Major Tyrosine Phosphorylation Sites Research Articles

Nucleocytoplasmic Shuttling of Bruton's Tyrosine Kinase

Bruton's tyrosine kinase (Btk), a nonreceptor cytoplasmic tyrosine kinase belonging to the Tec family of kinases, has been shown to be critical for B cell proliferation, differentiation, and signaling. Loss-of-function mutations in the Btk gene lead to X-linked agammaglobulinemia (XLA), a primary immunodeficiency in humans, and the less severe condition xid in mice. Although Btk is mainly localized in the cytoplasm under steady state conditions, it translocates to the plasma membrane upon growth factor stimulation and cross-linking of the B cell receptor. Nevertheless, in ectopically as well as endogenously Btk-expressing cells, it can also translocate to the nucleus. Deletion of the pleckstrin homology (PH) domain (DeltaPH1) leads, however, to an even redistribution of Btk within the nucleus and cytoplasm in the majority of transfected cells. In contrast, an SH3-deleted (DeltaSH3) mutant of Btk has been found to be predominantly nuclear. We also demonstrate that the nuclear accumulation of DeltaPH1 is dependent on Src expression. This nucleocytoplasmic shuttling is sensitive to the exportin 1/CRM1-inactivating drug, leptomycin B, indicating that Btk utilizes functional nuclear export signals. In addition, while the DeltaPH1 mutant of Btk was found to be active and tyrosine-phosphorylated in vivo, DeltaSH3 displayed decreased autokinase activity and was not phosphorylated. Our findings indicate that the nucleocytoplasmic shuttling of Btk has implications regarding potential targets inside the nucleus, which may be critical in gene regulation during B cell development and differentiation.

Tyrosine Phosphorylation of Paxillin α Is Involved in Temporospatial Regulation of Paxillin-containing Focal Adhesion Formation and F-actin Organization in Motile Cells

Tyrosine Phosphorylation of Paxillin α Is Involved in Temporospatial Regulation of Paxillin-containing Focal Adhesion Formation and F-actin Organization in Motile Cells

Negative Regulation of the Serine/Threonine Kinase B-Raf by Akt

Negative Regulation of the Serine/Threonine Kinase B-Raf by Akt

Cell-cell adhesion-mediated tyrosine phosphorylation of nectin-2delta, an immunoglobulin-like cell adhesion molecule at adherens junctions.

We have recently found a novel functional unit of cell-cell adhesion at cadherin-based adherens junctions, consisting of at least nectin, an immunoglobulin-like cell adhesion molecule, and afadin, an actin filament-binding protein which connects nectin to the actin cytoskeleton. Among the members of the nectin family, we have found here that nectin-2delta is tyrosine-phosphorylated in response to cell-cell adhesion. Expression of E-cadherin induced tyrosine phosphorylation of nectin-2delta, while disruption of cell-cell adhesion by an anti-E-cadherin antibody reduced the tyrosine phosphorylation of nectin-2delta. An inhibitor specific for Src family kinase or expression of Csk reduced tyrosine phosphorylation of nectin-2delta. In addition, Src kinase tyrosine phosphorylates the recombinant cytoplasmic region of nectin-2delta in vitro. The major tyrosine phosphorylation site of nectin-2delta was Tyr505 in the cytoplasmic region, because the mutant nectin-2delta, of which Tyr505 was replaced by Phe, showed a loss of tyrosine phosphorylation in vivo and in vitro. These results, together with our recent observations, indicate that the cadherin-catenin system and the nectin-afadin system are closely connected to each other. The cadherin-mediated cell-cell adhesion system may link to the activation of a Src family kinase, that is, at least in part, responsible for the tyrosine phosphorylation of the cytoplasmic region of nectin-2delta. Oncogene (2000) 19, 4022 - 4028.

Identification of major tyrosine phosphorylation sites in the human insulin receptor substrate Gab-1 by insulin receptor kinase in vitro.

