VIP stimulates proliferation and differentiation of the cultured retinal pigment epithelium with disparate potencies.

Previous studies showed that VIP modulates mediators of two signal transduction pathways, namely the adenylate cyclase and the nonreceptor tyrosine protein kinase pp60c-src in cultured chick retinal pigment epithelium (RPE). Here we show that VIP modulates simultaneously two disparate cellular events, namely the cell proliferation and differentiation of the RPE, however, with different potencies. The maximal effects on proliferation and differentiation are observed at 5 x 10(-9)M and 5 x 10(-7)M, respectively. Treatment with the maximally effective concentrations of VIP for 10 days increases the cell numbers and the melanin contents to 150% and 200% of the controls, respectively. The lowest concentrations of VIP showing significant stimulatory effect on cell proliferation and melanin synthesis are 5 x 10(-11) M and 5 x 10(-9)M, respectively.     

Structural and functional modification of pp60c-src associated with polyoma middle tumor antigen from infected or transformed cells.

The polyoma middle tumor antigen (MTAg) associates with the src proto-oncogene product pp60c-src in infected or transformed rodent cells. The tyrosine protein kinase activity of pp60c-src, as measured by in vitro phosphorylation of pp60c-src itself or the exogenous substrate enolase, was increased 10- to 20-fold in cells transformed or infected with transformation-competent polyoma virus compared with controls. pp60c-src associated with MTAg and precipitated with polyoma antitumor serum had a novel site(s) of in vitro tyrosine phosphorylation within its amino-terminal domain. These observations suggest that association of MTAg with pp60c-src alters the accessibility of pp60c-src tyrosine residues for phosphorylation in vitro and increases pp60c-src protein kinase activity. Several transformation-defective mutants of MTAg did not cause amino-terminal tyrosine phosphorylation of pp60c-src in vitro or enhance its protein kinase activity, suggesting that these properties correlate with the transforming ability of MTAg. However, one transformation-defective MTAg mutant, dl1015, did cause amino-terminal tyrosine phosphorylation of pp60c-src in vitro and did enhance its protein kinase activity. This suggests that properties of MTAg, in addition to modifying the structure and function of pp60c-src, may be important for transformation.     

Involvement of pp60c-src in platelet-activating factor-stimulated platelets. Evidence for translocation from cytosol to membrane.

We have investigated the characteristics of platelet-activating factor (PAF)-stimulated protein tyrosine phosphorylation in rabbit platelets and its relationship to pp60c-src. 32P-Labeled platelets were challenged with PAF (10(-7) M) for 15 s, the reaction was killed by lysis at 4 degrees C, and samples were loaded onto a phosphotyrosine monoclonal antibody (Tyr(P)-mAb)-agarose column. The column was eluted with 10 mM phenyl phosphate, and the fractions were collected. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by autoradiography of the column fractions, showed that PAF increased the radioactivity of about a dozen protein bands with predominant ones of approximate molecular masses of 50, 60, 71, 82, and 300 kDa. When Tyr(P)-mAb-agarose column fractions were subjected to immunoblotting with pp60v-src mAb, it was observed that PAF treatment increased the reactivity of 50- and 60-kDa protein species. Immunoprecipitation with pp60v-src mAb further confirmed that PAF treatment increased phosphorylation of the 60- and 50-kDa proteins. Polyclonal antibody to G-protein (alpha-subunit) did not exhibit any reactivity to the column fractions and thus ruled out this protein as substrate for the tyrosine kinase. We next attempted to localize the pp60c-src. Platelet membrane particulate and cytosol fractions were separated from control and PAF-treated platelets, and it was observed that the immunoreactivity to pp60v-src mAb dramatically increased in the particulate membrane fraction from PAF-treated platelets. A concomitant decrease in the immunoreactivity in the cytosol fraction of PAF-treated platelets was also noted. It is concluded that PAF stimulates phosphorylation of pp60c-src tyrosine kinase and causes its rapid translocation from cytosol to membranes in rabbit platelets.     

