Association of the 72-kDa protein-tyrosine kinase PTK72 with the B cell antigen receptor.

The antigen receptors on B lymphocytes are cell-surface immunoglobulins. Antibodies against surface IgM (sIgM) coimmunoprecipitate several sIgM-associated proteins. Incubation of anti-IgM complexes with [gamma-32P]ATP leads to the phosphorylation on tyrosine of IgM-associated proteins including MB-1 and a protein of 72 kDa. Peptide mapping and reimmunoprecipitation experiments indicate that the 72-kDa phosphoprotein is PTK72, a protein-tyrosine kinase that is expressed at highest levels in B lymphocytes. MB-1 is also phosphorylated in immune complexes prepared with antibodies to PTK72, indicating that components of the IgM complex are associated with PTK72. In addition, PTK72 is associated with sIgD complexes isolated from spleen B lymphocytes. The cross-linking of sIgM antigen receptors on B lymphocytes leads to the rapid phosphorylation of PTK72 on tyrosine and to the activation of PTK72 as measured by autophosphorylation and by the phosphorylation of an exogenous substrate in anti-PTK72 immune complexes. These results suggest that the signaling cascade initiated by engagement of the B cell antigen receptor involves the increased enzymatic activity of PTK72, which is already present in a preformed antigen receptor complex.     

Molecular cloning of a porcine gene syk that encodes a 72-kDa protein-tyrosine kinase showing high susceptibility to proteolysis

By using antibodies raised against a portion of N terminus of 40-kDa kinase (Kobayashi, T., Nakamura, S., Taniguchi, T., and Yamamura, H. (1990) Eur. J. Biochem. 188, 535-540), not only 40-kDa protein but also 72-kDa protein were detected on immunoblot analysis of porcine spleen homogenate. In splenocytes preparation, the antibodies could immunoprecipitate protein-tyrosine kinase activity of the 72-kDa protein but not detect the 40-kDa protein even on immunoblot. After incubation of crude spleen homogenate at 37 degrees C with or without various protease inhibitors, immunoblot analysis revealed proteolytic breakdown of the 72-kDa protein to 40-kDa fragment. Next, using oligonucleotides designed according to partially sequenced information of the 40-kDa kinase as a probe, we have isolated a clone containing entire coding sequence for the 40-kDa kinase from a porcine spleen cDNA library. This clone had a 1884-base-pair-long open reading frame encoding 628-amino-acid polypeptide with a calculated molecular weight of 71,618. The deduced amino acid sequence did not contain a ligand binding or membrane spanning region but did a well-conserved protein-tyrosine kinase domain and two src homology region 2 domains. The sequences of these domains showed 30-40% identity to those of other protein-tyrosine kinases, but those of remaining parts were quite unique. From these results, we concluded that the 40-kDa kinase was generated by proteolysis from the 72-kDa holoprotein which was a new member of nonreceptor protein-tyrosine kinase so far reported. We therefore designated this gene as syk after spleen tyrosine kinase.     

High-affinity IgE receptor-mediated stimulation of rat basophilic leukemia (RBL-2H3) cells induces early and late protein-tyrosine phosphorylations.

We reported previously that stimulation of RBL-2H3 cells through the high-affinity IgE receptor resulted in tyrosine phosphorylation of a 72-kDa protein (pp72) that was coupled to signal transduction. In the present study, although pp72 tyrosine phosphorylation was induced only by antigen triggering, stimulation of RBL-2H3 cells by either antigen or the calcium-ionophore A23187 led to increased tyrosine phosphorylation of a 110-kDa protein (pp110). This tyrosine phosphorylated protein was also observed when RBL-2H3 cells were transfected with the G protein-coupled m3 muscarinic receptor and then stimulated to secrete with carbachol. In contrast to tyrosine phosphorylation of pp72, antigen-induced pp110 tyrosine phosphorylation required extracellular calcium, was absent in cells depleted of protein kinase C, and was detected between 1 and 5 min after stimulation. The protein-tyrosine kinase inhibitor genistein blocked both histamine release and tyrosine phosphorylation induced by A23187. Altogether, the data suggest a role for pp110 in secretion. However, protein kinase C activation induced pp110 tyrosine phosphorylation but not histamine release demonstrating that pp110 tyrosine phosphorylation alone is not sufficient for degranulation. We conclude that tyrosine phosphorylation of pp72 is associated with the early steps of IgE receptor-generated signaling, whereas pp110 tyrosine phosphorylation occurs secondary to calcium influx and protein kinase C activation.     