Gab-1 (Grb2-associated binder-1), which appears to play a central role in cellular growth response, transformation, and apoptosis, is a member of the insulin receptor substrate (IRS) family. IRS proteins act downstream in the signaling pathways of different receptor tyrosine kinases, including the insulin receptor (IR). In this paper, we characterize the phosphorylation of recombinant human Gab-1 (hGab-1) by IR in vitro. Kinetic phosphorylation data revealed that hGab-1 is a high affinity substrate for the IR (K(M): 12.0 microM for native IR vs 23.3 microM for recombinant IR). To elucidate the IR-specific phosphorylation pattern of hGab-1, we used phosphopeptide mapping by two-dimensional HPLC analysis. Phosphorylated tyrosine residues were subsequently identified by sequencing the separated phosphopeptides by matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) and Edman degradation. Our results demonstrate that hGab-1 was phosphorylated by IR at eight tyrosine residues (Y242, Y285, Y373, Y447, Y472, Y619, Y657, and Y689). Seventy-five percent of the identified radioactivity was incorporated into tyrosine residues Y447, Y472, and Y619 exhibiting features (NYVPM motif) of potential binding sites for the regulatory subunit (p85) of phosphatidylinositol (PI)-3 kinase. Accordingly, pull down assays with human HepG2 cell lysates showed that IR-specific phosphorylation of wild-type hGab-1 strongly enhanced PI-3 kinase binding. This is still the case when a single tyrosine residue in the NYVPM motif was mutated to phenylalanine. In contrast, phosphorylation-dependent binding of PI-3 kinase was completely abolished by changing a second tyrosine residue in a NYVPM motif independent from its location. Recently, we identified a similar cohort of tyrosine phosphorylation sites for the epidermal growth factor receptor (EGFR) with a predominant phosphorylation of tyrosine residue Y657 and binding of Syp [Lehr, S. et al. (1999) Biochemistry 38, 151-159]. These differences in the phosphorylation pattern of hGab-1 may contribute to signaling specificity by different tyrosine kinase receptors engaging distinct SH2 signaling molecules.

Tyrosine Phosphorylation of Caldesmon Is Required for Binding to the Shc·Grb2 Complex

S3-v-erbB is a retroviral oncogene that encodes a ligand-independent, transforming mutant of the epidermal growth factor receptor. This oncogene has been shown to be sarcomagenic in vivo and to transform fibroblasts in vitro. Our previous studies (McManus, M. J., Lingle, W. L., Salisbury, J. L., and Maihle, N. J. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 11351-11356) showed that expression of S3-v-erbB in primary fibroblasts results in the tyrosine phosphorylation of caldesmon (CaD), an actin- and calmodulin-binding protein. This phosphorylation is transformation-associated, and the phosphorylated form of CaD is associated with a signaling complex consisting of Shc, Grb2, and Sos in transformed fibroblasts. To identify the tyrosine phosphorylation site(s) in the CaD molecule and to further elucidate the functional role of CaD tyrosine phosphorylation in S3-v-ErbB oncogenic signaling, we have generated a series of mutant CaDs in which one or more tyrosine residues have been replaced with phenylalanine. Using a CaD null cell line, DF1 cells (an immortalized chicken embryo fibroblast cell line), and transient transfection assays, we demonstrated that Tyr-27 and Tyr-393 are the major sites of tyrosine phosphorylation on CaD. Interestingly, Tyr-27 is located within the myosin binding domain of CaD, and Tyr-393 is adjacent to one of the major actin binding and actomyosin ATPase inhibitory domains. Our studies also show that the tyrosine phosphorylation of CaD enhances its binding to the Shc.Grb2 complex. Specifically, replacement of Tyr-27, but not of Tyr-165 or Tyr-393, significantly reduces the ability of CaD to interact with the Shc. Grb2 complex. Together, these studies demonstrate that the major sites of tyrosine phosphorylation on CaD are located in the myosin and actin binding domains of CaD and that Tyr-27 is the major tyrosine phosphorylation site through which CaD interacts with the Shc.Grb2 complex.

