Tyrosine phosphorylation of Crk-associated substrates by focal adhesion kinase. A putative mechanism for the integrin-mediated tyrosine phosphorylation of Crk-associated substrates

Integrin-ligand binding induces the tyrosine phosphorylation of various proteins including focal adhesion kinase (pp125(FAK)) and Crk-associated substrate (Cas). FAK is activated and autophosphorylated by the ligation of integrins, although the substrate of FAK has not been revealed. We show here that p130(Cas) and Cas-L are FAK substrates. FAK directly phosphorylates Cas proteins primarily at the YDYVHL sequence that is conserved among all Cas proteins. Furthermore, the phosphorylated YDYVHL sequence is a binding site for Src family protein-tyrosine kinases, and the recruited Src family kinase phosphorylates the other tyrosine residues within Cas. The Cas-L YDYVHL sequence is phosphorylated upon integrin-ligand binding, and this integrin-mediated tyrosine phosphorylation is inhibited by the cotransfection of the FAK COOH-terminal domain that does not contain a kinase domain. These findings strongly suggest that FAK initiates integrin-mediated tyrosine phosphorylation of Cas proteins; then, Src family tyrosine kinases, which are recruited to phosphorylated Cas and FAK, further phosphorylate Cas proteins.     

Role of c-Src tyrosine kinase in G protein-coupled receptor- and Gbetagamma subunit-mediated activation of mitogen-activated protein kinases

Several G protein-coupled receptors that interact with pertussis toxin-sensitive heterotrimeric G proteins mediate Ras-dependent activation of mitogen-activated protein (MAP) kinases. The mechanism involves Gbetagamma subunit-mediated increases in tyrosine phosphorylation of the Shc adapter protein, Shc*Grb2 complex formation, and recruitment of Ras guanine nucleotide exchange factor activity. We have investigated the role of the ubiquitous nonreceptor tyrosine kinase c-Src in activation of the MAP kinase pathway via endogenous G protein-coupled lysophosphatidic acid (LPA) receptors or by transient expression of Gbetagamma subunits in COS-7 cells. In vitro kinase assays of Shc immunoprecipitates following LPA stimulation demonstrated rapid, transient recruitment of tyrosine kinase activity into Shc immune complexes. Recruitment of tyrosine kinase activity was pertussis toxin-sensitive and mimicked by cellular expression of Gbetagamma subunits. Immunoblots for coprecipitated proteins in Shc immunoprecipitates revealed a transient association of Shc and c-Src following LPA stimulation, which coincided with increases in Shc-associated tyrosine kinase activity and Shc tyrosine phosphorylation. LPA stimulation or expression of Gbetagamma subunits resulted in c-Src activation, as assessed by increased c-Src autophosphorylation. Overexpression of wild-type or constitutively active mutant c-Src, but not kinase inactive mutant c-Src, lead to increased tyrosine kinase activity in Shc immunoprecipitates, increased Shc tyrosine phosphorylation, and Shc.Grb2 complex formation. MAP kinase activation resulting from LPA receptor stimulation, expression of Gbetagamma subunits, or expression of c-Src was sensitive to dominant negatives of mSos, Ras, and Raf. Coexpression of Csk, which inactivates Src family kinases by phosphorylating the regulatory C-terminal tyrosine residue, inhibited LPA stimulation of Shc tyrosine phosphorylation, Shc.Grb2 complex formation, and MAP kinase activation. These data suggest that Gbetagamma subunit-mediated formation of Shc.c-Src complexes and c-Src kinase activation are early events in Ras-dependent activation of MAP kinase via pertussis toxin-sensitive G protein-coupled receptors.     

