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PubMed2001

Src family kinase involvement in muscarinic receptor-induced tyrosine phosphorylation in differentiated SH-SY5Y cells.

Muscarinic receptor-mediated changes in protein tyrosine phosphorylation were examined in differentiated human neuroblastoma SH-SY5Y cells. Treatment of differentiated cells with 1 mM carbachol caused rapid increases in the tyrosine phosphorylation of focal adhesion kinase (FAK), Cas, and paxillin. The src family kinase-selective inhibitor PP1 reduced carbachol-stimulated tyrosine phosphorylation of FAK, Cas, and paxillin by 50 to 75%. In contrast, carbachol-stimulated activation of ERK1/2 was unaffected by PP1. Src family kinase activation by carbachol was further demonstrated by increased carbachol-induced tyrosine phosphorylation of the src-substrate, p120, and tyrosine phosphorylation of the src family kinase activation-associated autophosphorylation site. Site-specific FAK phosphotyrosine antibodies were used to determine that the carbachol-stimulated increase in the autophosphorylation of FAK was unaffected by pretreatment with PP1, whereas the carbachol-stimulated increase in the src family kinase-mediated phosphotyrosine of FAK was completely blocked by pretreatment with PP1. In SH-SY5Y cell lines stably overexpressing Fyn, the phosphotyrosine immunoreactivity of FAK was 625% that of control cells. Thus, muscarinic receptors activate protein tyrosine phosphorylation in differentiated cells, and the tyrosine phosphorylation of FAK, Cas, and paxillin, but not ERK1/2, is mediated by a src family tyrosine kinase activated in response to stimulation of muscarinic receptors.

Carbachol↗
PubMed2003

Protein phosphatase-2A regulates protein tyrosine phosphatase activity in Lewis lung carcinoma tumor variants.

Cellular adherence and motility are processes that are controlled by focal adhesion assembly and disassembly. Consequently, the dynamics of focal adhesions regulate tumor cell metastasis and are influenced by the tyrosine phosphorylation state of paxillin. Metastatic LLC cells are more migratory and have reduced paxillin tyrosine phosphorylation as compared to nonmetastatic LLC cells. In nonmetastatic Lewis lung carcinoma (LLC) tumor cells, inhibition of the serine/threonine protein phosphatase-2A (PP-2A) activity results in increased motility that is associated with a reduction in the phosphotyrosine content of paxillin. Studies to determine if PP-2A can regulate protein tyrosine phosphatase activity showed that blocking PP-2A activity of nonmetastatic LLC-C8 tumor cells with okadaic acid reduces protein tyrosine phosphatase activity. Among the tyrosine phosphatases whose activity was inhibited upon PP-2A inhibition is Shp-2. In contrast, protein levels of Shp-2 are unaffected by PP-2A inhibition. While these results do not fully identify how inhibition of PP-2A results in tyrosine dephosphorylation of paxillin, they do demonstrate that PP-2A can link serine/threonine and tyrosine signaling pathways by regulating protein tyrosine phosphatases.

Animals↗
PubMed1994

Lymphocyte antigen receptor activation of a focal adhesion kinase-related tyrosine kinase substrate.

One of the earliest responses of T and B lymphocytes to stimulation through their antigen receptors is the activation of protein tyrosine kinases and the tyrosine phosphorylation of multiple cellular substrates. Here we describe a tyrosine kinase substrate, fakB, a putative homologue of the focal adhesion kinase pp125FAK. Tyrosine phosphorylation of fakB was rapidly augmented in human T and B cells following antigen receptor cross-linking with antibody, while pp125FAK was nonresponsive. Costimulation of the T-cell antigen receptor (TCR/CD3) with either the CD2 or CD4 costimulatory receptors induced synergistic fakB tyrosine phosphorylation in normal human T cells. Engagement of TCR/CD3 induced the stable association of fakB with ZAP-70, the TCR/CD3 sigma-chain-associated tyrosine kinase involved in antigen receptor-induced T-cell activation. In addition, preformed complexes of fakB and ZAP-70 were observed in T-cell leukemia lines. Phosphorylation of fakB on serine, threonine, and tyrosine residues was observed both in vivo and in vitro, where a functional increase of in vitro kinase activity was observed following TCR/CD3 stimulation. fakB is thus a focal adhesion kinase-related tyrosine kinase substrate that is differentially regulated from that of pp125FAK and likely plays a role in antigen-induced lymphocyte signaling.