The effect of quercetin on the phosphorylation activity of the Rous sarcoma virus transforming gene product in vitro and in vivo

The phosphotransferase activity of the Rous sarcoma virus src gene product, pp60src, was inhibited both in vitro and in vivo by the bioflavonoid quercetin. The Ki for the inhibitory effect was in the range of 6-11 microM under conditions in vitro. The inhibitory effect of quercetin was competitive towards the nucleotides ATP and GTP as substrates for pp60src and was non-competitive towards alpha-casein as the protein substrate of this kinase activity. In contrast, studies in vitro of the phosphotransferase activity of the catalytic subunit of the cAMP-dependent protein kinase showed that this flavonoid did not inhibit the phosphorylation of physiological substrates of this enzyme. In cultured cells the half-maximal inhibition of tyrosine phosphorylation of pp60src as well as the phosphorylation of the Mr = 34000 protein, a physiological substrate of pp60src, was in the range 0.06-0.08 mM.     

Activation of the pp60c-src kinase by middle T antigen binding or by dephosphorylation.

The transforming protein of polyoma virus, middle T antigen, associates with the protein tyrosine kinase pp60c-src, and analysis of mutants of middle T suggests that this complex plays an important role in transformation by polyoma. It has recently been reported that pp60c-src from polyoma virus-transformed cells has enhanced tyrosine kinase activity in vitro. The data presented here confirm these findings and show that the enhanced kinase activity of pp60c-src is due to an increase in the Vmax of the enzyme. Sucrose density gradient analysis demonstrates that only the form of pp60c-src which is bound to middle T antigen is activated. The difference in enzyme activity between pp60c-src from normal and middle T-transformed cells is more marked when the enzyme is prepared from lysates containing the phosphotyrosine protein phosphatase inhibitor, sodium orthovanadate. pp60c-src from middle T transformed cells is unaffected, but pp60c-src from normal cells has reduced kinase activity if dephosphorylation is prevented. The kinase activity of pp60c-src thus appears to be regulated by its degree of phosphorylation at tyrosine, and data are presented which support this hypothesis. pp60c-src is the first example of a protein tyrosine kinase whose activity is inhibited by phosphorylation at tyrosine. Middle T antigen may increase the kinase activity of pp60c-src by preventing phosphorylation at this regulatory site.     

Cell transformation by pp60c-src mutated in the carboxy-terminal regulatory domain

We introduced two mutations into the carboxy-terminal regulatory region of chicken pp60c-src. One, F527, replaces tyrosine 527 with phenylalanine. The other, Am517, produces a truncated pp60c-src protein lacking the 17 carboxy-terminal amino acids. Both mutant proteins were phosphorylated at tyrosine 416 in vivo. The specific activity of the Am517 mutant protein kinase was similar to that of wild-type pp60c-src whereas that of the F527 mutant was 5- to 10-fold higher. Both mutant c-src genes induced focus formation on NIH 3T3 cells, but the foci appeared at lower frequency, and were smaller than foci induced by polyoma middle tumor antigen (mT). The wild-type or F527 pp60c-src formed a complex with mT, whereas the Am517 pp60c-src did not. The results suggest that one, inability to phosphorylate tyrosine 527 increases pp60c-src protein kinase activity and transforming ability; two, transformation by mT involves other events besides lack of phosphorylation at tyrosine 527 of pp60c-src; three, activation of the pp60c-src protein kinase may not be required for transformation by the Am517 mutant; and four, the carboxyl terminus of pp60c-src appears to be required for association with mT.     

