Phosphorylation-induced activation of tilapia nonspecific cytotoxic cells by serum cytokines.

Cytokines as soluble mediators of immunity are important in understanding immunological mechanisms against infectious organisms and during stress conditions. In the present study, the role of protein tyrosine phosphorylation is assessed in the activation of nonspecific cytotoxic cells (NCC) from tilapia Oreochromis niloticus by cytokine-like serum factors. NCC are the teleost equivalent of mammalian natural killer (NK) cells. In teleost fish, NCC are important mediators of innate immunity against bacterial and parasite insult and tumor growth. We have previously shown that exposure of tilapia (a tropical fish) to cold water temperatures (3 to 5 min at 5 to 10 degrees C) produces physiological stress responses characterized by immediate phenotypic and immunological changes. The serum obtained from stressed tilapia contains a 'stress activating serum factor' (SASF) which passively increases in vitro naive NCC cytotoxicity 2- to 4-fold over control levels. In an effort to identify the mechanisms of activation of cytotoxicity by SASF, the phosphorylation status of tyrosine residues in proteins from treated NCC was determined. NCC were incubated with heat-inactivated or untreated stress serum and Western blots of the cell lysates were probed with anti-phosphotyrosine monoclonal antibodies (mabs). The levels of tyrosine phosphorylation in several proteins of the SASF-activated NCC were higher than in control cells. Increased tyrosine phosphorylation was also induced by incubation of NCC in the presence of the tyrosine phosphatase inhibitor Na orthovanadate (vanadate). In every case, an increase in phosphorylation status shown by Western blotting was correlated with increases in cytotoxic activity of NCC against HL-60 target cells. The enzyme inhibitor Herbimycin A (HA) has been previously used to inhibit the activity of the src-family of tyrosine kinases. In the present study, a 4 h pretreatment of NCC with HA (2 microM), followed by treatment with SASF blocked the activation of cytotoxicity produced by SASF. These results suggested that activation of NCC by cytokine-like factors is mediated through activation of the src family of protein tyrosine kinases. Activation was associated with increased phosphorylation and higher cytotoxic effector functions.     

Activation-induced programmed cell death of nonspecific cytotoxic cells and inhibition by apoptosis regulatory factors

Nonspecific cytotoxic cells (NCC) are the teleost equivalent of mammalian lymphokine-activated natural killer cells. The cytotoxic activities of NCC are enhanced by stress-activated serum factors (SASF) present in tilapia acute-phase serum. In the present study purified NCC and xenogeneic target HL-60 tumor cells and nuclei were distinguishable in mixtures determined by flow cytometry. NCC activated by target HL-60 cells undergo activation-induced programmed cell death (AIPCD) during 12- to 16-h killing assays as shown by Annexin-V binding and nuclear DNA fragmentation results. Annexin-V binding studies also demonstrated that NCC kill HL-60 cells by an apoptotic mechanism. NCC are protected from AIPCD by 4-h preincubation in 50% SASF. Pretreatment also produced more than a fourfold increase in NCC cytotoxicity (effector/target (E:T) ratio = 100:1). In the absence of SASF preincubation, the percentage of apoptotic NCC increased from 8 to 91% at E:T ratios of 1:0 and 1:1, respectively. Kinetic studies (E:T = 10:1) demonstrated that the percentage of NCC exhibiting HL-60-dependent AIPCD increased between 0.1 and 12 h and then decreased inversely with total cell necrosis over the next 60 h. Preincubation of NCC with SASF protected NCC from AIPCD for over 72 h. Crosslinkage of the NCCRP-1 receptor with monoclonal antibody (mab) 5C6 produced AIPCD between 1 and 100 microg/mL mab concentrations. Preincubation with SASF completely protected NCC from mab 5C6-dependent AIPCD. SASF-mediated protection of NCC from AIPCD was dependent upon divalent cations, as demonstrated by increases in DNA hypoploidy of 38, 67, and 88% following preincubation in the presence of 10, 100, and 1000 microM EDTA, respectively. SASF also protected NCC from glucocorticoid- (i. e., dexamethasone) induced apoptosis. Combined, these results demonstrated that NCC activity is down-regulated by AIPCD. Release of SASF into the peripheral circulation may prevent negative regulation of NCC by AIPCD by increasing recycling capacity. Results are discussed in the context of the effects of acute stressors on innate immunity.     