Amino Acid Sequence↗
PubMed1995

Divergent insulin and platelet-derived growth factor regulation of focal adhesion kinase (pp125FAK) tyrosine phosphorylation, and rearrangement of actin stress fibers.

Insulin treatment of Chinese hamster ovary cells expressing high levels of the human insulin receptor resulted in the tyrosine dephosphorylation of the 125-kDa focal adhesion kinase (pp125FAK). The decrease in pp125FAK tyrosine phosphorylation paralleled a decrease in the cellular content of actin stress fibers, and these changes were independent of the extracellular matrix on which the cells were grown. The reduction in both pp125FAK tyrosine phosphorylation and actin stress fibers occurred in an insulin concentration-dependent manner, with significant effects at approximately 0.3 nM and a maximal effect at 3 nM. However, in the continuous presence of insulin, the decreases in the tyrosine phosphorylation state of pp125FAK and actin stress fiber content were transient. Maximal reduction of pp125FAK tyrosine phosphorylation was observed following 15 min of insulin treatment, with a return to unstimulated control levels by 60 min. Similarly, actin stress fiber content was maximally reduced by 15 min of insulin treatment and fully recovered by 60 min. In contrast to insulin, platelet-derived growth factor stimulation increased actin stress fiber content and enhanced pp125FAK tyrosine phosphorylation. These data demonstrate a novel signaling role for insulin in inducing the tyrosine dephosphorylation of pp125FAK and a concomitant reorganization of actin stress fibers, which underlies at least one aspect of signaling divergence between the insulin and platelet-derived growth factor receptor tyrosine kinases.

Actins↗
PubMed1995

The yeast immunophilin Fpr3 is a physiological substrate of the tyrosine-specific phosphoprotein phosphatase Ptp1.

The tyrosine-specific phosphoprotein phosphatase encoded by the Saccharomyces cerevisiae PTP1 gene dephosphorylates artificial substrates in vitro, but little is known about its functions and substrates in vivo. The presence of Ptp1 resulted in dephosphorylation of multiple tyrosine-phosphorylated proteins in yeast expressing a heterologous tyrosine-specific protein kinase, indicating that Ptp1 can dephosphorylate a broad range of substrates in vivo. Correspondingly, several proteins phosphorylated at tyrosine by endogenous protein kinases exhibited a marked increase in tyrosine phosphorylation in ptp1 mutant cells. One of these phosphotyrosyl proteins (p70) was also dephosphorylated in vitro when incubated with recombinant Ptp1. p70 was purified to homogeneity; analysis of four tryptic peptides revealed that p70 is identical to the recently described FPR3 gene product, a nucleolarly localized proline rotamase of the FK506- and rapamycin-binding family. The identity of p70 with Fpr3 was confirmed in the demonstration that the abundance of tyrosine-phosphorylated p70 in ptp1 mutants was strictly correlated with the level of FPR3 expression; immobilized phosphotyrosyl Fpr3 was directly dephosphorylated by recombinant Ptp1. Site-directed mutagenesis demonstrated that the site of tyrosine phosphorylation is Tyr-184, which resides within the nucleolin-like amino-terminal domain of Fpr3. Protein kinase activities from yeast cell extracts can bind to and phosphorylate the immobilized amino-terminal domain of Fpr3 on serine, threonine, and tyrosine. Fpr3 represents the first phosphotyrosyl protein identified in S. cerevisiae that is not itself a protein kinase and is as yet the only known physiological substrate of Ptp1.

Amino Acid Sequence↗
PubMed1996

Interdependence of calcium signaling and protein tyrosine phosphorylation in human endothelial cells.