Structural requirements for the efficient regulation of the Src protein tyrosine kinase by Csk

Protein tyrosine kinases of the Src family are negatively regulated by phosphorylation in the C-terminal tail of the molecule. A different protein tyrosine kinase, Csk, is largely responsible for this regulation. The phosphorylated tail of c-Src engages with the SH2 domain in a conformation that is associated with low kinase activity and which involves stabilization by the SH3 domain. Inducible expression of c-Src in fission yeast is lethal unless Csk is coexpressed. Using this assay we present evidence that Src regulation by C-terminal phosphorylation does not require the myristylation signal or the unique domain at the N-terminus of the Src protein. Mutagenesis of the SH3 and SH2 domains of Csk show that neither are necessary in yeast or in vitro for efficient regulation of Src. Mutation of Tyr416 of Src, a site of autophosphorylation common to most protein tyrosine kinases, abolished the ability of Src to arrest growth of phosphorylate endogenous proteins. Tyr416 had the same effect on a shorter form of Src consisting of the kinase domain only, indicating that the mutation affects a property intrinsic to the catalytic domain. The residual activity of full-length Src mutated at Tyr416 is efficiently repressed by Csk action, suggesting that regulation by C-terminal phosphorylation does not act by preventing phosphorylation at Tyr416.     

Regulation of connexin43 function by activated tyrosine protein kinases

Gap junctions are specialized membrane structures that are involved in the normal functioning of numerous mammalian tissues and implicated in several human disease processes. This mini-review focuses on the regulation of gap junctions through phosphorylation of connexin43 induced by the v-Src or epidermal growth factor receptor tyrosine kinases. These tyrosine kinases markedly disrupt gap junctional communication in mammalian cells. here, we describe work correlating the alteration of connexin43 function with the ability of the v-Src tyrosine kinase to phosphorylate connexin43 directly on two distinct tyrosine sites in mammalian cells (Y247 and Y265). We also present evidence that proline-rich regions and phosphotyrosine sites of connexin43 may mediate interactions with the SH3 and SH2 domains of v-Src. In contrast to v-Src, the activated epidermal growth factor receptor acts indirectly through activated MAP kinase which may stimulate phosphorylation of connexin43 exclusively on serine. This phosphorylation event is complex because MAP kinase phosphorylates three serine sites in connexin43 (S255, S279, and S282). These findings suggest novel interactions between connexin43, the v-Src tyrosine kinase, and activated MAP kinase that set the stage for future investigations into the regulation of gap junctions by protein phosphorylation.     

Effects of an antiallergic drug, pemirolast potassium on tyrosine phosphorylation and map kinase activation in antigen-stimulated rat basophilic leukemia (RBL-2H3) cells

Aggregation of high affinity IgE Fc receptors (Fc epsilon RI) on RBL-2H3 cells results in tyrosine phosphorylation of 33-, 42-, 44-, 72-, 80-, 90-, 125-kDa proteins. The 42 and 44 kDa proteins were identified as mitogen-activated protein (MAP) kinases with immunoblotting of anti-MAP kinase antibody. The effects of an antiallergic drug, pemirolast potassium (TBX) on Ag-induced protein tyrosine phosphorylation and MAP kinase activation were investigated. When RBL-2H3 cells were stimulated with Ag in the presence of TBX, tyrosine phosphorylation of three proteins (33, 42 and 44 kDa) was inhibited concentration-dependently (0.1-10 micrograms/ml). Inhibition of Ag-induced tyrosine phosphorylation of 33 kDa protein, which could be a beta subunit of Fc epsilon RI, suggests that TBX may prevent the activation of Fc epsilon RI. TBX suppressed activation of MAP kinases (42 and 44 kDa) in response to Ag as well as phorbol myristate acetate (100 nM) or calcium ionophore A23187 (500 nM), implying that the drug acts on signal transduction component(s) between the second messengers and MAP kinases. However, TBX had no effects on protein tyrosine phosphorylation and MAP kinase activation in MC3T3-E1 osteoblastic cells. These results indicate that TBX may affect Fc epsilon RI and also may act as a step distal of Ca2+ mobilization and protein kinase C activation leading to MAP kinase activation in RBL-2H3 cells.     