Platelet-derived growth factor-induced activation of sphingosine kinase requires phosphorylation of the PDGF receptor tyrosine residue responsible for binding of PLCgamma.

Sphingosine-1-phosphate, a sphingolipid metabolite, is involved in the mitogenic response of platelet-derived growth factor (PDGF) and is formed by activation of sphingosine kinase. We examined the effect of PDGF on sphingosine kinase activation in TRMP cells expressing wild-type or various mutant betaPDGF receptors. Sphingosine kinase was stimulated by PDGF in cells expressing wild-type receptors but not in cells expressing kinase-inactive receptors (R634). Cells expressing mutated PDGF receptors with phenylalanine substitutions at five major tyrosine phosphorylation sites 740/751/771/1009/1021 (F5 mutants), which are unable to associate with PLCgamma, phosphatidylinositol 3-kinase, Ras GTPase-activating protein, or protein tyrosine phosphatase SHP-2, not only failed to increase DNA synthesis in response to PDGF but also did not activate sphingosine kinase. Moreover, mutation of tyrosine-1021 of the PDGF receptor to phenylalanine, which impairs its association with PLCgamma, abrogated PDGF-induced activation of sphingosine kinase. In contrast, PDGF was still able to stimulate sphingosine kinase in cells expressing the PDGF receptor mutated at tyrosines 740/751 and 1009, responsible for binding of phosphatidylinositol 3-kinase and SHP-2, respectively. In agreement, PDGF did not stimulate sphingosine kinase activity in F5 receptor 'add-back' mutants in which association with the Ras GTPase-activating protein, phosphatidylinositol 3-kinase, or SHP-2 was individually restored. However, a mutant PDGF receptor that was able to bind PLCgamma (tyrosine-1021), but not other signaling proteins, restored sphingosine kinase sensitivity to PDGF. These data indicate that the tyrosine residue responsible for binding of PLCgamma is required for PDGF-induced activation of sphingosine kinase. Moreover, calcium mobilization downstream of PLCgamma, but not protein kinase C activation, appears to be required for stimulation of sphingosine kinase by PDGF.-Olivera, A., Edsall, J., Poulton, S., Kazlauskas, A., Spiegel, S. Platelet-derived growth factor-induced activation of sphingosine kinase requires phosphorylation of the PDGF receptor tyrosine residue responsible for binding of PLCgamma.

Association of Nck with tyrosine-phosphorylated SLP-76 in activated T lymphocytes.

The Nck adaptor protein links tyrosine kinases or their substrates to proteins containing proline-rich motifs. Here we show that in activated T cells two tyrosine phosphoproteins of 75 and 120 kDa are co-immunoprecipitated with polyclonal antibodies against Nck. Analysis of Nck immunoprecipitates with various candidate antibodies revealed that the 75-kDa tyrosine phosphoprotein is the SH2 domain-containing leukocyte protein referred to as SLP-76. In vitro experiments show that the interaction between Nck and SLP-76 is mediated via the Nck SH2 domain. Using specific phosphopeptides corresponding to the major tyrosine phosphorylation sites of SLP-76, it was found that Y113 and Y128 phosphopeptides could compete binding of SLP-76 to the SH2 domain of Nck. In addition, the 120-kDa tyrosine phosphoprotein was recognized by an antibody raised against Cbl, a proto-oncogene product that has been previously found to be associated with Nck. These results suggest that the Nck adaptor protein interacts with key signaling molecules and may play an important role in activation of T lymphocytes.

Decrease in protein tyrosine phosphorylation is associated with F-actin reorganization by retinoic acid in human endometrial adenocarcinoma (RL95-2) cells.