Myristoylation-regulated direct interaction between calcium-bound calmodulin and N-terminal region of pp60v-src

pp60v-src tyrosine protein kinase was suggested to interact with Ca2+-bound calmodulin (Ca2+/CaM) through the N-terminal region based on its structural similarities to CAP-23/NAP-22, a myristoylated neuron-specific protein, whose myristoyl group is essential for interaction with Ca2+/CaM; (1) the N terminus of pp60v-src is myristoylated like CAP-23/NAP-22; (2) both lysine residues are required for the myristoylation-dependent interaction and serine residues that are thought to regulate the interaction through the phosphorylations located in the N-terminal region of pp60v-src. To verify this possibility, we investigated the direct interaction between pp60v-src and Ca2+/CaM using a myristoylated peptide corresponding to the N-terminal region of pp60v-src. The binding assay indicated that only the myristoylated peptide binds to Ca2+/CaM, and the non-myristoylated peptide is not able to bind to Ca2+/CaM. Analyses of the binding kinetics revealed two independent reactions with the dissociation constants (KD) of 2.07 x 10(-9)M (KD1) and 3.93 x 10(-6)M (KD2), respectively. Two serine residues near the myristoyl moiety of the peptide (Ser2, Ser11) were phosphorylated by protein kinase C in vitro, and the phosphorylation drastically reduced the interaction. NMR experiments indicated that two molecules of the myristoylated peptide were bound around the hydrophobic clefts of a Ca2+/CaM molecule. The small-angle X-ray scattering analyses showed that the size of the peptide-Ca2+/CaM complex is 2-3A smaller than that of the known Ca2+/CaM-target molecule complexes. These results demonstrate clearly the direct interaction between pp60v-src and Ca2+/CaM in a novel manner different from that of known Ca2+/CaM, the target molecules, interactions.     

Differential inhibitory effects of TGF-beta on EGF-, PDGF-, and HBGF-1-stimulated MG63 human osteosarcoma cell growth: possible involvement of growth factor interactions at the receptor and postreceptor levels

The growth of MG63 human osteosarcoma cell line in 5% serum is stimulated by epidermal growth factor (EGF), platelet-derived growth factor (PDGF), or heparin-binding growth factor-1 (HBGF-1). The mitogenic effect of EGF and PDGF is completely blocked by TFG-beta at 1 ng per ml and the effect of HBGF-1 is attenuated by 75-80%. Treatment of MG63 cells with TGF-beta reduces HBGF-1 receptor binding affinity from 1.24 x 10(-11) M to 3.51 x 10(-11) M with no change on the receptor number (1.1 x 10(3) per cell). The receptor-binding affinity of EGF and PDGF is not altered by TGF-beta treatment; however, the number of EGF receptor is increased by 25%. Both EGF and PDGF stimulate MG63 cellular tyrosine kinase activity, and such stimulation is inhibited by TGF-beta pretreatment. No change in the cellular protein tyrosine phosphorylation pattern can be detected in HBGF-1-stimulated cells with and without TGF-beta pretreatment. These data suggest that TGF-beta inhibits EGF and PDGF mitogenicity by blocking EGF- and PDGF-stimulated tyrosine kinase activity and attenuates HBGF-1 mitogenicity by decreasing its receptor affinity.     

12-O-tetradecanoylphorbol-13-acetate stimulates phosphorylation of the 58,000-Mr form of polyomavirus middle T antigen in vivo: implications for a possible role of protein kinase C in middle T function.

The 58,000-Mr form (58K form) of the polyomavirus middle T antigen (mT) is a minor species distinguished by its phosphorylation in vivo on serine and by its efficient phosphorylation on tyrosine in immune complexes (B.S. Schaffhausen and T.L. Benjamin, J. Virol. 40:184-196, 1981). Here we report that the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, rapidly stimulates phosphorylation of this mT species when added to cultures of wild-type polyomavirus-infected or polyomavirus-transformed 3T3 cells. Incubation with TPA leads to an accumulation of the 58K mT species to levels 1.5- to 5-fold higher than that in untreated cells within 15 min. TPA specifically stimulates phosphorylation of the 58K mT species without affecting that of the 56K species. Mapping by partial proteolysis shows that TPA-stimulated phosphorylation occurs at or near the site in 58K mT that is normally phosphorylated in the absence of TPA. A synthetic diacyl glycerol, 1-oleoyl-2-acetyl-glycerol, also specifically stimulates phosphorylation of 58K mT in vivo, while an inactive phorbol analog does not. TPA fails to induce phosphorylation of a 58K mT species encoded by certain nontransforming virus mutants with altered mT proteins that normally fail to undergo phosphorylation at the 58K site. These results indicate that the 58K form of mT is phosphorylated by or through the action of protein kinase C. TPA treatment of infected cells also leads to increased levels of 58K mT as measured in the immune complex kinase reaction, in which mT becomes phosphorylated on tyrosine by pp60c-src. These results are discussed in terms of a possible role for protein kinase C in activating mT function(s), including the formation of stable complexes with pp60c-src.     