Phosphorylation of CrkII adaptor protein at tyrosine 221 by epidermal growth factor receptor

CrkII adaptor protein becomes tyrosine-phosphorylated upon various types of stimulation. We examined whether tyrosine 221, which has been shown to be phosphorylated by c-Abl, was phosphorylated also by other tyrosine kinases, such as epidermal growth factor (EGF) receptor. For this purpose, we developed an antibody that specifically recognizes Tyr221-phosphorylated CrkII, and we demonstrated that CrkII was phosphorylated on Tyr221 upon EGF stimulation. When NRK cells were stimulated with EGF, the tyrosine-phosphorylated CrkII was detected at the periphery of the cells, where ruffling is prominent, suggesting that signaling to CrkII may be involved in EGF-dependent cytoskeletal reorganization. The EGF-dependent phosphorylation of CrkII was also detected in a c-Abl-deficient cell line. Moreover, recombinant CrkII protein was phosphorylated in vitro by EGF receptor. These results strongly suggest that EGF receptor directly phosphorylates CrkII. Mutational analysis revealed that the src homology 2 domain was essential for the phosphorylation of CrkII by EGF receptor but not by c-Abl, arguing that these kinases phosphorylate CrkII by different phosphorylation mechanisms. Finally, we found that the CrkII protein phosphorylated upon EGF stimulation did not bind to the phosphotyrosine-containing peptide and that CrkII initiated dissociation from EGF receptor within 3 min even with the sustained tyrosine phosphorylation of EGF receptor. This result implicated phosphorylation of Tyr221 in the negative regulation of the src homology 2-mediated binding of CrkII to EGF receptor.     

Protein kinases of cultured chicken osteoblasts that phosphorylate extracellular bone proteins

Cytosolic and microsomal protein kinase preparations from cultured chicken osteoblasts were found to phosphorylate up to six major proteins with Mrs 66, 58, 50, 36, 32, and 22 kDa in chicken bone extract. Use of heparin led to the conclusion that these proteins were predominantly phosphorylated by factor-independent protein kinase (FIPK) present both in microsomal and cytosolic preparations. It was confirmed that microsomal preparation contained predominantly FIPK, whereas cytosolic preparation contained additional kinases, that can phosphorylate the bone proteins. Use of purified chicken bone osteopontin (OPN) (58 kDa) and recombinant OPN led to the same conclusions. The identify of the protein kinases was clearly established by using a series of synthetic peptide substrates. Quantitative analysis utilizing pure protein kinases and purified chicken bone OPN, recombinant mouse OPN, and bovine bone OPN and BSP led to introduction of approximately 9 moles of phosphate/mole of OPN and 6.6 moles phosphate/mole bovine bone sialoprotein (BSP) by casein kinase II. cGMP-dependent protein kinase and protein kinase C both introduced 0.5-1.2 moles phosphate/mole of OPN and BSP, whereas cAMP-dependent protein kinase led to no significant phosphorylation of OPN or BSP. Consistent with the above results, sites of phosphorylation identified for OPN (metabolically labeled) and BSP (labeled by casein kinase II) revealed that predominant phosphorylated sites have recognition sequences for FIPK.     

Roles of Lck, Syk and ZAP-70 tyrosine kinases in TCR-mediated phosphorylation of the adapter protein Shc

The adapter protein Shc has been implicated in mitogenic signaling via growth factor receptors, antigen receptors and cytokine receptors. Recent studies have suggested that tyrosine phosphorylation of Shc may play a key role in T lymphocyte proliferation via interaction of phosphorylated Shc with downstream molecules involved in activation of Ras and Myc proteins. However, the sites on Shc that are tyrosine phosphorylated in response to TCR engagement and the ability of different T cell tyrosine kinases to phosphorylate Shc have not been defined. In this report, we show that during TCR signaling, the tyrosines Y239, Y240 and Y317 of Shc are the primary sites of tyrosine phosphorylation. Mutation of all three tyrosines completely abolished tyrosine phosphorylation of Shc following TCR stimulation. Our data also suggest that multiple T cell tyrosine kinases contribute to tyrosine phosphorylation on Shc. In T cells, CD4/Lck-dependent tyrosine phosphorylation on Shc was markedly diminished when Y317 was mutated, suggesting a preference of Lck for the Y317 site. The syk-family kinases (Syk and ZAP-70) were able to phosphorylate the Y239 and Y240 sites, and less efficiently the Y317 site. Moreover, co-expression of Syk or ZAP-70 with Lck resulted in enhanced phosphorylation of Shc on all three sites, suggesting a synergy between the syk-family and scr-family kinases. Of the two potential Grb2 binding sites (Y239 and Y317), Y239 appears to play a greater role in recruiting Sos through Grb2. These studies have implications for Ras activation and mitogenic signaling during T cell activation.     