Identification of a putative antigen receptor on fish nonspecific cytotoxic cells with monoclonal antibodies

In the present study mAb were derived against flow cytometry (FCM) purified fish (Ictalurus punctatus) nonspecific cytotoxic cells (NCC). mAb 5C6.10.4 and 6D3.2.10 produced 60 to 65% inhibition of lysis of NC-37 target cells (a human B-lymphoblastoid cell line) by unfractionated NCC. mAb 2B2.4.9 and 6D3.4.4 were noninhibitors of cytotoxicity. All mAb were the same isotype (IgM) and were cloned by limiting dilution (2x). Inhibitory activity was specific for the effector cells because the mAb had no effect on NCC cytotoxicity when only the target cells were treated. Inhibition could be produced by preincubation of the mAb with NCC or by no preincubation, and inhibition was not reversible. Killing by FCM-sorted NCC of NC-37 target cells was inhibited almost 100% by mAb 5C6.10.4. Inhibitor mAb also significantly reduced NCC killing of MOLT-4, K562, P815, U937, Daudi, YAC-1, and HL-60 cells. Experiments also were conducted to determine at which stage of the lytic cycle the mAb acted. Both inhibitor mAb significantly inhibited conjugate formation between effector and NC-37 target cells. The technique of FCM was combined with competitive binding experiments to determine that the Ag recognized by both inhibitor and noninhibitor mAb was found on the membranes of the same cells. These results were confirmed by demonstrating (by using FCM) that FITC-labeled inhibitor and biotinylated noninhibitor mAb bound to the same cells. FCM also was next used to determine mAb binding to various effector cell populations. Inhibitor and noninhibitor mAb bound to approximately 25% (5C6.10.4) and 39% (6D3.4.4) of fish anterior kidney cells; to 42% (5C6.10.4) and 54% (6D3.4.4) of fish spleen cells; and to 2.5% (5C6.10.4 and 6D3.4.4) of fish peripheral blood. mAb were used to purify the target cell binding structure found on NCC. Con A-Sepharose purified mAb were used as the fixed ligand for Affi-Gel-10 affinity chromatography experiments. FCM-purified NCC were solubilized and the receptor was purified by using this technique. Analysis of the NCC-purified receptor by 12% SDS-PAGE indicated that the mAb purified structure may be composed of a dimeric molecule consisting of 41 kDa and 38 kDa proteins. The same dimer was purified by using either inhibitory (6D3.2.10) or noninhibitory (6D3.4.4) mAb. Similar results were obtained with immunoprecipitation experiments by using mAb 5C6.10.4. These studies demonstrate that the Ag-binding receptor structure on fish NCC may be comprised of a dimeric complex.     

Insulin-induced stimulation of protein phosphorylation in Neurospora crassa cells.

1) Insulin stimulated the phosphorylation of at least 14 discrete proteins in Neurospora crassa cells. Specific proteins were phosphorylated at serine, threonine, and tyrosine residues, as determined by phosphoamino acid analysis of discrete spots on two-dimensional gels. 2) Insulin stimulated the phosphorylation by [gamma-32P]ATP of at least six discrete proteins in solubilized N. crassa membrane preparations at serine and tyrosine residues. 3) A phosphotyrosine-containing protein of 38 kDa, pI 7.0-7.2, reacted by both immunoblotting and immunoprecipitation with antiserum to P2, a peptide from the human insulin receptor that contains an autophosphorylated tyrosine residue. In N. crassa cells, therefore, as in mammalian cells, insulin induces a variety of protein phosphorylations, some of which may be part of an evolutionarily conserved signal transduction pathway.     