The signal transduction cascade which initiates transmembraneous influx of Ca2+ into endothelial cells in response to the discharge of intracellular Ca2+ stores is thought to involve a step sensitive to tyrosine kinase inhibition. We investigated the interrelationship between Ca2+ signaling and protein tyrosine phosphorylation following cell stimulation with either the receptor-dependent agonist, bradykinin, or the protein-tyrosine phosphatase inhibitor, phenylarsine oxide. In cultured human endothelial cells phenylarsine oxide instigated a concentration-dependent increase in the intracellular concentration of free Ca2+ ([Ca2+]i). This increase in [Ca2+]i was not associated with the tyrosine phosphorylation of phospholipase C gamma, enhanced formation of inositol 1,4,5-trisphosphate, or the rapid depletion of intracellularly stored Ca2+ but was coincident with the enhanced and prolonged tyrosine phosphorylation of a number of cytoskeletal proteins. In bradykinin-stimulated cells the tyrosine phosphorylation of the same cytoskeletal proteins (most notably 85- and 100-kDa proteins) was transient when cells were stimulated in the presence of extracellular Ca2+, was maintained under Ca2+-free conditions, and was reversed following readdition of extracellular Ca2+. These data suggest that the tyrosine phosphorylation of 2 cytoskeletal proteins is determined by the level of Ca2+ present in intracellular stores thus indicating a critical role for tyrosine phosphorylation in the control of capacitative Ca2+ entry in endothelial cells.

Amino Acid Sequence↗
PubMed1997

Vascular endothelial growth factor stimulates tyrosine phosphorylation and recruitment to new focal adhesions of focal adhesion kinase and paxillin in endothelial cells.

Vascular endothelial growth factor (VEGF) stimulated the tyrosine phosphorylation of multiple components in confluent human umbilical vein endothelial cells (HUVECs) including bands of Mr 205,000, corresponding to the VEGF receptors Flt-1 and KDR, and Mr 145,000, 120,000, 97,000, and 65,000-70,000. VEGF caused a striking and transient increase in mitogen-activated protein (MAP) kinase activity and stimulated phospholipase C-gamma tyrosine phosphorylation, but it had no effect on phosphatidylinositol 3'-kinase activity. VEGF caused a marked increase in tyrosine phosphorylation of p125 focal adhesion kinase (p125(FAK)), which was both rapid and concentration-dependent. VEGF produced similar effects on p125(FAK) in the endothelial cell line ECV.304. VEGF stimulated tyrosine phosphorylation of the 68-kDa focal adhesion-associated component, paxillin, with similar kinetics and concentration dependence to that for p125(FAK). Thrombin and the phorbol ester, phorbol 12-myristate 13-acetate, also increased p125(FAK) tyrosine phosphorylation in HUVECs. The effect of VEGF on p125(FAK) tyrosine phosphorylation was completely inhibited by the actin filament-disrupting agent cytochalasin D and was partially inhibited by the protein kinase C inhibitor GF109203X. Inhibition of the MAP kinase pathway using a specific inhibitor of MAP kinase kinase had no effect on p125(FAK) tyrosine phosphorylation. VEGF stimulated migration and actin stress fiber formation in confluent HUVEC, and VEGF-induced p125(FAK)/paxillin tyrosine phosphorylation was accompanied by increased immunofluorescent staining of p125(FAK), paxillin, and phosphotyrosine in focal adhesions in confluent cultures of HUVECs. These findings identify p125(FAK) and paxillin as components in a VEGF-stimulated signaling pathway and suggest a novel mechanism for VEGF regulation of endothelial cell functions.

Cell Adhesion Molecules↗
PubMed1997

Tyrosine phosphorylation of RNA polymerase II carboxyl-terminal domain by the Abl-related gene product.