Transformed cells often express elevated levels of tyrosine-phosphorylated proteins. Inhibition of protein tyrosine kinases causes reversion of malignant cells to the normal phenotype. In the present study, we evaluated the possibility that the reversion of human endometrial adenocarcinoma RL95-2 cells to a stationary phenotype induced by retinoic acid was associated with inhibition of tyrosine phosphorylation of cellular proteins. We found that retinoic acid decreased the levels of tyrosine-phosphorylated proteins, as assessed by immunostaining and immunoprecipitations using specific anti-phosphotyrosine antibodies. In addition, the inhibitors of tyrosine kinases herbimycin A and tyrphostin mimicked retinoic acid, inducing F-actin reorganization and increasing the size of RL95-2 cells, as determined by measurement of cell perimeters. Because focal adhesions that connect actin filaments with the plasma membrane are major sites of tyrosine phosphorylation, we further investigated whether selected focal adhesion proteins were affected by retinoic acid. We found that retinoic acid altered the localization of focal adhesion kinase. All-trans retinoic acid was effective in reducing the levels of focal adhesion kinase and paxillin protein. Thirteen-cis retinoic acid increased the levels of vinculin protein in the cytosolic fraction of cells. These changes are consistent with actin reorganization and reversion toward a stationary phenotype induced by retinoic acid in endometrial adenocarcinoma RL95-2 cells. Our results indicate that the differentiating effects of retinoids on endometrial cells are associated with decreases in tyrosine phosphorylation and changes in the levels and distribution of focal adhesion proteins. These findings suggest that signaling pathways that involve tyrosine kinases are potential targets for drug design against endometrial cancer.

Identification of tyrosine phosphorylation sites in human Gab-1 protein by EGF receptor kinase in vitro.

Grb2-associated binder-1 (Gab-1) has been identified recently in a cDNA library of glioblastoma tumors and appears to play a central role in cellular growth response, transformation, and apoptosis. Structural and functional features indicate that Gab-1 is a multisubstrate docking protein downstream in the signaling pathways of different receptor tyrosine kinases, including the epidermal growth factor receptor (EGFR). Therefore, the aim of the study was to characterize the phosphorylation of recombinant human Gab-1 (hGab-1) protein by EGFR in vitro. Using the pGEX system to express the entire protein and different domains of hGab-1 as glutathione S-transferase proteins, kinetic data for phosphorylation of these proteins by wheat germ agglutinine-purified EGFR and the recombinant EGFR (rEGFR) receptor kinase domain were determined. Our data revealed similar affinities of hGab-1-C for both receptor preparations (KM = 2.7 microM for rEGFR vs 3.2 microM for WGA EGFR) as well as for the different recombinant hGab-1 domains. To identify the specific EGFR phosphorylation sites, hGab-1-C was sequenced by Edman degradation and mass spectrometry. The entire protein was phosphorylated by rEGFR at eight tyrosine residues (Y285, Y373, Y406, Y447, Y472, Y619, Y657, and Y689). Fifty percent of the identified radioactivity was incorporated in tyrosine Y657 as the predominant peak in HPLC analysis, a site exhibiting features of a potential Syp (PTP1D) binding site. Accordingly, GST-pull down assays with A431 and HepG2 cell lysates showed that phosphorylated intact hGab-1 was able to bind Syp. This binding appears to be specific, because it was abolished by changing the Y657 of hGab-1 to F657. These results demonstrate that hGab-1 is a high-affinity substrate for the EGFR and the major tyrosine phosphorylation site Y657 in the C terminus is a specific binding site for the tyrosine phosphatase Syp.

Identification of Vascular Endothelial Growth Factor Receptor-1 Tyrosine Phosphorylation Sites and Binding of SH2 Domain-containing Molecules

