Temperature-dependent tyrosine phosphorylation of microtubule-associated protein kinase in epidermal growth factor-stimulated human fibroblasts.

Treatment of normal human fibroblasts with epidermal growth factor (EGF) results in the rapid (0.5 min) and simultaneous tyrosine phosphorylation of the EGF receptor (EGFr) and several other proteins. An exception to this tyrosine phosphorylation wave was a protein (42 kDa) that became phosphorylated on tyrosine only after a short lag time (5 min). We identified this p42 kDa substrate as the microtubule-associated protein (MAP) kinase using a monoclonal antibody to a peptide corresponding to the C-terminus of the predicted protein (Science 249, 64-67, 1990). EGF treatment of human fibroblasts at 37 degrees C for 5 min resulted in the tyrosine phosphorylation of 60-70% of MAP kinase as determined by the percent that was immunoprecipitated with antiphosphotyrosine antibodies. Like other tyrosine kinase growth factor receptors, the EGFr is activated and phosphorylated at 4 degrees C but is not internalized. Whereas most other substrates were readily tyrosine phosphorylated at 4 degrees C, MAP kinase was not. When cells were first stimulated with EGF at 4 degrees C and then warmed to 37 degrees C without EGF, tyrosine phosphorylation of MAP kinase was again observed. Treatment of cells with the protein kinase C activator phorbol myristate acetate (PMA) also resulted in the tyrosine phosphorylation of MAP kinase, and again only at 37 degrees C. Tryptic phosphopeptide maps demonstrated that EGF and PMA both induced the phosphorylation of the same peptide on tyrosine and threonine. This temperature and PMA sensitivity distinguishes MAP kinase from most other tyrosine kinase substrates in activated human fibroblasts.     

Use of phosphoproteomics to study tyrosine kinase activity in capacitating boar sperm. Kinase activity and capacitation

It is generally accepted that sperm capacitation is associated with the protein kinase A-mediated appearance of tyrosine phosphoproteins, although the substrates and kinase(s) involved have not been identified. We described a Mr 32,000 tyrosine phosphoprotein, "p32", appearing in porcine sperm coincident with capacitation. We also discovered a tyrosine kinase-like enzyme in boar sperm of Mr 32,000 ("TK-32") with enhanced activity during capacitation. The present work was conducted to further characterize and to identify these capacitation-related protein(s). Fresh porcine sperm were incubated to induce capacitation then immunoprecipitation, immunoblotting and proteomic analysis revealed seven tyrosine-phosphorylated proteins aligned in the range of Mr 30,000 with different isoelectric pH values (pI). Therefore, p32 may be composed of several tyrosine phosphoproteins. Three were identified as acrosin-binding sp32 (pI 6.5), and two triosephosphate isomerase isoforms (pI 7.1 and 7.9). At present, however, proteonomic analysis has not revealed any kinase at Mr 32,000. Immunoprecipitation experiments show that p32 and TK-32 are different molecules, as TK-32 activity remains in the supernatant of the antiphosphotyrosine precipitates. Finally, in-gel renaturation and immunoblotting suggest that TK-32 is a mitogen-activated protein kinase (MAPK). The discovery of p32 and the MAPK-like TK-32 provides new insight regarding the mechanisms underlying capacitation in the pig.     

Tyrosine phosphorylations in vivo associated with v-fms transformation.

The role of tyrosine-specific phosphorylation in v-fms-mediated transformation was examined by immunoblotting techniques together with a high-affinity antibody that is specific for phosphotyrosine. This antiphosphotyrosine antibody detected phosphorylated tyrosine residues on the gp140v-fms molecule, but not gP180v-fms or gp120v-fms, in v-fms-transformed cells. This antibody also identified a number of cellular proteins that were either newly phosphorylated on tyrosine residues or showed enhanced phosphorylation on tyrosine residues as a result of v-fms transformation. However, the substrates of the v-fms-induced tyrosine kinase activity were not the characterized pp60v-src substrates. The phosphorylation of some of these cellular proteins and of the gp140fms molecule was found to correlate with the ability of v-fms/c-fms hybrids to transform cells. In addition, immunoblotting with the phosphotyrosine antibody allowed a comparison to be made of the substrates phosphorylated on tyrosine residues in various transformed cell lines. This study indicates that the pattern of tyrosine phosphorylation in v-fms-transformed cells is strikingly similar to that in v-sis-transformed cells.     