Depolarization-induced tyrosine phosphorylation in PC12h cells

Depolarization induced by KCl was found to induce tyrosine phosphorylation of cellular proteins in PC12h cells. By Western blotting with anti-phosphotyrosine antibody, we detected tyrosine phosphorylation of proteins with molecular weights of 120, 110, 105, 95, 75, 70, 66, 44, and 42 kDa in response to KCl. The immunoprecipitates from KCl-treated cells with the antibody contained large amounts of tyrosine-phosphorylated proteins and increased activity of tyrosine kinase. Incubation of the immunoprecipitates with [gamma-32P]ATP resulted in tyrosine phosphorylation of two proteins with the molecular weights of 120 and 140 kDa. These effects were completely abolished by the addition of EGTA before KCl treatment, suggesting that the depolarization-induced tyrosine phosphorylation may require calcium entry into the cells from the medium. Increased activity of tyrosine kinase phosphorylating the 120 and 140 kDa proteins was also recovered from cells stimulated with nerve growth factor, basic fibroblast growth factor, epidermal growth factor, and vasoactive intestinal peptide. Among them, depolarization by KCl elicited the strongest effect. These results indicate that a protein tyrosine kinase that phosphorylate the 120 and 140 kDa proteins is phosphorylated or activated in response to calcium ion, cAMP, and growth factors acting through tyrosine kinase receptors.     

Regulation of T-cell antigen receptor signalling by Syk tyrosine protein kinase

T-cell antigen receptor (TCR) signalling has been shown to involve two classes of tyrosine protein kinases: the Src-related kinases p56(lck) and p59(fyr), and the Zap-70/Syk family kinases. Lck and FynT are postulated to initiate TCR-triggered signal transduction by phosphorylating the CD3 and zeta subunits of the TCR complex. This modification permits the recruitment of Zap-70 and Syk, which are presumed to amplify the TCR-triggered signal, by phosphorylating additional intracellular proteins. While Zap-70 is expressed in all T cells, Syk is present in thymocytes and mature T-cell populations such as intraepithelial gammadelta T cells and naive alphabeta T cells. To better understand the role of Syk in these cells, its impact on the physiology of an antigen-specific T-cell line was tested. Our results showed that compared to Zap-70 alone, Syk was a strong positive regulator of antigen receptor-induced signals in BI-141 cells. Surprisingly, they indicated that, like Src family kinases, Syk augmented TCR-triggered tyrosine phosphorylation of CD3/zeta. Syk, but not Zap-70 alone, could also stimulate tyrosine phosphorylation of a zeta-bearing chimera in transiently transfected Cos-1 cells. Finally, evidence was provided that Syk has the capacity to directly phosphorylate a zeta-derived peptide in vitro. These findings suggested that Syk may have a unique role in T cells, as a consequence of its ability to efficiently phosphorylate multiple components of the TCR signalling cascade. Furthermore, they raised the possibility that Syk can regulate the initiation of TCR signalling, by promoting phosphorylation of the immunoreceptor tyrosine-based activation motifs of the TCR complex.     

Cell to substratum adhesion is involved in v-Src-induced cellular protein tyrosine phosphorylation: implication for the adhesion-regulated protein tyrosine phosphatase activity

Protein tyrosine phosphorylation accompanies the integrin-mediated cell to substratum adhesion, and is essential for the progression of G1/S phase of the cell-cycle in normal fibroblasts. To examine how cellular protein tyrosine phosphatase (PTPase) activity is involved in regulating the adhesion-dependent protein tyrosine phosphorylation, we employed fibroblast cells bearing an active form of a protein tyrosine kinase (PTK), v-Src. We found that the v-Src induced tyrosine phosphorylation in certain proteins such as tensin, talin, p120, p80/85 (cortactin) and paxillin was greatly reduced when the cell to substratum adhesion was lost. Readhesion of the cells onto fibronectin restored these phosphorylation events, while this was inhibited by the addition of RGD peptide. The kinase activity of the v-Src was unchanged by the loss of cell to substratum adhesion. On the other hand, treatment with a protein tyrosine phosphatase inhibitor vanadate caused much the same increase in the v-Src-mediated cellular tyrosine phosphorylation between cells adhered to the culture environments and cells kept in suspension. These data suggest that PTPase(s) appears to be more critical than the v-Src PTK in determining the cell adhesion-dependent protein tyrosine phosphorylation. Moreover, most of the protein tyrosine phosphorylations that are mediated by the v-Src but still dependent on the cell adhesion were indeed greatly reduced during an anchorage-independent growth of v-Src cells. Thus our data collectively indicate that the v-Src induced high level of tyrosine phosphorylation in certain types of proteins are still under the control of the integrin(s) or the cell adhesion to culture substratum, and most of these adhesion-regulated high levels of tyrosine phosphorylations are not essential for the transformed phenotype.     