Generation of an active protein-tyrosine kinase from lymphocytes by proteolysis

The major NaCl-stimulated protein-tyrosine kinase activity found in soluble thymus extracts, as measured by the phosphorylation of angiotensin I, is a 40-kDa enzyme known as p40 (Zioncheck, T. F., Harrison, M. L., and Geahlen, R. L. (1986) J. Biol. Chem. 261, 15637-15643). Antibodies prepared against p40 cross-react with a 72-kDa protein-tyrosine kinase (p72) from spleen or thymus that is closely related to p40 by peptide-mapping experiments. The recovery of p40 from spleen homogenates is reduced while the recovery of p72 is enhanced by the addition of high concentrations of leupeptin or soybean trypsin inhibitor to the homogenization media. The generation of p40 in spleen homogenates occurs with a concomitant increase in protein-tyrosine kinase activity. Activated catalytic fragments of 38-43 kDa can be generated by the treatment of partially purified p72 with trypsin or papain. The p72 protein-tyrosine kinase is found at the highest levels in spleen, thymus, and lung, tissues that also have high protein-tyrosine kinase activity and generate high levels of p40 following homogenization. p72 is also found in certain T and B cell-derived cell lines and in NIH3T3 cells.     

Protein-tyrosine phosphorylations induced by concanavalin A and N-formyl-methionyl-leucyl-phenylalanine in human neutrophils.

The ability of the lectin concanavalin A (ConA) and N-formyl-methionyl-leucyl-phenylalanine (fMLF) to induce protein-tyrosine phosphorylation in human neutrophils was examined by immunoblot analysis. ConA caused an increase in tyrosine phosphorylation of protein bands with apparent molecular masses of 120, 80, 76, 66 and 40 kDa; on the other hand, fMLF caused an increase in those of only 80-kDa and 40-kDa proteins. These protein-tyrosine phosphorylations were time- and dose-dependent. The tyrosine phosphorylation of 40-kDa protein induced by fMLF was suppressed but that by ConA was not suppressed by pertussis toxin pretreatment. At the same time, pertussis toxin pretreatment also inhibited lysozyme release and aggregation of neutrophils induced by fMLF but did not inhibit those responses induced by ConA. These results suggest that the tyrosine phosphorylation of 40-kDa protein may be involved in a part of neutrophil activation and be regulated via pleiotropic signal transduction pathways. In addition, immunoblot analysis employing antibodies against microtubule-associated protein 2 (MAP2) kinase suggested that this tyrosine-phosphorylated 40-kDa protein might be the MAP2 kinase.     

In vitro phosphorylation of the erythropoietin receptor and an associated protein, pp130.

The cytoplasmic domain of the cloned erythropoietin (EPO) receptor (EPOR) contains no protein kinase motif, yet addition of EPO to EPO-responsive cells causes an increase in protein-tyrosine phosphorylation. Here we show that addition of EPO or interleukin-3 (IL-3) to an IL-3-dependent cell line expressing the wild-type EPOR causes a small fraction (less than 5%) of total cellular EPOR to shift in gel mobility from 66 to 72 kDa, due at least in part to phosphorylation. Using biotinylated EPO as an affinity reagent, we show that the 72-kDa species is greatly enriched on the cell surface. To demonstrate that a protein kinase activity associates with cell surface EPOR, cells were incubated with biotinylated EPO and then cross-linked with a thiol-cleavable chemical cross-linker. The avidin-agarose-selected complexes were incubated with [gamma-32P]ATP. After in vitro phosphorylation and denaturation without reducing agent, both antiphosphotyrosine and anti-EPOR antibodies immunoprecipitated labeled 72-kDa EPOR and an unidentified 130-kDa phosphoprotein (pp130), indicating that a protein kinase is associated with cell surface EPOR and that a fraction of the EPOR was phosphorylated on tyrosine residues either in the cells or during the cell-free phosphorylation reaction. Under reducing conditions, the 72-kDa phosphorylated EPOR but not pp130 was immunoprecipitated with an anti-EPOR antibody, suggesting that the pp130 is bound to the EPOR by the thiol-cleavable chemical cross-linker. Previously, we showed that deletion of the 42 carboxy-terminal amino acids of the EPOR allows cells to grow in 1/10 the normal EPO concentration, without affecting receptor number or affinity. Two carboxy-terminal truncated EPO receptors that are hyperresponsive to EPO were poorly phosphorylated during the in vitro reaction, suggesting that the carboxy-terminal region of the EPOR contains a site for phosphorylation or a site for interaction with a protein kinase. Our data suggests that phosphorylation or interaction with a protein kinase in the carboxy-terminal region may down-modulate the proliferative action of the EPOR.     