Receptor tyrosine phosphorylation is crucial for signal transduction by creating high affinity binding sites for Src homology 2 domain-containing molecules. By expressing the intracellular domain of Flt-1/vascular endothelial growth factor receptor-1 in the baculosystem, we identified two major tyrosine phosphorylation sites at Tyr-1213 and Tyr-1242 and two minor tyrosine phosphorylation sites at Tyr-1327 and Tyr-1333 in this receptor. This pattern of phosphorylation of Flt-1 was also detected in vascular endothelial growth factor-stimulated cells expressing intact Flt-1. In vitro protein binding studies using synthetic peptides and immunoblotting showed that phospholipase C-gamma binds to both Y(p)1213 and Y(p)1333, whereas Grb2 and SH2-containing tyrosine protein phosphatase (SHP-2) bind to Y(p)1213, and Nck and Crk bind to Y(p)1333 in a phosphotyrosine-dependent manner. In addition, unidentified proteins with molecular masses around 74 and 27 kDa bound to Y(p)1213 and another of 75 kDa bound to Y(p)1333 in a phosphotyrosine-dependent manner. SHP-2, phospholipase C-gamma, and Grb2 could also be shown to bind to the intact Flt-1 intracellular domain. These results indicate that a spectrum of already known as well as novel phosphotyrosine-binding molecules are involved in signal transduction by Flt-1.

Characterization of the tyrosine phosphorylation and distribution of dystrobrevin isoforms

Dystrobrevin, a member of the dystrophin family of proteins, was initially identified as a major tyrosine phosphorylated synaptic protein in the electric organ of Torpedo californica. In this paper, we show that the major sites of tyrosine phosphorylation of Torpedo dystrobrevin are within its C-terminus, on Tyr-693 and Tyr-710. Cloning of the mammalian homologue of dystrobrevin has recently shown that this phosphotyrosine containing tail, or PYCT, is subject to alternative splicing. To compare the expression and distribution of PYCT − and PYCT + splice variants, we generated antibodies against different regions of dystrobrevin. Here we show that the PYCT − isoform of 62 kDa is expressed at high levels in all tissues examined. In contrast, PYCT + isoforms are expressed primarily in brain and muscle, where they are concentrated at synapses. Moreover, PYCT + isoforms associate more tightly with the membrane and with syntrophin, another synaptically enriched protein. These results suggest that PYCT + isoforms of dystrobrevin are specialized components of the dystroglycan complex which render the complex sensitive to regulation by tyrosine kinases.

Roles of the Complex Formation of SHPS-1 with SHP-2 in Insulin-stimulated Mitogen-activated Protein Kinase Activation

SHPS-1 is a receptor-like protein that undergoes tyrosine phosphorylation and binds SHP-2, an SH2 domain-containing protein tyrosine phosphatase, in response to insulin and other mitogens. The overexpression of wild-type SHPS-1, but not of a mutant SHPS-1 in which all four tyrosine residues in its cytoplasmic region were mutated to phenylalanine, markedly enhanced insulin-induced activation of mitogen-activated protein kinase in Chinese hamster ovary cells that overexpress the human insulin receptor. Mutation of each tyrosine residue individually revealed that the major sites of tyrosine phosphorylation of SHPS-1 in response to insulin are Tyr449 and Tyr473. In addition, mutation of either Tyr449 or Tyr473 abolished the insulin-induced tyrosine phosphorylation of SHPS-1 and its association with SHP-2. Surface plasmon resonance analysis showed that glutathione S-transferase fusion proteins containing the NH2-terminal or COOH-terminal SH2 domains of SHP-2 bound preferentially to phosphotyrosyl peptides corresponding to the sequences surrounding Tyr449 or Tyr473, respectively, of SHPS-1. Furthermore, phosphotyrosyl peptides containing Tyr449 or Tyr473 were effective substrates for the phosphatase activity of recombinant SHP-2 in vitro. Together, these results suggest that insulin may induce phosphorylation of SHPS-1 at Tyr449 and Tyr473, to which SHP-2 then binds through its NH2-terminal and COOH-terminal SH2 domains, respectively. SHPS-1 may play a crucial role both in the recruitment of SHP-2 from the cytosol to a site near the plasma membrane and in increasing its catalytic activity, thereby positively regulating the RAS-mitogen-activated protein kinase signaling cascade in response to insulin.