The largest subunit of RNA polymerase II contains a C-terminal repeated domain (CTD) that is the site of phosphorylation by serine (threonine) and tyrosine kinases. Phosphorylation of the CTD is correlated with transcription elongation. A number of different kinases have previously been shown to phosphorylate the CTD; among them is a nuclear tyrosine kinase encoded by the c-abl proto-oncogene. The processive and high stoichiometric phosphorylation of RNA polymerase II by c-Abl requires the tyrosine kinase, the SH2 domain, and a CTD-interacting domain (CTD-ID) in the Abl protein. The physiological tyrosine phosphorylation of RNA polymerase II by c-Abl in DNA damage response has previously been demonstrated. Basal tyrosine phosphorylation of RNA polymerase II, however, is observed in cells derived from abl-deficient mice, indicating the existence of other CTD tyrosine kinases. In this report, we show that the tyrosine kinase encoded by an Abl-related gene (Arg) also phosphorylates the CTD in vitro and in transfected cells. The SH2 and kinase domain of Arg are 95% identical to that of c-Abl. However, these two proteins share only 29% identity in the large C-terminal region. Interestingly, a CTD-ID is also found in the C-terminal region of Arg. Mapping studies and sequence analysis have led to the identification of the CTD-ID that is highly conserved among the divergent C-terminal regions of Abl and Arg. These results indicate that tyrosine phosphorylation of RNA polymerase II CTD could be catalyzed by either c-Abl or Arg kinase.

Amino Acid Sequence↗
PubMed1997

G protein-coupled receptors mediate two functionally distinct pathways of tyrosine phosphorylation in rat 1a fibroblasts. Shc phosphorylation and receptor endocytosis correlate with activation of Erk kinases.

The Ras-dependent activation of Erk kinases by G protein-coupled receptors (GPCRs) is thought to involve tyrosine phosphorylation of docking proteins that serve as scaffolds for the plasma membrane recruitment of Ras guanine nucleotide exchange factors, such as the Grb2-mSos complex. We have investigated the role of two GPCR-regulated tyrosine phosphoproteins, p125(FAK) (FAK) and Shc, in the Ras-dependent activation of Erk kinases by endogenously expressed GPCRs in Rat 1a fibroblasts. Several lines of evidence suggest that tyrosine phosphorylation of FAK and Shc are independently regulated. The GPCRs for lysophosphatidic acid (LPA), thrombin, and bombesin mediate equivalent increases in FAK tyrosine phosphorylation and FAK-Grb2 association. In contrast, only LPA and thrombin receptors significantly stimulate Shc tyrosine phosphorylation and Shc-Grb2 complex formation. Tyrosine phosphorylation of FAK is pertussis toxin-insensitive, can be mimicked by calcium ionophore, and is inhibited by treatment with cytochalasin D, which depolymerizes the actin cytoskeleton. In contrast, tyrosine phosphorylation of Shc is inhibited by pertussis toxin treatment, is not induced by calcium ionophore, and is insensitive to cytochalasin D. In each case, the rapid stimulation of Erk 1/2 correlates with tyrosine phosphorylation of Shc but not of FAK. The dissociation of FAK-Grb2 complex formation from receptor-mediated activation of Erk 1/2 indicates that recruitment of Grb2-mSos to the plasma membrane is not sufficient to mediate rapid Erk activation. Using four mechanistically distinct inhibitors of clathrin-mediated endocytosis, concanavalin A, hypertonic medium, depletion of intracellular potassium, and monodansylcadaverine, we find that GPCR-mediated Erk 1/2 activation is also endocytosis-dependent. Thus, we propose that an additional step involving vesicle-mediated endocytosis is required for the rapid, Ras-dependent activation of Erk kinases in fibroblasts.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Pervanadate stimulates amylase release and protein tyrosine phosphorylation of paxillin and p125(FAK) in differentiated AR4-2J pancreatic acinar cells.

We have studied the role of protein tyrosine phosphorylation in amylase secretion from differentiated AR4-2J cells. The secretagogue bombesin, the protein kinase C activator phorbol 12-myristate 13-acetate (PMA), and the protein-tyrosine phosphatase inhibitor pervanadate induced tyrosine phosphorylation of different proteins, including paxillin and p125(FAK), which was reduced or blocked by the tyrosine kinase inhibitors genistein and tyrphostin B56, respectively. Both PMA and pervanadate continuously increased amylase secretion with a similar time course, reaching the level of bombesin-induced amylase release after 60 min. Their effects were not additive and could be inhibited by preincubation of AR4-2J cells with genistein or tyrphostin B56, respectively. Inhibition of protein kinase C with Ro 31-8220 nearly abolished the effects of PMA, but had no effect on either pervanadate-induced protein tyrosine phosphorylation or amylase secretion. An increase in cytosolic free Ca2+ concentration by thapsigargin or A23187 caused a rapid increase in amylase release within the initial 5 min. In the presence of PMA or pervanadate, amylase secretion was further stimulated to levels comparable to those induced by bombesin after 30 min of stimulation. Inhibition of PMA-induced amylase secretion by Ro 31-8220 was less at elevated cytosolic free Ca2+ concentrations than without Ca2+. Furthermore, an increase in cytosolic free Ca2+ concentration had no effect on protein tyrosine phosphorylation in either the absence or presence of PMA or pervanadate. We therefore conclude that in the cascade of events that lead to bombesin-induced protein secretion from AR4-2J cells, protein tyrosine phosphorylation occurs downstream of protein kinase C activation. A further step in secretion that is Ca2+-dependent occurs distal to protein tyrosine phosphorylation.