Implication of Ca(2+)-dependent protein tyrosine phosphorylation in carbachol-induced phospholipase D activation in rat pheochromocytoma PC12 cells

The mechanism for carbachol (CCh)-induced phospholipase D (PLD) activation was investigated in [3H]palmitic acid-labeled pheochromocytoma PC12 cells with respect to the involvement of protein tyrosine phosphorylation and Ca2+. PLD activity was assessed by measuring the formation of [3H]phosphatidylbutanol in the presence of 0.3% butanol. Pretreatment of cells with the tyrosine kinase inhibitors herbimycin A, genistein, and tyrphostin inhibited PLD activation by CCh. Western blot analysis revealed several apparent tyrosine-phosphorylated protein bands (111, 91, 84, 74, 65-70, 44, and 42 kDa) in PC12 cells treated with CCh. Phosphorylation of the 111-, 91-, 84-, and 65-70-kDa proteins peaked within 1 min, and their time-dependent changes seemingly correlated with that of PLD activation. Others (74, 44MAPK, and 42MAPK kDa) were phosphorylated rather slowly, and maximal tyrosine phosphorylation was observed at 2 min. Herbimycin A inhibited PLD activity and tyrosine phosphorylation of four proteins (111, 91, 84, and 65-70 kDa) in a preincubation time- and concentration-dependent fashion. In Ca(2+)-free buffer, CCh-induced [3H]phosphatidylbutanol formation and protein tyrosine phosphorylation were abolished. A Ca2+ ionophore, A23187, caused PLD activation and tyrosine phosphorylation of four proteins of 111, 91, 84, and 65-70 kDa only in the presence of extracellular Ca2+. Extracellular Ca2+ dependency for CCh-induced PLD activation was well correlated with that for tyrosine phosphorylation of the four proteins listed above, especially the 111-kDa protein. These results suggest that Ca(2+)-dependent protein tyrosine phosphorylation is closely implicated in CCh-induced PLD activation in PC12 cells.     

Protein kinase C modulation of insulin receptor substrate-1 tyrosine phosphorylation requires serine 612

Activation of the endogenous protein kinase Cs in human kidney fibroblast (293) cells was found in the present study to inhibit the subsequent ability of insulin to stimulate the tyrosine phosphorylation of an expressed insulin receptor substrate-1. This inhibition was also observed in an in vitro phosphorylation reaction if the insulin receptor and its substrate were both isolated from cells in which the protein kinase C had been activated. To test whether serine phosphorylation of the insulin receptor substrate-1 was contributing to this process, serine 612 of this molecule was changed to an alanine. The insulin-stimulated tyrosine phosphorylation and the associated phosphatidylinositol 3-kinase activity of the expressed mutant were found to be comparable to those of the expressed wild-type substrate. However, unlike the wild-type protein, activation of protein kinase C did not inhibit the insulin-stimulated tyrosine phosphorylation of the S612A mutant nor its subsequent association with phosphatidylinositol 3-kinase. Tryptic peptide mapping of in vivo labeled IRS-1 and the S612A mutant revealed that PMA stimulates the phosphorylation of a peptide from wild-type IRS-1 that is absent from the tryptic peptide maps of the S612A mutant. Moreover, a synthetic peptide containing this phosphoserine and its nearby tyrosine was found to be phosphorylated by the insulin receptor to a much lower extent than the same peptide without the phosphoserine. Activation of protein kinase C was found to stimulate by 10-fold the ability of a cytosolic kinase to phosphorylate this synthetic peptide as well as the intact insulin receptor substrate-1. Finally, cytosolic extracts from the livers of ob/ob mice showed an 8-fold increase in a kinase activity capable of phosphorylating this synthetic peptide, compared to extracts of livers from lean litter mates. These results indicate that activation of protein kinase C stimulates a kinase which can phosphorylate insulin receptor substrate-1 at serine 612, resulting in an inhibition of insulin signaling in the cell, posing a potential mechanism for insulin resistance in some models of obesity.     