Lysosomal enzyme binding receptor protein from monkey brain and its phosphorylation.

The lysosomal enzyme binding receptor protein isolated from monkey brain by phosphomannan-Sepharose affinity chromatography was phosphorylated by [gamma-32P] ATP by protein kinases tightly associated with the receptor protein. A greater than 200 kDa protein was phosphorylated on both serine and tyrosine residues and a approximately 45 kDa protein was phosphorylated on only serine residues as evidenced by SDS-gel electrophoresis, autoradiography and phosphoamino acid analysis [(Panneerselvam, Ramamoorthy & Balasubramanian (1987) Biochem Biophys Res Commun, 147, 927-935)]. 125I-labelled lysosomal enzymes could be cross-linked to the receptor protein in the presence of disuccinimidyl suberate. Phosphorylation of the receptor on both serine and tyrosine residues was inhibited by quercetin, polylysine and polymyxin B. Catalytic subunit of cyclic AMP-dependent protein kinase preferentially phosphorylated the approximately 45 kDa protein. In the presence of Triton X-100, phosphorylation of a few additional protein bands on non-tyrosine residues was observed. There was a marked reduction in the efficiency of binding lysosomal enzymes by the phosphorylated receptor protein in comparison to the unphosphorylated receptor protein.     

Activation of protein kinase C decreases phosphorylation of c-Jun at sites that negatively regulate its DNA-binding activity.

In resting human epithelial and fibroblastic cells, c-Jun is phosphorylated on serine and threonine at five sites, three of which are phosphorylated in vitro by glycogen synthase kinase 3 (GSK-3). These three sites are nested within a single tryptic peptide located just upstream of the basic region of the c-Jun DNA-binding domain (residues 227-252). Activation of protein kinase C results in rapid, site-specific dephosphorylation of c-Jun at one or more of these three sites and is coincident with increased AP-1-binding activity. Phosphorylation of recombinant human c-Jun proteins in vitro by GSK-3 decreases their DNA-binding activity. Mutation of serine 243 to phenylalanine blocks phosphorylation of all three sites in vivo and increases the inherent trans-activation ability of c-Jun at least 10-fold. We propose that c-Jun is present in resting cells in a latent, phosphorylated form that can be activated by site-specific dephosphorylation in response to protein kinase C activation.     

Identification of phosphoproteins associated with maintenance of transformed state in temperature-sensitive Rous sarcoma-virus infected cells by proteomic analysis

To identify phosphotyrosine-containing proteins essential for maintaining the transformed state, we studied the tyrosine phosphorylation profile of temperature-sensitive mutant of Rous sarcoma virus, tsNY68, infected cells (68N7). Shifting the temperature from 39 degrees C (nonpermissive) to 32 degrees C (permissive) markedly increased the expression of phosphotyrosine-containing cell membrane proteins of approximately 40kDa, as assessed by SDS-PAGE. Membrane and nuclear proteins were separated by two-dimensional gel electrophoresis and immunoblotted with anti-phosphotyrosine antibody. Proteins showing temperature-dependent changes in phosphorylation profile were subjected to in-gel digestion with trypsin and analyzed by mass spectrometry. Five proteins were identified: heterogeneous nuclear ribonucleoprotein (hnRNP) A3, hnRNP A2, annexin II, phosphoglycerate mutase 1, and triosephosphate isomerase 1. hnRNP A3 was phosphorylated at serine residues and had both serine and tyrosine phosphorylated sites. These results suggest an important complementary role for proteomics in identifying molecular abnormalities associated with tumor progression that may be attractive candidates for tumor diagnosis.     

Phosphorylation of the 48-kDa subunit of the glycine receptor by protein kinase C.