Amylases↗
PubMed2001

Palmitoylation of caveolin-1 at a single site (Cys-156) controls its coupling to the c-Src tyrosine kinase: targeting of dually acylated molecules (GPI-linked, transmembrane, or cytoplasmic) to caveolae effectively uncouples c-Src and caveolin-1 (TYR-14).

Caveolin-1 was initially identified as a phosphoprotein in Rous sarcoma virus-transformed cells. Previous studies have shown that caveolin-1 is phosphorylated on tyrosine 14 by c-Src and that lipid modification of c-Src is required for this phosphorylation event to occur in vivo. Phosphocaveolin-1 (Tyr(P)-14) localizes within caveolae near focal adhesions and, through its interaction with Grb7, augments anchorage-independent growth and epidermal growth factor-stimulated cell migration. However, the cellular factors that govern the coupling of caveolin-1 to the c-Src tyrosine kinase remain largely unknown. Here, we show that palmitoylation of caveolin-1 at a single site (Cys-156) is required for coupling caveolin-1 to the c-Src tyrosine kinase. Furthermore, upon evaluating a battery of nonreceptor and receptor tyrosine kinases, we demonstrate that the tyrosine phosphorylation of caveolin-1 by c-Src is a highly selective event. We show that Src-induced tyrosine phosphorylation of caveolin-1 can be inhibited or uncoupled by targeting dually acylated proteins (namely carcinoembryonic antigen (CEA), CD36, and the NH(2)-terminal domain of Galpha(i1)) to the exoplasmic, transmembrane, and cytoplasmic regions of the caveolae membrane, respectively. Conversely, when these proteins are not properly targeted or lipid-modified, the ability of c-Src to phosphorylate caveolin-1 remains unaffected. In addition, when purified caveolae preparations are preincubated with a myristoylated peptide derived from the extreme N terminus of c-Src, the tyrosine phosphorylation of caveolin-1 is abrogated; the same peptide lacking myristoylation has no inhibitory activity. However, an analogous myristoylated peptide derived from c-Yes also has no inhibitory activity. Thus, the inhibitory effects of the myristoylated c-Src peptide are both myristoylation-dependent and sequence-specific. Finally, we investigated whether phosphocaveolin-1 (Tyr(P)-14) interacts with the Src homology 2 and/or phosphotyrosine binding domains of Grb7, the only characterized downstream mediator of its function. Taken together, our data identify a series of novel lipid-lipid-based interactions as important regulatory factors for coupling caveolin-1 to the c-Src tyrosine kinase in vivo.

Animals↗
PubMed2001

Transcription factor STAT5A is a substrate of Bruton's tyrosine kinase in B cells.