Thyrotropin and insulin-like growth factor I regulation of tyrosine phosphorylation in FRTL-5 cells. Interaction between cAMP-dependent and growth factor-dependent signal transduction.

Pretreatment of rat FRTL-5 thyroid cells with thyrotropin (TSH) markedly potentiated the mitogenic response to insulin-like growth factor I (IGF-I) (Tramontano, D., Moses, A. C., Veneziani, B. M., and Ingbar, S. H. (1988) Endocrinology 122, 127-132; Takahashi, S.-I., Conti, M., and Van Wyk, J. J. (1990) Endocrinology 126, 736-745). The present study was undertaken to determine whether this synergism between TSH and IGF-I in FRTL-5 cells was correlated with changes in tyrosine phosphorylation of intracellular proteins. Tyrosine phosphorylation in intact cells was determined by gel electrophoresis and immunoblotting using monospecific anti-phosphotyrosine antibodies. Cells were preincubated for up to 24 h with TSH, dibutyryl cAMP, forskolin, or cholera toxin and then incubated for an additional 1 min in the absence or presence of IGF-I. As reported by others, IGF-I rapidly increased tyrosine phosphorylation of a 175-kDa protein as well as a less intense band of 90-100 kDa. Pretreatment for 6-12 h with either TSH or other agents that elevate intracellular cAMP potentiated the IGF-I-dependent tyrosine phosphorylation of the 175-kDa substrate by 3-5-fold. Since TSH did not increase IGF receptor number of kinase activity, the effect of TSH is assumed to be exerted at a step distal to IGF receptor tyrosine kinase. Surprisingly, IGF-I-independent tyrosine phosphorylation was also increased by pretreatment with TSH. When intact cells were analyzed TSH produced a time- and concentration-dependent increase in tyrosine phosphorylation of a prominent 120-125-kDa substrate and less prominent 100- and 80-kDa substrates. Assays using Triton X-100-soluble extracts incubated with MgCl2, ATP, and orthovanadate demonstrated that TSH pretreatment increased tyrosine phosphorylation over that observed in untreated cells. In this cell-free assay, TSH pretreatment enhanced the phosphorylation of multiple substrates. These studies suggest that a cAMP stimulus that initiates a trophic effect can be propagated indirectly through multiple pathways including enhancement of tyrosine phosphorylation.     

Lymphocyte lineage-restricted tyrosine-phosphorylated proteins that bind PLC gamma 1 SH2 domains.

Epidermal growth factor (EGF) or platelet-derived growth factor binding to their receptor on fibroblasts induces tyrosine phosphorylation of PLC gamma 1 and stable association of PLC gamma 1 with the receptor protein tyrosine kinase. Similarly in lymphocytes, cross-linking of antigen receptors induces the formation of molecular complexes incorporating PLC gamma 1; however, associated kinase activity is thought to be mediated through cytoplasmic protein tyrosine kinase(s). In this report, we generated a fusion protein containing the SH2 domains of human PLC gamma 1 and human IgG1 heavy chain constant region to identify lymphocyte phosphoprotein-binding PLC gamma 1 SH2 domains following cellular activation. As in EGF- or platelet-derived growth factor-stimulated fibroblasts, PLC gamma 1 is coprecipitated in activated lymphocytes, complexed with associated tyrosine-phosphorylated proteins. One of these, a 35/36-kDa protein found prominently in T cells and at lower levels in B cells, bound to the fusion protein in immunoprecipitation experiments. The fusion protein showed lineage restricted association with a 74-kDa phosphoprotein in T cells and a 93-kDa phosphoprotein in B cells. It bound to activated EGF receptor in fibroblasts as expected, and protein tyrosine kinase activity was precipitated from EGF-stimulated cells. However, PLC gamma 1-associated protein tyrosine kinase activity was not detected in activated lymphocytes. These data suggest that lymphocyte PLC gamma 1 SH2-binding proteins are cell lineage specific and may be transiently associated with activated PLC gamma 1.     