The postsynaptic glycine receptor purified from rat spinal cord is rapidly and specifically phosphorylated by protein kinase C. The target for phosphorylation is the strychnine-binding subunit of the receptor (molecular mass of approximately 48 kDa), which is phosphorylated on serine residues to a final stoichiometry of approximately 0.8 mol of phosphate/mol of subunit. The 48-kDa phosphoprotein was analyzed by proteolytic cleavage and peptide mapping in order to localize the site of phosphorylation within the receptor molecule. Examination of the 32P-labeled receptor fragments generated by digestion with N-chlorosuccinimide, cyanogen bromide, and endoproteinase lysine C and of the deduced amino acid sequence of the 48-kDa protein (Grenningloh, G., Rienitz, A., Schmitt, B., Methfessel, C., Zensen, M., Beyreuther, K., Gundelfinger, E. D., and Betz, H. (1987) Nature 328, 215-220) indicates that the phosphorylation site is located in a region corresponding to the major intracellular loop of the predicted structure of the glycine receptor subunit and suggests serine 391 as the phosphorylated residue. In fact, a synthetic peptide corresponding to residues 384-392 of the 48-kDa subunit was specifically phosphorylated by protein kinase C. Moreover, tryptic digests of this phosphopeptide and of the phosphorylated 48-kDa subunit of the glycine receptor migrated to the same position in two-dimensional peptide mapping. Furthermore, antibodies elicited against peptide 384-392 were shown to inhibit the protein kinase C-dependent phosphorylation of the 48-kDa polypeptide. Interestingly, the relative position of the phosphorylated domain is similar to those known or proposed to be phosphorylated in other ligand-gated ion channel receptor subunits, thus suggesting further the existence of a homologous regulatory region in these receptor proteins.     

Rapid increase of the human IFN-gamma receptor phosphorylation in response to human IFN-gamma and phorbol myristate acetate. Involvement of different serine/threonine kinases

Various cell surface receptors are phosphorylated upon binding of their ligand, and this phosphorylation seems to be involved in the signal transduction or in the feedback regulation of this signal. The possibility of a phosphorylation of the human IFN-gamma receptor (hu-IFN-gamma-R) has been investigated with 32P-labeled whole Raji cells and receptor purification either by immunoprecipitation with an anti-hu-IFN-gamma-R polyclonal antiserum or by affinity chromatography. The hu-IFN-gamma-R was found to be phosphorylated at a basal level. Upon incubation of the cells with recombinant hu-IFN-gamma, a dose-dependent two-fold increase of this phosphorylation was observed. Phosphoamino acid analysis by TLC showed that the same amino acids, serine and threonine, are phosphorylated at a basal level and after incubation with hu-IFN-gamma. Protein kinase C and Ca2+/calmodulin-dependent kinase pathways have been reported in some cases to be involved in the signal transduction pathway of hu-IFN-gamma. Both pathways involved the activation of a serine/threonine kinase and therefore we have investigated the possibility of hu-IFN-gamma-R phosphorylation by these kinases. PMA, an activator of protein kinase C, induced a rapid increase of the receptor phosphorylation in Raji cells, whereas the Ca2+ ionophore A23187 did not. PMA-induced hu-IFN-gamma-R phosphorylation was not associated with any effect on expression or inactivation of the receptor. PMA alone did not mimic the hu-IFN-gamma effect in Raji cells as measured by induction of IP-10 gene expression, a high specific marker of hu-IFN-gamma response. But the protein kinase C inhibitors, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) and staurosporine, reduced this IFN-gamma-induced expression. However, H7 and staurosporine treatment as well as protein kinase C depletion suppressed PMA-induced receptor phosphorylation, whereas constitutive and hu-IFN-gamma-induced phosphorylation remained unchanged. Our results suggest that the serine/threonine kinase involved in the hu-IFN-gamma-R phosphorylation induced by IFN-gamma is different from protein kinase C.     

Tyrosine phosphorylation of two cytosolic proteins of 50 kDa and 35 kDa in rat liver by insulin-receptor kinase in vitro.