STAT5A is a molecular regulator of proliferation, differentiation, and apoptosis in lymphohematopoietic cells. Here we show that STAT5A can serve as a functional substrate of Bruton's tyrosine kinase (BTK). Purified recombinant BTK was capable of directly binding purified recombinant STAT5A with high affinity (K(d) = 44 nm), as determined by surface plasmon resonance using a BIAcore biosensor system. BTK was also capable of tyrosine-phosphorylating ectopically expressed recombinant STAT5A on Tyr(694) both in vitro and in vivo in a Janus kinase 3-independent fashion. BTK phosphorylated the Y665F, Y668F, and Y682F,Y683F mutants but not the Y694F mutant of STAT5A. STAT5A mutations in the Src homology 2 (SH2) and SH3 domains did not alter the BTK-mediated tyrosine phosphorylation. Recombinant BTK proteins with mutant pleckstrin homology, SH2, or SH3 domains were capable of phosphorylating STAT5A, whereas recombinant BTK proteins with SH1/kinase domain mutations were not. In pull-down experiments, only full-length BTK and its SH1/kinase domain (but not the pleckstrin homology, SH2, or SH3 domains) were capable of binding STAT5A. Ectopically expressed BTK kinase domain was capable of tyrosine-phosphorylating STAT5A both in vitro and in vivo. BTK-mediated tyrosine phosphorylation of ectopically expressed wild type (but not Tyr(694) mutant) STAT5A enhanced its DNA binding activity. In BTK-competent chicken B cells, anti-IgM-stimulated tyrosine phosphorylation of STAT5 protein was prevented by pretreatment with the BTK inhibitor LFM-A13 but not by pretreatment with the JAK3 inhibitor HI-P131. B cell antigen receptor ligation resulted in enhanced tyrosine phosphorylation of STAT5 in BTK-deficient chicken B cells reconstituted with wild type human BTK but not in BTK-deficient chicken B cells reconstituted with kinase-inactive mutant BTK. Similarly, anti-IgM stimulation resulted in enhanced tyrosine phosphorylation of STAT5A in BTK-competent B cells from wild type mice but not in BTK-deficient B cells from XID mice. In contrast to B cells from XID mice, B cells from JAK3 knockout mice showed a normal STAT5A phosphorylation response to anti-IgM stimulation. These findings provide unprecedented experimental evidence that BTK plays a nonredundant and pivotal role in B cell antigen receptor-mediated STAT5A activation in B cells.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed2006

Role of protein-tyrosine phosphatase SHP2 in focal adhesion kinase down-regulation during neutrophil cathepsin G-induced cardiomyocytes anoikis.

Inflammatory cells and their proteases contribute to tissue reparation at site of inflammation. Although beneficial at early stages, excessive inflammatory reaction leads to cell death and tissue damage. Cathepsin G (Cat.G), a neutrophil-derived serine protease, has been shown to induce neonatal rat cardiomyocyte detachment and apoptosis by anoikis through caspase-3 dependent pathway. However the early mechanisms that trigger Cat.G-induced caspase-3 activation are not known. This study identifies focal adhesion kinase (FAK) tyrosine dephosphorylation as an early mechanism that regulates Cat.G-induced anoikis in cardiomyocytes. Both FAK tyrosine phosphorylation at Tyr-397 and kinase activity decrease rapidly upon Cat.G treatment and was associated with a decrease of FAK association with adapter and cytoskeletal proteins, p130(Cas) and paxillin, respectively. FAK-decreased tyrosine phosphorylation is required for Cat.G-induced myocyte anoikis as concurrent expression of phosphorylation-deficient FAK mutated at Tyr-397 or pretreatment with a protein-tyrosine phosphatase (PTP) inhibitor, pervanadate, blocks Cat.G-induced FAK tyrosine dephosphorylation, caspase-3 activation and DNA fragmentation. Analysis of PTPs activation shows that Cat.G treatment induces an increase of SHP2 and PTEN phosphorylation; however, only SHP2 forms a complex with FAK in response to Cat.G. Expression of dominant negative SHP2 mutant markedly attenuates FAK tyrosine dephosphorylation induced by Cat.G and protects myocytes to undergo apoptosis. In contrast, increased SHP2 expression exacerbates Cat.G-induced FAK tyrosine dephosphorylation and myocyte apoptosis. Taken together, these results show that Cat.G induces SHP2 activation that leads to FAK tyrosine dephosphorylation and promotes cardiomyocyte anoikis.

Adenoviridae↗
PubMed2004

Fyn and PTP-PEST-mediated regulation of Wiskott-Aldrich syndrome protein (WASp) tyrosine phosphorylation is required for coupling T cell antigen receptor engagement to WASp effector function and T cell activation.