Thrombin and collagen induce rapid phosphorylation of a common set of cellular proteins on tyrosine in human platelets

The ability of thrombin and collagen to induce protein-tyrosine phosphorylation in intact human platelets was assessed by using antibodies to phosphotyrosine in conjugation with immunoblots. Upon stimulation by thrombin there was an increase in the amount of protein-tyrosine phosphorylation of three bands with molecular masses of 135, 124, and 76 kDa in a time-dependent manner. The tyrosine phosphorylation in these three proteins increased in a concurrent fashion and reached a maximum level in 10 s and then a plateau or a slight decrease. Stimulation by collagen was also followed by an increase in tyrosine phosphorylation of 135- and 124-kDa proteins. Unlike stimulation by thrombin, collagen induced no obvious tyrosine phosphorylation of 76-kDa protein. The time courses for thrombin- or collagen-induced protein-tyrosine phosphorylation were similar to that for [14C] serotonin release. These results suggest that 135- and 124-kDa proteins are a common set of proteins that become phosphorylated on tyrosine residue during platelet activation.     

Tyrosine phosphorylation of a 22-kDa protein is correlated with transformation by Rous sarcoma virus

Recent studies from this laboratory have identified novel cytoskeletal proteins that are phosphorylated on tyrosine in vivo in Rous sarcoma virus-transformed chick fibroblasts (Glenney, J. R., Jr., and Zokas, L. (1989) J. Cell Biol. 108, 2401-2408). In the present report, the phosphorylation of these proteins was examined in cells expressing the nonmyristylated mutants of src that are not transformed. A good correlation was found between transformation and the tyrosine phosphorylation of a 22-kDa protein. Tyrosine phosphorylation of the 22-kDa protein was reduced more than 95% in cells expressing the nonmyristylated mutants of src. Size fractionation revealed that the 22-kDa phosphoprotein in transformed chick fibroblasts is found in a Mr 150,000 complex. Monoclonal antibodies were used to screen various chicken tissues where the 22-kDa protein was found at high levels in muscle and lung with low levels in epithelial cells and brain. The 22-kDa protein becomes an excellent candidate for a mediator of transformation by the tyrosine kinase class of oncogenes.     

Protein-tyrosine phosphatase-1B negatively regulates insulin signaling in l6 myocytes and Fao hepatoma cells

Insulin signaling is regulated by tyrosine phosphorylation of the signaling molecules, such as the insulin receptor and insulin receptor substrates (IRSs). Therefore, the balance between protein-tyrosine kinases and protein-tyrosine phosphatase activities is thought to be important in the modulation of insulin signaling in insulin-resistant states. We thus employed the adenovirus-mediated gene transfer technique, and we analyzed the effect of overexpression of a wild-type protein-tyrosine phosphatase-1B (PTP1B) on insulin signaling in both L6 myocytes and Fao cells. In both cells, PTP1B overexpression blocked insulin-stimulated tyrosine phosphorylation of the insulin receptor and IRS-1 by more than 70% and resulted in a significant inhibition of the association between IRS-1 and the p85 subunit of phosphatidylinositol 3-kinase and Akt phosphorylation as well as mitogen-activated protein kinase phosphorylation. Moreover, insulin-stimulated glycogen synthesis was also inhibited by PTP1B overexpression in both cells. These effects were specific for insulin signaling, because platelet-derived growth factor (PDGF)-stimulated PDGF receptor tyrosine phosphorylation and Akt phosphorylation were not inhibited by PTP1B overexpression. The present findings demonstrate that PTP1B negatively regulates insulin signaling in L6 and Fao cells, suggesting that PTP1B plays an important role in insulin resistance in muscle and liver.     