Insulin-receptor tyrosine kinase can phosphorylate a variety of artificial substrates in vitro. Its physiological substrate(s), however, remains unknown. In the present study, we show that immobilized insulin receptors phosphorylate tyrosine residues of two cytosolic proteins of 50 kDa and 35 kDa in rat liver. Phosphorylation of these two proteins required Mn2+- or Mg2+-ATP as the phosphate donor. Phosphorylation was time- and temperature-dependent. Furthermore, the rate of phosphorylation of the two proteins was related to the autophosphorylated state of the insulin receptor. The pI of the phosphorylated 50 kDa and 35 kDa proteins was 5.4 and 5.6 respectively. These proteins were present in low abundance. They were not related to each other, nor to the insulin receptor, as demonstrated by in-gel proteolytic digestion and by immunoprecipitation using antibodies produced against them. They were specific substrates for the insulin receptor kinase, since they were not phosphorylated by epidermal-growth-factor-receptor kinase. These observations suggest that the 50 kDa and 35 kDa cytosolic proteins may be endogenous substrates for the insulin-receptor kinase.     

Protein serine and threonine phosphorylation,hyperactivation and acrosome reaction in in vitro capacitated hamster spermatozoa

Monoclonal antibodies against phosphoserine and phosphothreonine were used in the present study to investigate the changes in serine and threonine phosphorylation respectectively during capacitation of hamster spermatozoa. Immunoblot analysis of hamster spermatozoa capacitated in TALP, a medium that supports capacitation, showed that a set of four proteins of molecular weight 56, 63, 66, and 100 kDa was phosphorylated both at the serine and threonine residues. In addition, five other proteins of molecular weight 32, 39, 45, 53, and 61 kDa were phosphorylated specifically at the threonine residues. Of these nine proteins, the 100 kDa protein showed a time dependent or capacitation-dependent decrease in intensity which coincided with the percentage acrosome-reacted spermatozoa. In contrast, the 49 and 63 kDa threonine phosphorylated proteins showed increased phosphorylation coinciding with capacitation. H8 (a serine and threonine kinase inhibitor) had a transient effect on the phosphorylation of these two phosphothreonine proteins but inhibited acrosome reaction substantially all through the treatment period. Okadaic acid (OA) (a serine and threonine protein phosphatase inhibitor) inhibited hyperactivation but had no effect on acrosome reaction. In fact, OA stimulated acrosome reaction. Finally the immunofluorescence studies indicated localization of the serine phosphorylated proteins in tail as well as in head of the capacitated hamster spermatozoa whereas the threonine phosphorylated proteins were localized mostly in the tail of the spermatozoa. The findings of the present study suggest that serine/threonine phosphorylation and the enzymes responsible for regulating the level of phosphorylation play an important role in capacitation and capacitation-associated events namely hyperactivation and acrosome reaction. However, further studies are needed in order to establish the exact role of these proteins in capacitation of spermatozoa.     

Specific phosphorylation of a COOH-terminal site on the full-length form of the alpha 1 subunit of the skeletal muscle calcium channel by cAMP-dependent protein kinase.

The primary (alpha 1) subunit of purified skeletal muscle dihydropyridine-sensitive calcium channels is present in full-length (212 kDa) and truncated (190 kDa) forms which are both phosphorylated by cAMP-dependent protein kinase (cA-PK) in vitro. In the present study, phosphorylation of the purified calcium channel by cA-PK followed by immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and two-dimensional phosphopeptide mapping revealed differential phosphorylation of the related 190- and 212-kDa forms. The 190-kDa form of the alpha 1 subunit was phosphorylated on three major and three minor tryptic phosphopeptides; the 212-kDa form was phosphorylated on all six of these phosphopeptides plus two that were unique. Time course experiments showed that a single site on the COOH-terminal portion of the full-length form of the alpha 1 subunit is most intensely and rapidly (within 10 s) phosphorylated. Phosphorylation occurs almost exclusively on this COOH-terminal site unless harsh conditions such as treatment with denaturing detergents are employed to expose phosphorylation sites within the 190-kDa segment of the molecule. Elution of phosphopeptides from the second dimension chromatograph followed by immunoprecipitation with an anti-peptide antibody (anti-CP1) directed against the COOH-terminal amino acid sequence enabled us to identify this major phosphorylation site as serine 1854. The nearby consensus sites for cA-PK phosphorylation at serines 1757 and 1772 were phosphorylated only after denaturation or proteolytic cleavage. Phosphorylation of serine 1854 may play a pivotal role in the regulation of calcium channel function by cA-PK.     