Tyrosine phosphorylation of a 120-kilodalton pp60src substrate upon epidermal growth factor and platelet-derived growth factor receptor stimulation and in polyomavirus middle-T-antigen-transformed cells.

The monoclonal antibody 2B12 is directed toward p120, a 120-kDa cellular protein originally identified as a protein tyrosine kinase substrate in cells expressing membrane-associated oncogenic variants of pp60src. In this report, we show that p120 was tyrosine phosphorylated in avian cells expressing membrane-associated, enzymatically activated variants of c-src, including variants having structural alterations in the src homology regions 2 and 3. In contrast, p120 was not tyrosine phosphorylated in cells expressing enzymatically activated, nonmyristylated pp60src. Furthermore, p120 was tyrosine phosphorylated in avian cells expressing middle T antigen, the transforming protein of polyomavirus, as well as in rodent cells stimulated with either epidermal growth factor (EGF) or platelet-derived growth factor. Analysis of the time course of p120 tyrosine phosphorylation in EGF-stimulated cells revealed a rapid onset of tyrosine phosphorylation. In addition, both the extent and duration of p120 phosphorylation increased when cells overexpressing the EGF receptor were stimulated with EGF. Biochemical analysis showed that p120 (in both normal and src-transformed cells) was membrane associated, was myristylated, and was phosphorylated on serine and threonine residues. Hence, p120 appears to be a substrate of both nonreceptor- and ligand-activated transmembrane receptor tyrosine kinases and of serine/threonine kinases and is perhaps a component of both mitogen-stimulated and tyrosine kinase oncogene-induced signaling pathways.     

Comparison of baculovirus-expressed c-Abl and BCR/ABL protein tyrosine kinases

Mouse c-Abl type IV and human BCR/ABL proteins have been expressed in insect cells using the baculovirus system. The proteins were expressed as full-length polypeptides as judged by electrophoresis in denaturing gels. They were identified by immunoprecipitation and immunoblotting with antibodies against ABL peptides and, for BCR/ABL, against a BCR peptide. In these immunoprecipitates both proteins gave autophosphorylation principally on tyrosine. Both proteins were active tyrosine kinases, phosphorylating a variety of tyrosine-containing substrates. In fresh extracts both proteins contained phosphotyrosine as shown by Western blots with antiphosphotyrosine antibodies. Partial purification could be achieved readily using ion exchange columns, and the BCR/ABL protein, p210^BCR/ABL, could be further purified to near-homogeneity using an antiphosphotyrosine column. Both enzymes required a divalent metal ion for activity. At low concentrations of ATP (2 μM) and with angiotensin II as substrate both enzymes were activated by Mn²⁺ or by Mg²⁺. No major differences in catalytic properties were found between the two isolated enzymes in solution. The oncogenic properties of p210^BCR/ABL may be due to its different subcellular location, or to the presence of an intracellular inhibitor of c-Abl that does not inhibit BCR/ABL, or to altered substrate-specificity such that it can phosphorylate a unique substrate which is not recognised by c-Abl.     

Granulocyte macrophage-colony stimulating factor stimulates both association and activation of phosphoinositide 3OH-kinase and src-related tyrosine kinase(s) in human myeloid derived cells.

The signalling pathways used by the GM-CSF receptor are currently unknown. Here we show that in human myeloid derived cells GM-CSF can stimulate; (i) the accumulation of PtdIns(3,4,5)P3; (ii) increases in p53/p56lyn and p62c-yes directed protein tyrosine kinase activities in anti-lyn and anti-c-yes antibody directed immunoprecipitates, respectively and; (iii) increases in phosphoinositide 3OH-kinase activity in antiphosphotyrosine, anti-p53/p56lyn and anti-p62c-yes antibody directed immunoprecipitates. These results suggest that GM-CSF can stimulate formation of protein tyrosine kinase co-ordinated signalling complexes, that contain p53/p56lyn, p62c-yes and an activated PtdInsP2 directed phosphoinositide 3OH-kinase, which can drive the accumulation of the putative second-messenger PtdIns(3,4,5)P3.     