Novel yeast protein kinase (YPK1 gene product) is a 40-kilodalton phosphotyrosyl protein associated with protein-tyrosine kinase activity.

Extracts of bakers' yeast (Saccharomyces cerevisiae) contain protein-tyrosine kinase activity that can be detected with a synthetic Glu-Tyr copolymer as substrate (G. Schieven, J. Thorner, and G.S. Martin, Science 231:390-393, 1986). By using this assay in conjunction with ion-exchange and affinity chromatography, a soluble tyrosine kinase activity was purified over 8,000-fold from yeast extracts. The purified activity did not utilize typical substrates for mammalian protein-tyrosine kinases (enolase, casein, and histones). The level of tyrosine kinase activity at all steps of each preparation correlated with the content of a 40-kDa protein (p40). Upon incubation of the most highly purified fractions with Mn-ATP or Mg-ATP, p40 was the only protein phosphorylated on tyrosine. Immunoblotting of purified p40 or total yeast extracts with antiphosphotyrosine antibodies and phosphoamino acid analysis of 32P-labeled yeast proteins fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the 40-kDa protein is normally phosphorylated at tyrosine in vivo. 32P-labeled p40 immunoprecipitated from extracts of metabolically labeled cells by affinity-purified anti-p40 antibodies contained both phosphoserine and phosphotyrosine. The gene encoding p40 (YPK1) was cloned from a yeast genomic library by using oligonucleotide probes designed on the basis of the sequence of purified peptides. As deduced from the nucleotide sequence of YPK1, p40 is homologous to known protein kinases, with features that resemble known protein-serine kinases more than known protein-tyrosine kinases. Thus, p40 is a protein kinase which is phosphorylated in vivo and in vitro at both tyrosine and serine residues; it may be a novel type of autophosphorylating tyrosine kinase, a bifunctional (serine/tyrosine-specific) protein kinase, or a serine kinase that is a substrate for an associated tyrosine kinase.     

Phospholipase C-gamma 1 and phospholipase C-gamma 2 are substrates of the B cell antigen receptor associated protein tyrosine kinase.

Cross-linking the antigen receptor on B cells results in a rapid increase in protein tyrosine kinase activity as detected by increased phosphorylation on tyrosine residues of multiple proteins. Although the identity of most of this substrates remains unknown, some have been proposed. One possible substrate of the antigen receptor-associated kinase is phospholipase C (PLC). Since multiple isoforms of PLC have been identified, we have studied which isoforms are targets of the antigen receptor. PLC-gamma 1 and PLC-gamma 2 but not PLC-beta 1 or PLC-delta 1 were detected in human B cells. Immunoprecipitation with antibodies against PLC-gamma 1 or PLC-gamma 2 and subsequent Western blotting with anti-phosphotyrosine antibodies revealed that both PLC-gamma 1 and PLC-gamma 2 are tyrosine phosphorylated in stimulated but not in resting B cells. This was confirmed by experiments whereby B cell lysates were immunoprecipitated with anti-phosphotyrosine antibody and subsequently blotted with antibodies against PLC-gamma 1 or PLC-gamma 2. Further, the specific protein tyrosine kinase inhibitors, tyrphostins, which block phospholipase-C activation and proliferation of B cells also inhibited tyrosine phosphorylation on both PLC-gamma 1 and PLC-gamma 2. We conclude that both isoforms PLC-gamma 1 and PLC-gamma 2 are targets of the antigen receptor-associated protein tyrosine kinase.     

The lectin wheat germ agglutinin stimulates a protein-tyrosine kinase activity of p72syk in porcine splenocytes

We previously reported a molecular cloning of porcine gene syk encoding a non-receptor type 72-kDa protein-tyrosine kinase and a generation of anti-CPTK40 antibodies which could immunoprecipitate the activity of p72syk (Taniguchi et al. (1991) J. Biol. Chem. 266, 15790-15796). In this study, we have demonstrated that wheat germ agglutinin caused an increase in the autophosphorylation activity of p72syk which preceded an increase of protein-tyrosine phosphorylation observed at the same time in porcine splenocyte. The increase of p72syk activity was dose dependent and was inhibited by the coexistence of N-acetyl-D-glucosamine. Upon stimulation by the combination of 4 beta-phorbol 12-myristate 13-acetate and ionophore A23187, we could not detect the increase of activity of p72syk suggesting that the activation of p72syk was independent of protein kinase C and calcium ions.     