Dephosphin, a 96,000 Da substrate of protein kinase C in synaptosomal cytosol, is phosphorylated in intact synaptosomes

A 96,000 dalton phosphoprotein, called dephosphin, is phosphorylated in intact synaptosomes from rat brain and is rapidly dephosphorylated upon depolarisation-dependent calcium entry. A 96,000 dalton phosphoprotein is also a substrate of protein kinase C in synaptosomal cytosol, and the aim of the study was to determine whether the two proteins may be the same. Dephosphin in intact synaptosomes and the 96,000 dalton protein kinase C substrate comigrated on polyacrylamide gels. Both phosphoproteins had identical phosphopeptide maps after digestion with V8 protease. Both phosphoproteins ran on isoelectric focussing gels with a pI of 6.3-6.7 and focussed as a series of 5-6 spots. Both proteins were phosphorylated exclusively on serine. Both proteins could be resolved into a doublet on longer polyacrylamide gels. The two subunits were of 96 and 93 kDa in both phosphorylation conditions and had dissimilar phosphopeptide maps. However, phosphopeptide maps of either the 96 or 93 kDa subunits were identical in intact synaptosomes compared with synaptosomal cytosol. These results show that a phosphoprotein phosphorylated in intact synaptosomes and a 96,000 dalton protein kinase C substrate from rat brain synaptosomal cytosol are the same, and raise the possibility that protein kinase C is the protein kinase responsible for dephosphin phosphorylation in intact synaptosomes.     

Functional characterization of a maize ribosomal S6 protein kinase (ZmS6K), a plant ortholog of metazoan p70(S6K)

Ribosomal protein S6 (S6rp) is phosphorylated by the p70S6K enzyme in mammals, under mitogen/IGF regulation. This event has been correlated with an increase in 5'TOP mRNA translation. In this research, a maize S6 kinase (ZmS6K) was isolated from maize (Zea mays L.) embryonic axes by human p70S6K antibody immunoprecipitation. This enzyme, a 62 kDa peptide, proved to be specific for S6rp phosphorylation, as revealed by in vivo and in vitro kinase activity using either the 40S ribosomal subunit or the RSK synthetic peptide as the substrates. ZmS6K activation was achieved by phosphorylation on serine/threonine residues. Specific phospho-Threo recognition by the p70S6K antibody directed to target phospho-Threo residue 389 correlated with ZmS6K activation. The ZmS6K protein content remained almost steady during maize seed germination, whereas the ZmS6K activity increased during this process, consistent with Zm6SK phosphorylation. Addition of insulin to germinating maize axes proved to increase ZmS6K activity and the extent of S6rp phosphorylation. These events were blocked by rapamycin, an inhibitor of the insulin signal transduction pathway in mammals, at the TOR (target of rapamycin) enzyme level. We conclude that ZmS6K is a kinase, structurally and functionally ortholog of the mammalian p70S6K, responsible for in vivo S6rp phosphorylation in maize. Its activation is induced by insulin in a TOR-dependent manner by phosphorylation on conserved serine/threonine residues.     

