperactive disorder (ADHD). There are two key components of response inhibition; reactive inhibition where one attempts to Twelve adolescents with ADHD and 12 age-matched healthy controls (age range 9–18) were imaged while performing the stop signal task (SST).Reactive inhibition activated right inferior frontal gyrus (IFG) in both groups. ADHD subjects activated IFG bilaterally. In controls, prospective inhibition invoked preactivation of the same part of right IFG that activated during reactive inhibition. In ADHD subjects, prospective inhibition was associated with deactivation in this region. Controls also deactivated the medial prefrontal cortex (MPFC) during prospective inhibition, whereas ADHD subjects activated the same area.This pattern of activity changes in the same structures, but in opposite directions, was also evident across all phases of the task in various task-specific areas like the superior and middle temporal gyrus and other frontal areas.Differences between ADHD and control participants in task-specific and default mode structures (IFG and MPFC) were evident during prospective, but not during reactive inhibition. We isolated prospective inhibition from motor activities during the go phase of the task by contrasting right- and left-hand responses in order to preferentially conserve common cognitive activities while suppressing those specific to the handedness of the response. The resulting estimate of prospective inhibition activities that precede the appearance of stop signals could then be used to isolate activities underlying reactive inhibition that occurs after the presentation of stop signals. Using this approach in a group of healthy adults  who had been taking stimulant medication were asked to stop 24 h prior to the scan in order to eliminate drug-induced BOLD changes  between the ages of 9–18 years. Participants gave informed, written consent and the Hospital for Sick Children institutional research ethics board approved the study. ADHD participants . Control subjects were assessed in a comparable manner and reported no psychiatric or medical disorders. All subjects were right-handed and had normal vision and hearing.The stop-signal task (SST) involves a primary choice reaction time task and a secondary stop task. Each trial began with a fixation point which appeared in the center of a black screen for 500 ms, followed by the go stimulus for 1000 ms. Participants were instructed to respond as quickly and as accurately as possible with their left thumb using a fMRI-compatible response box when the letter “X” appears on the screen or with their right thumb when the letter “O” appeared. In 33% of the trials, a stop signal (background color change from black to red) followed the go stimulus by some delay. Participants were told to stop their response if they saw the stop signal. They were told not to wait for stop signals. The initial stop-signal delay was 250 ms and was dynamically adjusted following each stop signal. When subjects successfully inhibited a response, the delay was increased by 50 ms on the next stop trial and when they failed to stop a response, the delay was decreased by 50 ms. Dynamic adjustment tracks to the delay and on average, individuals can stop 50% of responses when a stop signal is presented. Intertrial interval (ITI) was jittered such that trials were either 2.5 or 3.5 sec to ensure no multicollinearity of event types. The trials were jittered using random combinations of spread-spectrum binary coding sequences to maximize the number of independent equations in the deconvolution analysis, which enhances the separation of the event types in the experimental design. In order to establish a well-defined baseline of neural activity, every fourteenth trial was followed by a 17.5 sec rest period in which no stimuli were presented. This also ensured consistency with the bounds established by Ollinger et al.  for the Trial order was pseudorandomized so that the current type of trial did not predict the subsequent kind of trial. The task involved 224 trials, requiring a total scan time of 15 min. The mean go response time (RT) was observable from the 67% of trials in which no stop signal appeared. The stop-signal reaction time (SSRT) was estimated by subtracting the mean delay on stop-signal trials from the mean go RT on no-signal trials.All behavioral data were analyzed using SPSS . We assessed group differences in behavioral measures , and percentage of successful inhibition (PSI). For adequate performance, the PSI was required to be approximately in the 50th percentile and PCR was required to be above 95 percent.3. Behavioral data were collected using a Lumitouch fibre-optic button box  interfaced to a laptop running the stop task paradigm.Imaging was done with a GE LX 1.5T MRI scanner . Anatomical data were acquired with a standard high-quality SPGR sequence . Functional data were collected using a GRE-EPI sequence with a custom 8-channel head coil . These images were reconstructed to a 64 × 64 pixel resolution and final voxel size of 3.75 × 3.75 × 6 mmFunctional data were analyzed using AFNI Cox . Images common prospective inhibition, which is present in every trial of the SST. Activity associated with reactive inhibition on successful stop trials could then be estimated by removing the prospective inhibitory activity from successful stop trials using the contrast [stop–½(X + O)].Prospective inhibition activity common to both left- and right-hand responses was isolated using the contrast [½(X + O)]. This contrast suppresses activity specific to left- and right-hand responses, which only occur on a subset of trials trial, while enhancing neural activity that reflects 3 resolution. The single subject activation maps were passed on to a random effects ANOVA analysis that was conducted separately for the ADHD and healthy control groups in order to identify the general pattern of whole brain corrected activity for each group. Maps for ADHD and healthy controls were examined to identify qualitative differences in their patterns of activity.Intensity maps for the relevant contrasts (prospective inhibition = [½(X + O)] and reactive inhibition = [stop–½(X + O)]) were generated for individual subjects by taking the area under the HRF, warped into Talairach space, Gaussian blurred (6-mm FWHM), and resampled at 1 mmGroup difference maps were generated using a nested repeated-measures 3-factor ANOVA  in order to identify significantly different activities between healthy and ADHD adolescents. Group difference (Control-ADHD) for prospective and reactive inhibition activities from the ANOVA output were distributed as a t* statistic with 138° of freedom due to the number of regressors and subjects in the study.Z scores and corrected for multiple comparisons using AFNI's AlphaSim program as in Chevrier et al. (3) with a minimum Z score of 2.32, for an overall α of 0.046.Output from all the analyses  were converted to raw r et al. . AlphaSir et al. .This anaP = 0.06) and Go RT (P = 0.08). There was no difference in IQ or in Stop Task performance as indexed by percentage correct response (PCR) (P = 0.42) and percentage of successful inhibition (PSI) (P = 0.38). Behavioral data confirmed that there were no differences in either the performance of the task or in the speed of the going process other than in the latency of the response inhibition process or SSRT  and deactivation in the right medial prefrontal cortex (MPFC) . We also observed positive activation in the right middle frontal gyri , left superior frontal gyrus , right superior temporal gyrus , and inferior parietal lobe (IPL) . Deactivations were observed in the anterior temporal lobe , bilateral posterior insula , left supramarginal/post central gyrus , left precuneus , and various cerebellar areas.During prospective inhibition, the control group exhibited positive activity in the right IFG , in addition to positive activity in left IPL , left fusiform , right posterior cingulate cortex (PCC) , and left cerebellum . They also showed a deactivation in the right cingulate gyrus .During reactive inhibition, the control group elicited positive activity in the right inferior frontal gyrus (IFG)  as well as a deactivation in the right superior frontal , left presupplementary area , left superior temporal gyrus , right middle temporal gyrus , posterior insula , PCC , and precuneus areas . ADHD subjects also showed positive activation in the inferior temporal gyrus  and right cingulate gyrus .During prospective inhibition, the ADHD group exhibited deactivation in the right IFG , right superior frontal , right middle frontal , right  and left caudate , right IPL , and cuneus  There were no significant negative activations in this map  on this plot (in SPSS) was capable of classifying subjects based on their prospective withholding activity in these two regions alone. The discriminant analysis assigns a posterior probability of being in a particular group. A loading factor for the variates above 0.30 is generally considered to be a meaningful contributor to the discriminant score. In our study, the two discriminants, that is, IFG and MPFC activities had loadings of -0.59 and 0.76, respectively.Medial prefrontal cortex and IFG activities in normal controls and ADHD groups during prospective withholding are plotted in Figure We used the stop-signal task to separate neural activity related to prospective inhibition of a response from activity associated with reactive inhibition in adolescents with and without ADHD. The approach provided an estimate of prospective inhibition-related activity during the go phase of the stop task by explicitly accounting for the handedness of responses in the deconvolution analysis and by using a statistical