Antibodies to the ATP-binding site of the human epidermal growth factor (EGF) receptor as specific inhibitors of EGF-stimulated protein-tyrosine kinase activity

A region of the primary amino acid sequence of the epidermal growth factor receptor (EGF) protein-tyrosine kinase, which is involved in ATP binding, was identified using chemical modification and immunological techniques. EGF receptor was 14C-labelled with the ATP analogue 5'-p-fluorosulphonylbenzoyladenosine and from a tryptic digest a single radiolabelled peptide was isolated. The amino acid sequence was determined to be residues 716-724 and hence lysine residue 721 is located within the ATP-binding site. Antisera were elicited in rabbits to a synthetic peptide identical to residues 716-727 of the EGF receptor and the homologous sequence in v-erb B transforming protein from avian erythroblastosis virus. The affinity-purified antibodies precipitated human ECF receptor from A431 cells and placenta, and the v-erb B protein from erythroblasts. The antibodies inhibited EGF-stimulated receptor protein-tyrosine kinase autophosphorylation and phosphorylation of an exogenous peptide substrate containing tyrosine. The antibodies did not immunoprecipitate the transforming proteins pp60v-src or P120gag-abl or cAMP-dependent protein kinase, proteins which have homologous but not identical sequences surrounding the lysine residue within the ATP-binding site, nor did they react with the platelet-derived growth factor receptor. The antibodies had no effect on the kinase activity of purified v-abl protein in solution. The antibodies may therefore be a specific inhibitor of the tyrosine kinase of the EGF receptor.     

Relationship of site-specific beta subunit tyrosine autophosphorylation to insulin activation of the insulin receptor (tyrosine) protein kinase activity

The ability of insulin to activate the insulin receptor protein kinase is shown to be completely dependent on prior beta subunit tyrosine autophosphorylation. Autophosphorylation in the presence of insulin is a highly concerted reaction; tryptic digestion of insulin receptor beta subunits derived from preparations whose kinase activation ranges from under 5% to 100% of maximal yields the same array of [32P]Tyr(P)-containing peptides over the entire range. Of special note is the significant contribution of multiply phosphorylated forms of tryptic peptides corresponding to proreceptor residues 1144-1152 (from the "tyrosine kinase" domain) and 1314-1329 (near the carboxyl terminus) to overall beta subunit phosphorylation at kinase activations of 5% and under. Thus, partially activated/autophosphorylated receptor preparations consist of mixtures of unactivated unphosphorylated receptors and activated fully (or nearly fully) phosphorylated receptors. The latter can be selectively removed by adsorption to antiphosphotyrosine antibodies. This abrupt multiple phosphorylation of individual receptor molecules explains why, in the presence of insulin, overall beta subunit tyrosine phosphorylation tracks closely with kinase, up to approximately 90% activation. Insulin stimulates phosphorylation into all domains (involving at least 6 of the 13 tyrosines on the intracellular portion of the beta subunit) but does not cause the appearance of "new" 32P-labeled species. Rather, insulin directs 32P incorporation preferentially into those domains most productive of kinase activation. Phosphorylation of the tyrosine residues at 1146, 1150, and 1151 correlates most closely with kinase activation. These residues show the largest 32P incorporation during rapid kinase activation; moreover, in comparisons of receptors with similar overall autophosphorylation but very different activations (or similar activations but different extents of autophosphorylation), achieved by omitting insulin or varying [ATP], the phosphorylation of peptide 1144-1152 tracks closely with kinase activation, and phosphorylation of sites and Mr 4000-5000 tryptic peptide (presumably Tyr 953 and/or 960) tract nearly as well. By contrast the extent of phosphorylation of the carboxy-terminal peptide is frequently dissociated from the extent of kinase activation. Phosphorylation of this latter domain probably underlies a beta subunit function other than tyrosine kinase activity.     

Interleukin 2- and polyomavirus middle T antigen-induced modification of phosphatidylinositol 3-kinase activity in activated T lymphocytes.