Phosphorylation of Glial Fibrillary Acidic Protein and Vimentin by Cytoskeletal-Associated Intermediate Filament Protein Kinase Activity in Astrocytes

These studies describe a cytoskeletal-associated protein kinase activity in astrocytes that phosphorylated the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin and that appeared to be distinct from protein kinase C (PK-C) and the cyclic AMP-dependent protein kinase (PK-A). The cytoskeletal-associated kinase activity phosphorylated intermediate filament proteins in the presence of 10 mM MgCl2 and produced an even greater increase in 32P incorporation into these proteins in the presence of calcium/calmodulin. Tryptic peptide mapping of phosphorylated intermediate filament proteins showed that the intermediate filament protein kinase activity produced unique phosphopeptide maps, in both the presence and the absence of calcium/calmodulin, as compared to that of PK-C and PK-A, although there were some common sites of phosphorylation among the kinases. In addition, it was determined that the intermediate filament protein kinase activity phosphorylated both serine and threonine residues of the intermediate filament proteins, vimentin and GFAP. However, the relative proportion of serine and threonine residues phosphorylated varied depending on the presence or absence of calcium/calmodulin. The magnesium-dependent activity produced the highest proportion of threonine phosphorylation, suggesting that the calcium/calmodulin-dependent kinase activity acts mainly at serine residues. PK-A and PK-C phosphorylated mainly serine residues. Also, the intermediate filament protein kinase activity phosphorylated both the N-and the C-terminal domains of vimentin and the N-terminal domain of GFAP. In contrast, both PK-C and PK-A are known to phosphorylate the N-terminal domains of both proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

The phosphorylation and activation of B-raf in PC12 cells stimulated by nerve growth factor.

Treatment of PC12 cells with nerve growth factor does not alter the levels of B-raf mRNA, but does induce rapid phosphorylation of B-raf proteins. Phosphorylation was observed after 1.5 min and reached a maximum by 10-15 min. B-raf protein was phosphorylated almost exclusively on serine residues; no tyrosine phosphorylation was detected. Nerve growth factor-induced phosphorylation was not affected by depletion of protein kinase C or by removal of extracellular calcium but was inhibited by K-252a. Concomitant with the increase in serine phosphorylation, nerve growth factor treatment also increased the serine/threonine kinase activity of B-raf protein within 1-2 min.     

Phosphorylation of the acetylcholine receptor by protein kinase C and identification of the phosphorylation site within the receptor delta subunit

Purified acetylcholine receptor is rapidly and specifically phosphorylated by partially purified protein kinase C, the Ca2+/phospholipid-dependent enzyme. The receptor delta subunit is the major target for phosphorylation and is phosphorylated on serine residues to a final stoichiometry of 0.4 mol of phosphate/mol of subunit. Phosphorylation is dose-dependent with a Km value of 0.2 microM. Proteolytic digestion of the delta subunit phosphorylated by either protein kinase C or the cAMP-dependent protein kinase yielded a similar pattern of phosphorylated fragments. The amino acids phosphorylated by either kinase co-localized within a 15-kDa proteolytic fragment of the delta subunit. This fragment was visualized by immunoblotting with antibodies against a synthetic peptide corresponding to residues 354-367 of the receptor delta subunit. This sequence, which contains 3 consecutive serine residues, was recently shown to include the cAMP-dependent protein kinase phosphorylation site (Souroujon, M. C., Neumann, D., Pizzighella, S., Fridkin, M., and Fuchs, S. (1986) EMBO J. 5, 543-546). Concomitantly, the synthetic peptide 354-367 was specifically phosphorylated in a Ca2+- and phospholipid-dependent manner by protein kinase C. Furthermore, antibodies directed against this peptide inhibited phosphorylation of the intact receptor by protein kinase C. We thus conclude that both the cAMP-dependent protein kinase and protein kinase C phosphorylation sites reside in very close proximity within the 3 adjacent serine residues at positions 360, 361, and 362 of the delta subunit of the acetylcholine receptor.     