contrast that enhanced cognitive activities common to left- and right-hand responses while suppressing activities specific to the lateralized motor response. This contrast also provides an optimal baseline from which to estimate activity attributable to reactive inhibition. Stop trials involve prospective inhibition activity that is similar to that on go trials, but do not actually contain an overt motor response. Therefore, estimating go-phase activity on stop trials using a contrast that suppresses hand-specific response-related activity is preferable to using a single regressor for all go trials because common and hand-specific go-phase activities are not statistically independent and should be accounted for in a simultaneous regression analysis. Therefore, both theoretical and empirical evidence support the use of the current method for separating prospective inhibition from reactive inhibition  (Corbetta and Shulman Inferior frontal gyrus activation in prospective inhibition indicates a broader role for IFG as suggested by Hampshire et al. . The rigParticipants with ADHD had significantly longer SSRT, which indicated deficient inhibitory control (Verbruggen and Logan negative activation in the IFG and activation in the MPFC during inhibition but comparable activity to controls in IFG during inhibition. This pattern is in contrast to previous studies that consistently report negative activation in the IFG during inhibition (Pliszka et al. Attention deficit hyperactive disorder participants showed Negative activation in the MPFC in control groups during prospective inhibition supports the role of the ACC in default mode network processing (Gusnard et al. Studies of default mode network support the hypothesis of atypical preparation in ADHD. The default mode network is associated with spontaneous attentional fluctuations (Sonuga-Barke and Castellanos disengage default mode networks and engage task-related networks during prospective inhibition, but further demonstrate that the ADHD subjects are in fact doing the opposite during prospective inhibition: namely, disengaging task-related circuits while engaging default mode circuits more intensely.Consistent with previous findings, our results indicate a failure to transition from the default mode to task-oriented networks in the ADHD group which in turn may affect their ability to inhibit a motor response. In fact, our results not only show a failure to properly This interpretation is supported by the observation that in our sample, the ADHD group showed negative activity and controls showed positive activity in areas that are critical for efficient task completion. For example, during the go phase, the control group exhibited positive activity and the ADHD group displayed negative activity in the superior and middle temporal regions and anterior insula. Superior and middle temporal regions are involved in mental preparation (Kounios et al. In short, the pattern of preparatory activity in the right structures, but in the wrong direction compared to baseline, suggests that the inhibitory control deficit in ADHD could be associated with inappropriate tuning of networks involved in preparedness and attention to the task at hand.These activity differences between ADHD and controls, consistently being the result of activity changes in opposite directions with respect to baseline, can perhaps be explained through the dysfunctional dopamine modulation in ADHD. Several studies have identified abnormalities in dopaminergic neurotransmission systems in an ADHD population (Rowe et al. This is the first study to differentiate neural activity during reactive inhibition and prospective inhibition in ADHD and normal control adolescents. We adopted a methodology that makes it possible to investigate these cognitive processes during inhibition within the confines of a single task.It should be noted that while the current approach separates prospective from reactive inhibition, it is currently not possible to distinguish between prospective inhibition, monitoring, and the wave of various simultaneous cognitive processes and central response activations that precede the lateralization of the motor response that also resides in the go phase. We chose to focus on one aspect of the cognitive activity that lives in the go phase and future advancements in analytical techniques may make it possible to extricate the influence of events that occur almost simultaneously in time.The next step in our research is to investigate the reinforcement-learning signals that arise following errors in an ADHD sample. The aim is to provide a clearer view of how processes like error detection and the subsequent modification after failed stop trials affect inhibitory control in normal and ADHD groups and the role of dopamine-regulated activities in models of stop-signal task performance in health and disease.