Fyn, Yes, and Syk Phosphorylation Sites in c-Cbl Map to the Same Tyrosine Residues That Become Phosphorylated in Activated T Cells

Protooncogenic protein c-Cbl undergoes tyrosine phosphorylation in response to stimulation through the receptors for antigens, immunoglobulins, cytokines, and growth factors as well as through the integrins. Tyrosine phosphorylation of c-Cbl may play a functional role in signal transduction, since c-Cbl interacts with many crucial signaling molecules including protein-tyrosine kinases, adaptor proteins, and phosphatidylinositol 3'-kinase. Therefore, it is essential for our understanding of the functions of c-Cbl in signal transduction to identify its tyrosine phosphorylation sites, to determine the protein-tyrosine kinases that phosphorylate these sites, and to elucidate the role of these sites in the interactions of c-Cbl with other signaling proteins. In this report, we demonstrate that tyrosines 700, 731, and 774 are the major tyrosine phosphorylation sites of c-Cbl in T cells in response to pervanadate treatment, as well as in response to TcR/CD3 ligation. Coexpression experiments in COS cells demonstrate that among T cell-expressed Src- and Syk-related protein-tyrosine kinases, Fyn, Yes, and Syk appear to play a major role in phosphorylation of c-Cbl, whereas Lck and Zap phosphorylate c-Cbl ineffectively. Fyn, Yes, and Syk phosphorylate the same sites of c-Cbl that become phosphorylated in stimulated T cells. Among these kinases, Fyn and Yes demonstrate strong binding to c-Cbl, which involves both phosphotyrosine-dependent and phosphotyrosine-independent mechanisms.

Adhesion of fibroblasts to fibronectin stimulates both serine and tyrosine phosphorylation of paxillin.

Tyrosine phosphorylation of paxillin by the focal adhesion kinase (FAK) has been implicated as a signal transduction mechanism associated with cell adhesion and cytoskeletal reorganization. The potential role of serine phosphorylation of paxillin in these events has not been well characterized. In this study we have examined the phosphorylation profile of paxillin both in vitro and in vivo. By using glutathione S-transferase-paxillin fusion proteins in precipitation-kinase assays in vitro we observed that a fusion protein spanning amino acid residues 54-313 of paxillin, and containing a FAK-binding site, precipitated substantial serine kinase activity as well as FAK activity from a smooth-muscle lysate. Together these kinases phosphorylated paxillin on tyrosine residue 118, a site that has been identified previously as a target for FAK phosphorylation, and on serine residues 188 and/or 190. The binding site for the serine kinase, the identity of which is currently unknown, was further mapped to residues 168-191 of paxillin. To assess the physiological relevance of these sites phosphorylated in vitro, the profile of paxillin phosphorylation in vivo stimulated by seeding fibroblasts on fibronectin was characterized. As expected, plating cells on fibronectin enhanced the tyrosine phosphorylation of paxillin. However, 96% of the phosphorylation of paxillin occurred on serine residues. Comparison by two-dimensional phosphopeptide analyses indicated that the major sites of tyrosine and serine phosphorylation detected in the assays in vitro co-migrate with phosphopeptides derived from paxillin phosphorylated in vivo in response to plating cells on fibronectin. These findings support a role for both tyrosine and serine kinases in the signal transduction pathway linking integrin activation to paxillin phosphorylation.

The Shc adaptor protein is highly phosphorylated at conserved, twin tyrosine residues (Y239/240) that mediate protein–protein interactions