Effects of protein kinase C activation after epidermal growth factor binding on epidermal growth factor receptor phosphorylation

The possible role of epidermal growth factor (EGF) receptor phosphorylation at threonine 654 in modulating the protein-tyrosine kinase activity of EGF-treated A431 cells has been studied. It has been suggested that EGF could indirectly activate a protein-serine/threonine kinase, protein kinase C, that can phosphorylate the EGF receptor at threonine 654. Protein kinase C is known to be activated, and threonine 654 is phosphorylated, when A431 cells are exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA). The protein-tyrosine kinase activity of EGF receptors is normally evidenced in EGF-treated cells by phosphorylation of the receptor at tyrosine. This is inhibited when TPA-treated cells are exposed to EGF. We now show that receptor phosphorylation at threonine 654 can also be detected in EGF-treated A431 cells, presumably due to indirect stimulation of protein kinase C or a similar kinase. Some receptor molecules are phosphorylated both at threonine 654 and at tyrosine. Since prior phosphorylation at threonine 654 inhibits autophosphorylation, we propose that protein kinase C can phosphorylate the threonine 654 of autophosphorylated receptors. This provides evidence for models in which protein kinase C activation, consequent upon EGF binding, could reduce the protein-tyrosine kinase activity of the EGF receptor. Indeed, we find that 12-O-tetradecanoylphorbol-13-acetate, added 10 min after EGF, further increases threonine 654 phosphorylation and induces the loss of tyrosine phosphate from A431 cell EGF receptors.     

Tyrosine phosphorylation is an essential event in the stimulation of B lymphocytes by Staphylococcus aureus Cowan I.

Staphylococcus aureus Cowan I (SAC) is a potent mitogen for purified human B cells. By using Western blotting with antiphosphotyrosine antibodies, we demonstrated that the mitogenic effect of SAC is associated with rapid tyrosine phosphorylation of proteins of 45, 68, 75, 97, and 145 kDa. This tyrosine phosphorylation was detected within 30 s of the addition of SAC; it reached a maximum within 10 min, after which it declined gradually. In contrast to SAC, most soluble anti-IgM antibodies do not induce proliferation of isolated human B cells. As indicated by Western blotting, soluble anti-IgM antibodies induced a similar pattern of tyrosine phosphorylation, with the exception of the 68-kDa protein, which was the most heavily phosphorylated protein in SAC-treated cells. A similar but less intense 68-kDa band was also induced by mitogenic anti-IgM bound to beads. This suggested that tyrosine phosphorylation, especially of p68, may play an important role in B cell mitogenesis. To test this hypothesis, we determined the effect of specific tyrosine kinase inhibitors (tyrphostins) on SAC-induced tyrosine phosphorylation, oncogene expression, and B cell proliferation. The concentration dependencies of inhibition of these processes suggested that they were linked. Nonspecific toxic effects of the tyrphostins were ruled out by the demonstration that the tyrphostins did not alter cell viability and did not inhibit B cell proliferation induced by phorbol esters, which do not induce tyrosine phosphorylation. For maximal inhibition of SAC-induced cell proliferation, the tyrophostins needed to be added before or shortly after addition of SAC. Taken together, these data indicate that tyrosine phosphorylation is an obligatory early signal in B cell proliferation.     

Mitogen-induced tyrosine-phosphorylated 41- and 43-kDa proteins are family members of extracellular signal-regulated kinases/microtubule-associated protein 2 kinases.