Phosphorylation of highly purified growth hormone receptors by a growth hormone receptor-associated tyrosine kinase

We have shown previously that growth hormone (GH) promotes the phosphorylation of its receptor on tyrosyl residues (Foster, C. M., Shafer, J. A., Rozsa, F. W., Wang, X., Lewis, S. D., Renken, D. A., Natale, J. E., Schwartz, J., and Carter-Su, C. (1988) Biochemistry 27, 326-334). In the present study, we investigated the possibility that a tyrosine kinase is specifically associated with the GH receptor. GH-receptor complexes were first partially purified from GH-treated 3T3-F442A fibroblasts, a GH-responsive cell, by immunoprecipitation using anti-GH antiserum. 35S-Labeled proteins of Mr = 105,000-125,000 were observed in the immunoprecipitate from GH-treated cells labeled metabolically with 35S-amino-acids. These proteins were not observed in immunoprecipitates from cells not exposed to GH or when non-immune serum replaced the anti-GH antiserum, consistent with the proteins being GH receptors. GH receptors appeared to be phosphorylated, as evidenced by the presence of 32P-labeled bands, comigrating with the 105-125 kDa 35S-labeled proteins, in the immunoprecipitate of GH-treated cells labeled metabolically with [32P]Pi. When partially purified GH receptor preparation was incubated with [gamma-32P]ATP (7-15 microM) for 10 min at 30 degrees C in the presence of MnCl2, a protein of Mr = 121,000 was phosphorylated exclusively on tyrosyl residues. As expected for the GH receptor, this protein was not observed in immunoprecipitates when cells had not been treated with GH nor when non-immune serum replaced the anti-GH antiserum. GH-receptor complexes were also purified to near homogeneity by sequential immunoprecipitation with phosphotyrosyl-binding antibody followed by anti-GH antiserum. When cells were labeled metabolically with 35S-amino acids, the 35S label migrated almost exclusively as an Mr = 105,000-125,000 protein. This protein also incorporated 32P into tyrosyl residues when incubated in solution with [gamma-32P]ATP. These results show that highly purified GH receptor preparations undergo tyrosyl phosphorylation, suggesting that either the GH receptor itself is a tyrosine kinase or is tightly associated with a tyrosine kinase.     

Interleukin-3 stimulates the tyrosine phosphorylation of the 140-kilodalton interleukin-3 receptor

Murine interleukin-3 (mIL-3) stimulates the rapid and transient tyrosine phosphorylation of a number of proteins in mIL-3-dependent B6SUtA1 cells. Two of these proteins, p68 and p140, are maximally phosphorylated at tyrosine residues within 2 min of addition of mIL-3. Because 125I-mIL-3 can be cross-linked to both 70- and 140-kDa proteins on intact B6SUtA1 cells, we investigated whether the tyrosine phosphorylated p68 and p140 were these two mIL-3 receptor proteins. Addition of antiphosphotyrosine antibodies (alpha PTyr Abs) to cell lysates from B6SUtA1 cells, to which 125I-mIL-3 had been disuccinimidyl suberate-cross-linked, resulted in the immunoprecipitation of 125I-mIL-3 complexed to both 70- and 140-kDa proteins. To determine if the observed immunoprecipitation pattern was due to the direct interaction of alpha-PTyr Abs with these two mIL-3 receptor proteins or with tyrosine-phosphorylated proteins that were associated with the receptor proteins, cell lysates were treated with 2% sodium dodecyl sulfate, 5% 2-mercaptoethanol, and boiled for 1 min. After removal of sodium dodecyl sulfate and 2-mercaptoethanol, alpha PTyr Abs immunoprecipitated 125I-mIL-3 cross-linked to only the 140-kDa protein. To confirm this finding, 32P-labeled B6SUtA1 cells were treated with biotinylated or fluoresceinated mIL-3. Addition of immobilized streptavidin or antifluorescein antibodies, respectively, to cell lysates from these cells resulted in the enrichment of only a 140-kDa tyrosine phosphorylated protein. Taken together, these results strongly suggest that only the 140-kDa receptor protein is tyrosine phosphorylated upon mIL-3 binding.