Background Signal transduction initiated by a wide variety of extracellular signals involves the activation of protein-tyrosine kinases. Phosphorylated tyrosine residues in activated receptors or docking proteins then function as binding sites for the Src homology 2 (SH2) or phosphotyrosine-binding (PTB) domains of cytoplasmic signalling proteins. Shc is an adaptor protein that contains both PTB and SH2 domains and becomes phosphorylated on tyrosine in response to many different extracellular stimuli. These results have suggested that Shc is a prominent effector of protein-tyrosine kinase signalling. Thus far, only a single Shc phosphorylation site, the tyrosine at position 317 (Y317) has been identified. Phosphorylation of Y317 has been implicated in Grb2 binding and activation of the Ras pathway.Results Here, we report the identification of two major and novel Shc tyrosine phosphorylation sites, Y239 and Y240. These residues are present in the central proline-rich (CH1) region and are conserved in all isoforms of Shc. Y239/240 are co-ordinately phosphorylated by the Src protein-tyrosine kinase in vitro, and in response to epidermal growth factor stimulation or in v-src-transformed cells in vivo. Mutagenesis studies indicate that Y239/240 make an important contribution to the association of Shc with Grb2. Phosphopeptide-binding studies suggest that these two tyrosine residues may be involved in interactions with a number of cellular proteins.Conclusions Shc is the most prominent general substrate for protein-tyrosine kinases in vivo. The identification of two novel Shc phosphorylation sites indicates that Shc has the potential to interact with multiple downstream effectors. Shc Y239/240 are highly conserved in evolution, suggesting that the phosphorylation of these residues is of fundamental importance. We propose that distinct Shc phosphorylation isomers form different signalling complexes and thereby activate separate downstream signalling cascades.

A transmembrane-anchored chimeric focal adhesion kinase is constitutively activated and phosphorylated at tyrosine residues identical to pp125FAK.

Focal adhesion kinase, pp125FAK, is a nonmyristylated cytosolic tyrosine kinase unrelated to protein-tyrosine kinase families categorized to date. The kinase activity and tyrosine phosphorylation of pp125FAK are induced by beta 1 and beta 3 integrin-mediated cell adherence or aggregation. pp125FAK is also a tyrosine phosphorylation substrate in v-src-transformed cells and is localized to focal adhesion contracts of adherent fibroblasts and carcinoma cells. In this report, we have transiently expressed in COS cells a transmembrane-anchored chimeric receptor kinase, CD2FAK, consisting of CD2 and pp125FAK. We analyzed its kinase activity and tyrosine phosphorylation and compared to those of pp125FAK. We found that CD2FAK exhibited constitutive kinase activity and a high basal tyrosine phosphorylation level when COS transfectants were suspended in serum-free media. The kinase activity of CD2FAK was similarly up-regulated upon beta 1 integrin-mediated cell adherence as the endogenous pp125FAK. Both CD2FAK and pp125FAK appeared to be active as autophosphorylating kinases as shown by mutation of the ATP binding site. We determined the major tyrosine phosphorylation site, Tyr397, identical for both the constitutively activated CD2FAK and pp125FAK in response to beta 1 integrin-mediated cell adherence by site-directed mutagenesis. Deletions of the NH2- or the COOH-terminal noncatalytic domain of FAK, including Tyr397 did not lead to abolition of the kinase activity of pp125FAK or CD2FAK. Taken together, CD2FAK exhibits properties of an activated pp125FAK and the kinase activity does not appear to require tyrosine phosphorylation in vitro or in vivo.

Phosphorylation and identification of a major tyrosine phosphorylation site in protein tyrosine phosphatase 1C.