Stimulation of activated T lymphocytes with interleukin 2 (IL-2) results in rapid increases in intracellular protein tyrosine phosphorylation. Both the identity of the protein tyrosine kinase (PTK) activated by IL-2 receptor ligation and the identities of the critical target proteins for this PTK remain largely undefined. In this article, we demonstrate that stimulation of activated murine or human T cells with IL-2 for 10 to 30 min induces two- to threefold increases in the level of phosphatidylinositol (PtdIns) 3-kinase activity present in antiphosphotyrosine (p-Tyr) antibody immunoprecipitates from these cells. Furthermore, substantial levels of PtdIns 3-kinase activity were coprecipitated from IL-2-deprived T cells by antibodies to the src-related PTK p59fyn. Cellular stimulation with IL-2 induced a two- to threefold increase in the level of p59fyn-associated PtdIns 3-kinase activity. To examine the effect of a constitutive increase in PtdIns 3-kinase activity on the growth factor responsiveness of activated T cells, murine CTLL-2 cells were transfected with a polyomavirus middle T antigen (MTAg) expression vector. Anti-p-Tyr and anti-p59fyn immunoprecipitates from MTAg-transfected CTLL-2 cells contained three- to sixfold higher levels of PtdIns 3-kinase activity than wild-type cells. Immune complex kinase assays revealed that MTAg expression concomitantly induced a constitutive threefold increase in the PTK activity of p59fyn in these cells. However, stable MTAg expression did not abrogate the dependence of CTLL-2 cells on exogenous IL-2 for continued growth and proliferation.     

An insulin-stimulated kemptide kinase purified from rat liver is deactivated by phosphatase 2A.

Insulin action leads to the rapid stimulation of a cytosolic Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) kinase (KIK) that has been recently purified to near homogeneity (Klarlund, J. K., Bradford, A. P., Milla, M. G., and Czech, M. P. (1990) J. Biol. Chem. 265, 227-234). To examine its activation mechanism, purified KIK was treated with purified protein phosphatases. The catalytic subunit of phosphatase 2A inhibited the activity of control KIK by about 50% and abolished the 5-fold elevation in KIK activity due to insulin action. The catalytic subunit of phosphatase 1 with equivalent activity based on dephosphorylation of 32P-labeled phosphorylase alpha had no effect on either control or insulin-stimulated KIK activity. The deactivation of insulin-stimulated KIK by phosphatase 2A was time- and concentration-dependent and was blocked by phosphatase inhibitors. The purified native complexes of phosphatase 2A, phosphatase 2A1, and phosphatase 2A2 similarly deactivated KIK. Analyis of control or insulin-stimulated KIK with two antiphosphotyrosine antibodies by immunoblotting and immunoprecipitation failed to detect the presence of phosphotyrosine in the kinase. These results indicate that KIK is activated by phosphorylation as part of a kinase cascade emanating from insulin receptor stimulation.     

Postnatal Age and Protein Tyrosine Phosphorylation at Synapses in the Developing Rat Brain

The relationship between postnatal age and protein tyrosine kinase activity in synaptosomes prepared from the rat forebrain was studied. Synaptosomal particulate and soluble fractions, as well as total homogenates, the cell soluble fraction, and P3, were prepared from rats ranging in postnatal age from 5 to 60 days and analyzed for (a) tyrosine kinase activity using polyglutamyltyrosine (4:1) as the substrate, (b) the presence of endogenous substrates for tyrosine phosphorylation using polyclonal antibodies specific for phosphotyrosine, and (c) levels of pp60src. Enzyme activity, expressed per milligram of protein, in the total homogenate, P3, and both the cell and synaptosomal soluble fractions was highest in the brains of young animals (postnatal days 5-10) and decreased thereafter to adult levels. In contrast, tyrosine kinase activity in the synaptosomal particulate fraction exhibited a unique biphasic developmental profile, increasing to maxima at postnatal days 10 and 20 before decreasing to adult values. Endogenous substrates for tyrosine phosphorylation were identified by incubating subcellular fractions with 2 mM ATP in the presence of sodium orthovanadate and probing nitrocellulose blots of proteins separated by gel electrophoresis with antiphosphotyrosine antibodies. Several phosphotyrosine-containing proteins were detected in the synaptosomal particulate and P3 fractions, including proteins of Mr 180K, 145K, 120K, 100K, 77K, 68K, 62K, 54K, 52K, and 42K. In the cell soluble fraction a protein doublet of Mr 54/52K and a 120K protein were the major phosphotyrosine-containing proteins. The 54/52K doublet was the major protein tyrosine kinase substrate in the synaptosomal soluble fraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemoattractant-induced tyrosine phosphorylation and activation of microtubule-associated protein kinase in human neutrophils.