Involvement of the Wiskott-Aldrich syndrome protein (WASp) in promoting cell activation requires its release from autoinhibitory structural constraints and has been attributed to WASp association with activated cdc42. Here, however, we show that T cell development and T cell receptor (TCR)-induced proliferation and actin polymerization proceed normally in WASp-/- mice expressing a WASp transgene lacking the cdc42 binding domain. By contrast, mutation of tyrosine residue Y291, identified here as the major site of TCR-induced WASp tyrosine phosphorylation, abrogated induction of WASp tyrosine phosphorylation and its effector activities, including nuclear factor of activated T cell transcriptional activity, actin polymerization, and immunological synapse formation. TCR-induced WASp tyrosine phosphorylation was also disrupted in T cells lacking Fyn, a kinase shown here to bind, colocalize with, and phosphorylate WASp. By contrast, WASp was tyrosine dephosphorylated by protein tyrosine phosphatase (PTP)-PEST, a tyrosine phosphatase shown here to interact with WASp via proline, serine, threonine phosphatase interacting protein (PSTPIP)1 binding. Although Fyn enhanced WASp-mediated Arp2/3 activation and was required for synapse formation, PTP-PEST combined with PSTPIP1 inhibited WASp-driven actin polymerization and synapse formation. These observations identify key roles for Fyn and PTP-PEST in regulating WASp and imply that inducible WASp tyrosine phosphorylation can occur independently of cdc42 binding, but unlike the cdc42 interaction, is absolutely required for WASp contributions to T cell activation.

Animals↗
PubMed2003

Identification of Fer tyrosine kinase localized on microtubules as a platelet endothelial cell adhesion molecule-1 phosphorylating kinase in vascular endothelial cells.

Platelet endothelial adhesion molecule-1 (PECAM-1) is a part of intercellular junctions and triggers intracellular signaling cascades upon homophilic binding. The intracellular domain of PECAM-1 is tyrosine phosphorylated upon homophilic engagement. However, it remains unclear which tyrosine kinase phosphorylates PECAM-1. We sought to isolate tyrosine kinases responsible for PECAM-1 phosphorylation and identified Fer as a candidate, based on expression cloning. Fer kinase specifically phosphorylated PECAM-1 at the immunoreceptor tyrosine-based inhibitory motif. Notably, Fer induced tyrosine phosphorylation of SHP-2, which is known to bind to the immunoreceptor tyrosine-based inhibitory motif of PECAM-1, and Fer also induced tyrosine phosphorylation of Gab1 (Grb2-associated binder-1). Engagement-dependent PECAM-1 phosphorylation was inhibited by the overexpression of a kinase-inactive mutant of Fer, suggesting that Fer is responsible for the tyrosine phosphorylation upon PECAM-1 engagement. Furthermore, by using green fluorescent protein-tagged Fer and a time-lapse fluorescent microscope, we found that Fer localized at microtubules in polarized and motile vascular endothelial cells. Fer was dynamically associated with growing microtubules in the direction of cell-cell contacts, where p120catenin, which is known to associate with Fer, colocalized with PECAM-1. These results suggest that Fer localized on microtubules may play an important role in phosphorylation of PECAM-1, possibly through its association with p120catenin at nascent cell-cell contacts.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Peroxynitrite modulates tyrosine-dependent signal transduction pathway of human erythrocyte band 3.