Restriction of the in vitro and in vivo tyrosine protein kinase activities of pp60c-src relative to pp60v-src.

The tyrosine protein kinase activities of pp60c-src and pp60v-src were compared. The activities were qualitatively similar in vitro when the src proteins were bound in an immune complex with monoclonal antibody; both proteins utilized either ATP or GTP as phosphate donors, preferred Mn2+ to Mg2+, and had similar exogenous substrate specificities. The specific activity of pp60c-src was about 10-fold lower than that of pp60v-src for exogenous substrate phosphorylation but was only 1.1- to 2-fold lower than that of pp60v-src for autophosphorylation. Six glycolytic enzymes, including three not previously identified as substrates for pp60src phosphorylation, were phosphorylated by both pp60c-src and pp60v-src. Levels of pp60c-src fourfold higher than the amount of pp60v-src in src-plasmid-transformed cells did not detectably alter the level of phosphotyrosine in cellular proteins, but increasing the expression of pp60c-src another twofold (which induces cells to form foci in monolayer culture (P.J. Johnson, P.M. Coussens, A.V. Danko, and D. Shalloway, Mol. Cell. Biol. 5:1073-1083, 1985) resulted in a threefold increase in the level of cellular protein phosphotyrosine. Immunoprecipitation and analysis of the alkali-stable phosphoproteins by two-dimensional electrophoresis showed that, in contrast to pp60v-src-transformed cells, pp36 and enolase are only weakly phosphorylated in these high-level pp60c-src overexpresser cells. Even allowing for the in vitro differences in specific activities of phosphorylation, these results suggest that the pp60c-src tyrosine protein phosphorylating activity may be restricted relative to that of pp60v-src by additional in vivo mechanisms.     

Biological and biochemical properties of the c-src+ gene product overexpressed in chicken embryo fibroblasts.

The c-src protein isolated from neuronal cells (pp60c-src+) displays a higher level of protein kinase activity than does pp60c-src from nonneural tissues. There are two structural alterations present in the amino-terminal half of pp60c-src+ expressed in neurons which could contribute to the enhanced activity of this form of pp60c-src: (i) a hexapeptide insert located at amino acid 114 of avian pp60c-src+ and (ii) a novel site(s) of serine phosphorylation. We characterized pp60c-src+ expressed in a nonneuronal cell type to identify factors that regulate the activity of the c-src+ protein and the importance of the neuronal environment on this regulation. The c-src+ protein overexpressed in chicken embryo fibroblasts (CEFs) displayed higher kinase activity than did pp60c-src. The major sites of phosphorylation of the c-src+ protein were Ser-17 and Tyr-527. The unique site(s) of serine phosphorylation originally identified in pp60c-src+ expressed in neurons was not detected in the c-src+ protein overexpressed in CEFs. Therefore, the hexapeptide insert is sufficient to cause an elevation in the tyrosine protein kinase activity of pp60c-src+. Our data also indicate that CEFs infected with the Rous sarcoma virus (RSV)c-src+ display phenotypic changes that distinguish them from cultures producing pp60c-src and that pp60c-src+-expressing cells are better able to grow in an anchorage-independent manner. The level of total cellular tyrosine phosphorylation in RSVc-src+-infected cultures was moderately higher than the level observed in cultures infected with RSVc-src. This level was not as pronounced as that observed in cells infected with RSVv-src or oncogenic variants of RSVc-src. Thus, pp60c-src+ could be considered a partially activated c-src variant protein much like other c-src proteins that contain mutations in the amino-terminal domain.     