Protein tyrosine phosphatase 1C (PTP1C) was the first member of the protein tyrosine phosphatase family demonstrated to contain the src homology 2 (SH2) domain. This enzyme is believed to play a role in regulating downstream signaling in hematopoietic cells since it was predominantly expressed in these cells. However, recent studies have revealed that the protein is expressed in other tissues as well. This report describes both the phosphorylation of PTP1C in non-hematopoietic cells treated with growth factors (in vivo) and incubation of purified PTP1C with a variety of protein kinases (in vitro). PTP1C was transiently phosphorylated in A431 and 293 cells and also when the purified enzyme was incubated with receptor protein tyrosine kinases. In vitro, the tyrosine-phosphorylated PTP1C underwent rapid auto-dephosphorylation, an effect which could be blocked by the addition of sodium vanadate. On the other hand, cells containing a PTP1C in which the catalytic site had been inactivated through mutagenesis, stably phosphorylated the phosphatase. These results suggested that PTP1C was responsible for its own auto-dephosphorylation. The sites of tyrosine phosphorylation were characterized from purified enzyme following treatment with insulin receptor kinase and from PTP1C expressed in 293 cells which had been stimulated with platelet-derived growth factor. Through the techniques of peptide mapping and microsequencing, Tyr538 was determined to be the major phosphorylation site. This result was confirmed in vivo through site-specific mutagenesis of PTP1C expressed in 293 cells; changing Tyr538 to Phe538 completely abolished tyrosine phosphorylation of the molecule. In addition, Tyr538 lies within the sequence ESEYGNI which can be correlated with the consensus sequence pYXNX associated with GRB2 binding. These results suggest that PTP1C plays a prominent role in growth factor receptor-mediated signal transduction within both hematopoietic cells and tissues of non-lymphoid origin.

Autophosphorylation of the focal adhesion kinase, pp125FAK, directs SH2-dependent binding of pp60src.

The phosphorylation of protein tyrosine kinases (PTKs) on tyrosine residues is a critical regulatory event that modulates catalytic activity and triggers the physical association of PTKs with Src homology 2 (SH2)-containing proteins. The integrin-linked focal adhesion kinase, pp125FAK, exhibits extracellular matrix-dependent phosphorylation on tyrosine and physically associates with two nonreceptor PTKs, pp60src and pp59fyn, via their SH2 domains. Herein, we identify Tyr-397 as the major site of tyrosine phosphorylation on pp125FAK both in vivo and in vitro. Tyrosine 397 is located at the juncture of the N-terminal and catalytic domains, a novel site for PTK autophosphorylation. Mutation of Tyr-397 to a nonphosphorylatable residue dramatically impairs the phosphorylation of pp125FAK on tyrosine in vivo and in vitro. The mutation of Tyr-397 to Phe also inhibits the formation of stable complexes with pp60src in cells expressing Src and FAK397F, suggesting that autophosphorylation of pp125FAK may regulate the association of pp125FAK with Src family kinases in vivo. The identification of Tyr-397 as a major site for FAK autophosphorylation provides one of the first examples of a cellular protein containing a high-affinity binding site for a Src family kinase SH2 domain. This finding has implications for models describing the mechanisms of action of pp125FAK, the regulation of the Src family of PTKs, and signal transduction through the integrins.

Autophosphorylation of the focal adhesion kinase, pp125FAK, directs SH2-dependent binding of pp60src.

The phosphorylation of protein tyrosine kinases (PTKs) on tyrosine residues is a critical regulatory event that modulates catalytic activity and triggers the physical association of PTKs with Src homology 2 (SH2)-containing proteins. The integrin-linked focal adhesion kinase, pp125FAK, exhibits extracellular matrix-dependent phosphorylation on tyrosine and physically associates with two nonreceptor PTKs, pp60src and pp59fyn, via their SH2 domains. Herein, we identify Tyr-397 as the major site of tyrosine phosphorylation on pp125FAK both in vivo and in vitro. Tyrosine 397 is located at the juncture of the N-terminal and catalytic domains, a novel site for PTK autophosphorylation. Mutation of Tyr-397 to a nonphosphorylatable residue dramatically impairs the phosphorylation of pp125FAK on tyrosine in vivo and in vitro. The mutation of Tyr-397 to Phe also inhibits the formation of stable complexes with pp60src in cells expressing Src and FAK397F, suggesting that autophosphorylation of pp125FAK may regulate the association of pp125FAK with Src family kinases in vivo. The identification of Tyr-397 as a major site for FAK autophosphorylation provides one of the first examples of a cellular protein containing a high-affinity binding site for a Src family kinase SH2 domain. This finding has implications for models describing the mechanisms of action of pp125FAK, the regulation of the Src family of PTKs, and signal transduction through the integrins.