Influence of tyrosine phosphorylation on protein interaction with FcgammaRIIa

The cytoplasmic tail of Fc(gamma)RIIa present on human neutrophils shares with other antigen receptors a common amino acid sequence called ITAM (Immunoreceptor Tyrosine-based Activation Motif). After receptor ligation, the tyrosine residues within this motif become phosphorylated. We prepared a recombinant fusion protein of the cytoplasmic tail of Fc(gamma)RIIa (containing the ITAM) with glutathione-S-Transferase (GST-CT) to characterize the phosphorylation of Fc(gamma)RIIa and its ability to interact with other proteins involved in signal transduction. The GST-CT became phosphorylated in the presence of Lyn, Hck and Syk (immunoprecipitated from human neutrophils), but not in the presence of Fgr. Of the active kinases, only Lyn (mainly present in the membrane fraction) was found to associate with the GST-CT in the absence of ATP. This association was also observed in immunoprecipitates of Fc(gamma)RIIa from resting neutrophils, suggesting that Lyn might be the kinase responsible for the initial Fc(gamma)RIIa phosphorylation. Moreover, we observed specific association of Syk and the p85 subunit of PI 3-kinase after incubation of the GST-CT with neutrophil cytosol. This interaction was dependent on tyrosine phosphorylation of the GST-CT. Substitution of 269Tyr by Phe almost completely abolished tyrosine phosphorylation of the fusion protein. Substitution of either 253Tyr or 269Tyr eliminated Syk binding, but only 253Tyr appeared to be essential for p85 binding. We hypothesize that, upon activation, the membrane-associated Lyn is responsible for the initial tyrosine phosphorylation of Fc(gamma)RIIa, thus creating a docking site for Syk and PI 3-kinase.     

Effects of protein kinase inhibitors on in vitro protein phosphorylation and cellular differentiation of Streptomyces griseus

In vitro phosphorylation reactions using extracts of Streptomyces griseus cells and gamma-[32P]ATP revealed the presence of multiple phosphorylated proteins. Most of the phosphorylations were distinctly inhibited by staurosporine and K-252a which are known to be eukaryotic protein kinase inhibitors. The in vitro experiments also showed that phosphorylation was greatly enhanced by manganese and inhibition of phosphorylation by staurosporine and K-252a was partially circumvented by 10 mM manganese. A calcium-activated protein kinase(s) was little affected by these inhibitors. Herbimycin and radicicol, known to be tyrosine kinase inhibitors, completely inhibited the phosphorylation of one protein. Consistent with their in vitro effects the protein kinase inhibitors inhibited aerial mycelium formation and pigment production by S. griseus. All these data suggest that S. griseus possesses several protein kinases of eukaryotic type which are essential for morphogenesis and secondary metabolism. In vitro phosphorylation of some proteins in a staurosporine-producing Streptomyces sp. was also inhibited by staurosporine, K-252a and herbimycin, which suggests the presence of a mechanism for self-protection in this microorganism.     

A dual participation of ZAP-70 and scr protein tyrosine kinases is required for TCR-induced tyrosine phosphorylation of Sam68 in Jurkat T cells