Activation of human neutrophils by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP) induces tyrosine phosphorylation of several polypeptides, including a prominent band of approximately 41 kDa. A polypeptide of identical electrophoretic mobility was recognized by a monoclonal antibody raised against a sequence corresponding to amino acids 325-345 of ERK-1, one of a family of mitogen-activated protein (MAP) kinases. To establish the possible identity of these polypeptides, extracts from control and fMLP-treated cells were immunoprecipitated with immobilized antiphosphotyrosine antibodies. Reactivity with anti-ERK-1 antibodies was observed only in the precipitate of chemoattractant-stimulated cells. These data imply that a MAP kinase constitutes at least part of the tyrosine-phosphorylated 41-kDa polypeptide. By using an in vitro renaturation assay, treatment of intact cells with fMLP was found to stimulate several protein kinases, including one of approximately 41 kDa. Renaturation of samples immunoprecipitated with antiphosphotyrosine antibodies revealed the presence of an active protein kinase in chemoattractant-stimulated, but not in control cells. The immunoprecipitated kinase comigrated with the 41-kDa tyrosine phosphorylated polypeptide and the anti-ERK-1 reactive band. We conclude that a MAP kinase closely related or identical to ERK-1 is tyrosine phosphorylated and activated when human neutrophils are stimulated by chemotactic peptides. The rapid phosphorylation of this kinase, which is apparent within seconds, is compatible with a role in the activation of the respiratory burst and/or other neutrophil responses.     

VCP, the mammalian homolog of cdc48, is tyrosine phosphorylated in response to T cell antigen receptor activation.

Activation of T cells through the T cell antigen receptor (TCR) results in the rapid tyrosine phosphorylation of a number of cellular proteins, one of the earliest being a 100 kDa protein. We have sought to identify this 100 kDa substrate by partially purifying the protein by antiphosphotyrosine (APT) affinity purification, in order to obtain amino acid sequence data and, using this information, to isolate the cDNA clone encoding the molecule. We report here that the amino acid sequence data showed pp100 to be the murine equivalent of porcine valosin containing protein (VCP), a finding confirmed from the cloning and sequencing of the murine pp100 cDNA. Sequence analysis has shown VCP to be a member of a family of ATP binding, homo-oligomeric proteins, and the mammalian homolog of Saccharomyces cerevisiae cdc48p, a protein essential to the completion of mitosis in yeast. We also provide proof that both endogenous and expressed murine VCP are tyrosine phosphorylated in response to T cell activation. Thus we have identified a novel component of the TCR mediated tyrosine kinase activation pathway that may provide a link between TCR ligation and cell cycle control.     

Identification of JAK protein tyrosine kinases as signaling molecules for prolactin. Functional analysis of prolactin receptor and prolactin-erythropoietin receptor chimera expressed in lymphoid cells.

The mechanism of action of prolactin (PRL) was studied in murine lymphoid BAF-3 cells transfected with either the long form of the PRL receptor (PRL-R), or a chimeric receptor consisting of the extracellular domain of the PRL-R and the transmembrane and intracellular domain of the erythropoietin receptor (PRL/EPO-R). PRL sustained normal and long-term proliferation of BAF-3 cells expressing either the PRL-R or the hybrid PRL/EPO-R. Upon [125I]PRL cross-linking, both types of BAF-3 transfectants were shown to express two [125I]PRL cross-linked species differing in size by 20 kDa. These cross-linked complexes, after denaturation, were recognized by antibody against the PRL-R, indicating that they contain the transfected receptor. PRL induced rapid and transient tyrosine phosphorylation of both the PRL-R and the PRL/EPO-R in BAF-3 transfectants. Furthermore, PRL induced rapid tyrosine phosphorylation of the Janus kinase 2 (JAK2) which was already physically associated with the PRL-R or the PRL/EPO-R in the absence of ligand. JAK1 was also associated with PRL-R and PRL/EPO-R in the absence of ligand. However, only in BAF-3 cells expressing the PRL-R does PRL induce rapid and transient tyrosine phosphorylation of JAK1. These results demonstrate that JAK protein tyrosine kinases couple PRL binding to tyrosine phosphorylation and proliferation.     

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.     