Two antipeptide antibodies, one against the peptide corresponding to residues 307-327 (alpha Y91) and one against the peptide corresponding to the C-terminal portion (alpha C92) of the deduced amino acid sequence of the extracellular signal-regulated kinase 1 (ERK1), precipitated two 41-kDa and/or two 43-kDa phospho-proteins from mitogen-stimulated Swiss 3T3 cells. Electrophoretic mobilities on two-dimensional gels of the immunoprecipitated 41- and 43-kDa phosphoproteins were similar to those of the 41- and 43-kDa cytosol proteins, whose increased tyrosine phosphorylation we and others had originally identified in various mitogen-stimulated cells (Cooper, J. A., Sefton, B. M., and Hunter, T. (1984) Mol. Cell. Biol. 4, 30-37; Kohno, M. (1985) J. Biol. Chem. 260, 1771-1779); phosphopeptide map analysis revealed that they were respectively identical molecules. All those phosphoproteins contained phosphotyrosine, and the more acidic forms contained additional phosphothreonine. Immunoprecipitated 41- and 43-kDa phosphoproteins had serine/threonine kinase activity toward myelin basic protein (MBP) and microtuble-associated protein 2 (MAP2). With the combination of two-dimensional gel electrophoresis and the kinase assay in MBP-containing polyacrylamide gels of the alpha Y91 immunoprecipitates, with or without phosphatase 2A treatment, we showed that only their acidic forms were active. These results clearly indicate that 41- and 43-kDa proteins, the increased tyrosine phosphorylation of which is rapidly and commonly induced by mitogen stimulation of fibroblasts, are family members of ERKs/MAP2 kinases and that phosphorylation both on tyrosine and threonine residues is necessary for their activation.     

H(2)O(2)-induced tyrosine phosphorylation of protein kinase cdelta by a mechanism independent of inhibition of protein-tyrosine phosphatase in CHO and COS-7 cells

It has been proposed that H(2)O(2) increases tyrosine phosphorylation of cellular proteins by inhibiting protein-tyrosine phosphatase through oxidation of the cysteine residue of the enzyme essential for its catalytic activity. Tyrosine phosphorylation of the delta isoform of protein kinase C (PKC) was induced by H(2)O(2) in CHO and COS-7 cells. H(2)O(2) also induced activation of mitogen-activated protein kinase. Vanadate and molybdate, which inhibit protein-tyrosine phosphatase by binding to its active site, did not induce tyrosine phosphorylation of PKCdelta, but enhanced H(2)O(2)-induced tyrosine phosphorylation of PKCdelta in the cell. The oxoanions, however, generated the active form of mitogen-activated protein kinase. Another protein-tyrosine phosphatase inhibitor, phenylarsine oxide, which bridges the thiol residues of the enzyme, induced tyrosine phosphorylation of PKCdelta, and the reaction was enhanced by vanadate. These results suggest that inhibition of protein-tyrosine phosphatase is insufficient for induction of tyrosine phosphorylation of PKCdelta in the cells, and that presumably activation of protein-tyrosine kinase may be essential for tyrosine phosphorylation of the PKC isoform.     

Glucose stimulates the tyrosine phosphorylation of Crk-associated substrate in pancreatic beta-cells

Several years ago, we demonstrated that glucose induced tyrosine phosphorylation of a 125-kDa protein (p125) in pancreatic beta-cells (Konrad, R. J., Dean, R. M., Young, R. A., Bilings, P. C., and Wolf, B. A. (1996) J. Biol. Chem. 271, 24179-24186). Glucose induced p125 tyrosine phosphorylation in beta-TC3 insulinoma cells, beta-HC9 cells, and in freshly isolated rat islets, whereas increased tyrosine phosphorylation was not observed with other fuel secretagogues. Initial efforts to identify p125 were unsuccessful, so a new approach was taken. The protein was purified from betaTC6,F7 cells via an immunodepletion method. After electrophoresis and colloidal Coomassie Blue staining, the area of the gel corresponding to p125 was excised and subjected to tryptic digestion. Afterward, mass spectrometry was performed and the presence of Crk-associated substrate (Cas) was detected. Commercially available antibodies against Cas were obtained and tested directly in beta-cells, confirming glucose-induced tyrosine phosphorylation of Cas. Further experiments demonstrated that in beta-cells the glucose-induced increase in Cas tyrosine phosphorylation occurs immediately and is not accompanied by increased focal adhesion kinase tyrosine phosphorylation. Finally, it is also demonstrated via Western blotting that Cas is present in normal isolated rat islets. Together, these results show that the identity of the previously described p125 beta-cell protein is Cas and that Cas undergoes rapid glucose-induced tyrosine phosphorylation in beta-cells.