Peroxynitrite, the product of the reaction between nitric oxide and superoxide anion, is able to nitrate protein tyrosines. If this modification occurs on phosphotyrosine kinase substrates, it can down-regulate cell signaling. We investigated the effects of peroxynitrite on band 3-mediated signal transduction of human erythrocytes. Peroxynitrite treatment induced two different responses. At low concentrations (10-100 microM) it stimulated a metabolic response, leading to 1) a reversible inhibition of phosphotyrosine phosphatase activity, 2) a rise of tyrosine phosphorylation in the 22K cytoplasmic domain of band 3, 3) the release of glyceraldehyde 3-phosphate dehydrogenase from the membrane, and 4) the enhancement of lactate production. At high concentrations (200-1000 microM), peroxynitrite induced 1) cross-linking of membrane proteins, 2) inhibition of band 3 tyrosine phosphorylation, 3) nitration of tyrosines in the 22K cytoplasmic domain of band 3, 4) binding of hemoglobin to the membrane, 5) irreversible inhibition of phosphotyrosine kinase activity, 6) massive methemoglobin production, and 7) irreversible inhibition of lactate production. Our results demonstrate that at concentrations that could conceivably be achieved in vivo (10-100 microM), peroxynitrite behaves like other oxidants, i.e., it stimulates band 3 tyrosine phosphorylation and increases glucose metabolism. Thus, one plausible physiologic effect of peroxynitrite is the up-regulation of signaling through the reversible inhibition of phosphotyrosine phosphatase activity. At high concentrations of peroxynitrite, the tyrosine phosphorylation ceases in parallel with the nitration of band 3 tyrosines, but at these concentrations phosphotyrosine kinase activity and glycolysis are also irreversibly inhibited. Thus, at least in red blood cells, the postulated down-regulation of signaling by peroxynitrite cannot merely be ascribed to the nitration of tyrosine kinase targets.

Amino Acid Sequence↗
PubMed1990

Activation of p56lck through mutation of a regulatory carboxy-terminal tyrosine residue requires intact sites of autophosphorylation and myristylation.

Mutation of the major site of in vivo tyrosine phosphorylation of p56lck (tyrosine 505) to a phenylalanine constitutively enhances the p56lck-associated tyrosine-specific protein kinase activity. The mutant polypeptide is extensively phosphorylated in vivo at the site of in vitro Lck autophosphorylation (tyrosine 394) and is capable of oncogenic transformation of rodent fibroblasts. These observations have suggested that phosphorylation at Tyr-505 down regulates the tyrosine protein kinase activity of p56lck. Herein we have attempted to examine whether other posttranslational modifications may be involved in regulation of the enzymatic function of p56lck. The results indicated that activation of p56lck by mutation of Tyr-505 was prevented by a tyrosine-to-phenylalanine substitution at position 394. Furthermore, activation of p56lck by mutation of the carboxy-terminal tyrosine residue was rendered less efficient by substituting an alanine residue for the amino-terminal glycine. This second mutation prevented p56lck myristylation and stable membrane association and was associated with decreased in vivo phosphorylation at Tyr-394. Taken together, these findings imply that lack of phosphorylation at Tyr-505 may be insufficient for enhancement of the p56lck-associated tyrosine protein kinase activity. Our data suggest that activation of p56lck may be dependent on phosphorylation at Tyr-394 and that this process may be facilitated by myristylation, membrane association, or both.

Animals↗
PubMed1995

Identification of Tyr-397 as the primary site of tyrosine phosphorylation and pp60src association in the focal adhesion kinase, pp125FAK.

A number of cellular processes, such as proliferation, differentiation, and transformation, are regulated by cell-extracellular matrix interactions. Previous studies have identified a novel tyrosine kinase, the focal adhesion kinase p125FAK, as a component of cell adhesion plaques. p125FAK was identified as a 125-kDa tyrosine-phosphorylated protein in cells transformed by the v-src oncogene. p125FAK is an intracellular protein composed of three domains: a central domain with homology to protein tyrosine kinases, flanked by two noncatalytic domains of 400 amino acids which bear no significant homology to previously cloned proteins. p125FAK is believed to play an important regulatory role in cell adhesion because it localizes to cell adhesion plaques and because its phosphorylation on tyrosine residues is regulated by binding of cell surface integrins to the extracellular matrix. Recent studies have shown that Src, through its SH2 domain, stably associates with pp125FAK and that this association prevents dephosphorylation of pp125FAK in vitro by protein tyrosine phosphatases. In this report, we identify Tyr-397 as the primary in vivo and in vitro site of p125FAK tyrosine phosphorylation and association with Src. Substituting phenylalanine for tyrosine at position 397 significantly reduces p125FAK tyrosine phosphorylation and association with Src but does not abolish p125FAK kinase activity. In addition, p125FAK kinase is able to trans-phosphorylate Tyr-397 in vitro in a kinase-deficient p125FAK variant. Phosphorylation of Tyr-397 provides a site [Y(P)AEI] that fits the consensus sequence for the binding of Src.

Amino Acid Sequence↗