The protein-tyrosine kinase fer associates with signaling complexes containing insulin receptor substrate-1 and phosphatidylinositol 3-kinase

In a screen for 3T3-F442A adipocyte proteins that bind SH2 domains, we isolated a cDNA encoding Fer, a nonreceptor protein-tyrosine kinase of the Fes/Fps family that contains a functional SH2 domain. A truncated splicing variant, iFer, was also cloned. iFer is devoid of both the tyrosine kinase domain and a functional SH2 domain but displays a unique 42-residue C terminus and retains the ability to form oligomers with Fer. Expression of both Fer and iFer proteins are strikingly increased upon differentiation of 3T3-L1 fibroblasts to adipocytes. Platelet-derived growth factor treatment of the cultured adipocytes caused rapid tyrosine phosphorylation of Fer and its recruitment to complexes containing platelet-derived growth factor receptor and the p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase. Insulin treatment of 3T3-L1 adipocytes stimulated association of Fer with complexes containing tyrosine phosphorylated IRS-1 and PI 3-kinase but did not stimulate tyrosine phosphorylation of Fer. PI 3-kinase activity in anti-Fer immunoprecipitates was also acutely activated by insulin treatment of cultured adipocytes. These data demonstrate the presence of Fer tyrosine kinase in insulin signaling complexes, suggesting a role of Fer in insulin action.     

IL-2 regulation of tyrosine kinase activity is mediated through the p70-75 beta-subunit of the IL-2 receptor

The stimulation of activated human T lymphocytes with IL-2 results in increased tyrosine kinase activity. IL-2 treatment of Tac+ T cells stimulates the rapid phosphorylation of multiple protein substrates at M of 116, 100, 92, 70 to 75, 60, 56, 55, 33, and 32 kDa. Phosphorylation on tyrosine residues was detected by immunoaffinity purification of protein substrates with Sepharose linked antiphosphotyrosine mAb, 1G2. Although phorbol ester stimulated serine phosphorylation of the IL-2R alpha (p55) subunit recognized by alpha TAC mAb, IL-2 did not stimulate any detectable phosphorylation of IL-2R alpha or associated coimmune precipitated proteins. In fact, the tyrosine phosphorylated proteins did not coprecipitate with alpha Tac antibody and similar phosphoproteins were stimulated by IL-2 in IL-2R alpha- human large granular lymphocytes which express only the 70 to 75 kDa IL-2R beta subunit of the high affinity IL-2R. Anti-Tac mAb could inhibit IL-2-stimulated tyrosine phosphorylation in activated T cells, which express both IL-2R subunits that together form the high affinity receptor complex, but not in large granular lymphocytes expressing only the IL-2R beta subunit. The data suggest that IL-2 stimulation of tyrosine kinase activities requires only the IL-2R beta subunit.     

Human c-fgr induces a monocyte-specific enzyme in NIH 3T3 cells.

The mutant c-fgr protein (p58c-fgr/F523) containing Phe-523 instead of Tyr-523 exhibited transforming activity in NIH 3T3 cells like other protein-tyrosine kinases of the src family, but normal p58c-fgr (p58c-fgr/wt) did not. The mutant protein showed tyrosine kinase activity threefold higher than that of the normal protein in vitro. Surprisingly, transfection of the normal c-fgr gene into NIH 3T3 cells resulted in induction of sodium fluoride (NaF)-sensitive alpha-naphthyl butyrate esterase (alpha-NBE), a marker enzyme of cells of monocytic origin, which was not induced in v-src-, v-fgr-, or lyn-transfected NIH 3T3 cells. The NaF-sensitive alpha-NBE induced in c-fgr transfectants was shown by isoelectric focusing to have a pI of 5.2 to 5.4, a range which was the same as those for thioglycolate-induced murine peritoneal macrophages and 1 alpha,25-dihydroxyvitamin D3-treated WEHI-3B cells. Immunoblotting studies with antiphosphotyrosine antibodies revealed that 58-, 62-, 75-, 120-, 200-, and 230-kDa proteins were commonly phosphorylated at tyrosine residues in NIH 3T3 cells transfected with normal and mutated c-fgr, while 95-kDa protein was significantly phosphorylated at tyrosine residues in cells transfected with the mutated c-fgr. These findings suggest that tyrosine phosphorylation of specific cellular substrate proteins is important in induction of NaF-sensitive alpha-NBE and cell transformation by p58c-fgr.