Sam68 has been initially described as a substrate of src kinases during mitosis in fibroblasts. Recent evidence suggests that in T lymphocytes Sam68 may act as an adaptor protein and participate in the early biochemical cascade triggered after CD3 stimulation. A direct interaction between Sam68 and the two src kinases involved in T cell activation, p59(fyn) and p56(lck), as well as a partnership of Sam68 with various key downstream signaling molecules, like phospholipase Cgamma-1 and Grb2, has been shown. In this study we analyze the contribution of p56(lck), as well as the role of ZAP-70, the second class of protein tyrosine kinase involved in T cell activation, in Sam68 tyrosine phosphorylation in the human Jurkat T cell line. Using the src inhibitor PP1 [4-amino-5-(4-methylphenyl)7-(t-butyl) pyrazolo [3,4-d] pyrymidine] and cell variants with defective expression of p56(lck) or expressing a dominant negative form of ZAP-70, we demonstrate that, while both p56(lck) and ZAP-70 are dispensable for the low constitutive phosphorylation of Sam68 observed in Jurkat cells, a cooperation between the two kinases is required to increase its rapid phosphorylation observed in vivo after CD3 stimulation. We also show that recombinant forms of both p56(lck) and ZAP-70 phosphorylate Sam68 in vitro. However, using CD2 stimulated cells, we observe that p56(lck) activation by itself does not induce Sam68 tyrosine phosphorylation. We conclude that p59(fyn) and p56(lck) differently participate in regulating the phosphorylation state of Sam68 in T cells and that ZAP-70 may contribute to Sam68 tyrosine phosphorylation and to the specific recruitment of this molecule after CD3 stimulation.     

The roles of protein phosphorylation/dephosphorylation in tumor necrosis factor antitumor effects

The roles of protein phosphorylation and dephosphorylation in the tumor necrosis factor (TNF) cytotoxic and antiproliferative effects on L-929-transformed fibroblasts were explored. Genistein and erbstatin, specific inhibitors of tyrosine kinase, had antiproliferative but not cytotoxic effects on the cells by themselves and synergistically enhanced the cytotoxic and antiproliferative effects of TNF-alpha. Immunoblot analysis with a monoclonal antiphosphotyrosine antibody revealed that TNF, administered for 5-180 min, induced tyrosine dephosphorylation of two pairs of membranal proteins, 34-36 kDa and 50-52 kDa, and potentiated tyrosine phosphorylation of a 115-kDa protein in both the cytosolic and membranal fractions of the cells. A very brief exposure (30 sec) to TNF induced rapid phosphorylation of several proteins, whereas genistein, but not inhibitors of other protein kinases, enhanced this effect of TNF. The results suggest that TNF activity could be potentiated by the inhibition of tyrosine phosphorylation and point to specific proteins that are dephosphorylated on tyrosine in response to TNF.     

Constitutive activation of the Janus kinase-STAT pathway in T lymphoma overexpressing the Lck protein tyrosine kinase

The Lck protein, a Src family tyrosine kinase, plays a critical role in T cell maturation and activation. Dysregulation of Lck expression or Lck kinase activity has been implicated in T cell leukemias from mice to humans, although the mechanism underlying Lck-mediated oncogenesis is still largely unclear. We report here that both DNA binding activities and tyrosine phosphorylation of STAT3 and STAT5, but not STAT1, are constitutively enhanced in the mouse T cell lymphoma LSTRA, which is a well-characterized cell line that overexpresses Lck protein and exhibits high levels of Lck kinase activity. Furthermore, Janus kinase 1 (jak1) and Jak2 protein tyrosine kinases are constantly activated in these cells, as determined by their autophosphorylation in an in vitro kinase assay and increased levels of tyrosine phosphorylation on immunoblots. Therefore, like Src-transformed cells, Lck-overexpressing LSTRA cells also exhibit constitutive activation of distinct Jak and STAT proteins.     

The ezrin-like family of tyrosine kinase substrates: receptor-specific pattern of tyrosine phosphorylation and relationship to malignant transformation

A method for the isolation of tyrosine kinases substrates was developed. The method takes advantage of immuno-affinity purification of an entire set of proteins phosphorylated by tyrosine kinases, followed by generation of antisera against the purified protein pool and immunological screening of bacterial expression libraries with these antisera. By applying this methodology to the study of the phosphorylation events triggered by activation of the epidermal growth factor receptors, we have isolated several cDNAs encoding novel putative tyrosine kinase substrates. One of these cDNAs encodes radixin, a protein belonging to the band 4.1 family of proteins and highly related to ezrin and moesin. We demonstrated that, despite a high degree of relatedness, these three proteins exhibit a distinct receptor-specific pattern of phosphorylation, raising the possibility that they might mediate receptor-specific cellular changes. In addition the generation of antibodies specific for either radixin, ezrin or moesin allowed us to show that a previously described tumor transplantation antigen is indeed ezrin, thus implicating this protein in the determination of the biological phenotype of certain tumors.