Regulation of the oncogenic activity of the cellular src protein requires the correct spacing between the kinase domain and the C-terminal phosphorylated tyrosine (Tyr-527).

Repression of the tyrosine kinase activity of the cellular src protein (pp60c-src) depends on the phosphorylation of a tyrosine residue (Tyr-527) near the carboxy terminus. Tyr-527 is located 11 residues C terminal from the genetically defined end of the kinase domain (Leu-516) and is therefore in a negative regulatory region. Because the precise sequence of amino acids surrounding Tyr-527 appears to be unimportant for regulation, we hypothesized that the conformational constraints induced by phosphorylated Tyr-527 may require the correct spacing between the kinase domain (Leu-516) and Tyr-527. In this report, we show that deletions at residue 518 of two, four, or seven amino acids or insertions at this residue of two or four amino acids activated the kinase activity and thus the transforming potential of pp60c-src. As is the case for the prototype transforming variant, pp60527F, activation caused by these deletions or insertions was abolished when Tyr-416 (the autophosphorylation site) was changed to phenylalanine. In comparison with wild-type pp60c-src, the src proteins containing the alterations at residue 518 showed a lower phosphorylation state at Tyr-527 regardless of whether residue 416 was a tyrosine or a phenylalanine. Mechanisms dealing with the importance of spacing between the kinase domain and Tyr-527 are discussed.     

Genistein differentially inhibits postreceptor effects of insulin in rat adipocytes without inhibiting the insulin receptor kinase.

Genistein, an isoflavone putative tyrosine kinase inhibitor, was used to investigate the coupling of insulin receptor tyrosine kinase activation to four metabolic effects of insulin in the isolated rat adipocyte. Genistein inhibited insulin-stimulated glucose oxidation in a concentration-dependent manner with an ID50 of 25 micrograms/ml and complete inhibition at 100 micrograms/ml. Genistein also prevented insulin's (10(-9) M) inhibition of isoproterenol-stimulated lipolysis with an ID50 of 15 micrograms/ml and a complete effect at 50 micrograms/ml. The effect of genistein (25 micrograms/ml) was not reversed by supraphysiological (10(-7) M) insulin levels. In contrast, genistein up to 100 micrograms/ml had no effect on insulin's (10(-9) M) stimulation of either pyruvate dehydrogenase or glycogen synthase activity. We determined whether genistein influenced insulin receptor beta-subunit autophosphorylation or tyrosine kinase substrate phosphorylation either in vivo or in vitro by anti-phosphotyrosine immunoblotting. Genistein at 100 micrograms/ml did not inhibit insulin's (10(-7) M) stimulation of insulin receptor tyrosine autophosphorylation or tyrosine phosphorylation of the cellular substrates pp185 and pp60. Also, genistein did not prevent insulin-stimulated autophosphorylation of partially purified human insulin receptors from NIH 3T3/HIR 3.5 cells or the phosphorylation of histones by the activated receptor tyrosine kinase. In control experiments using either NIH 3T3 fibroblasts or partially purified membranes from these cells, genistein did inhibit platelet-derived growth factor's stimulation of its receptor autophosphorylation. These findings indicate the following: (a) Genistein can inhibit certain responses to insulin without blocking insulin's stimulation of its receptor tyrosine autophosphorylation or of the receptor kinase substrate tyrosine phosphorylation. (b) In adipocytes genistein must block the stimulation of glucose oxidation and the antilipolytic effects of insulin at site(s) downstream from the insulin receptor tyrosine kinase. (c) The inhibitory effects of genistein on hormonal signal transduction cannot necessarily be attributed to inhibition of tyrosine kinase activity, unless specifically demonstrated.     

Activation of cyclic AMP-dependent protein kinases by vasoactive intestinal peptide (VIP) in isolated intestinal epithelial cells from rat.

VIP stimulates protein kinase activity in intestinal epithelial cells isolated from rat jejuno-ileum. The stimulation is time-dependent and is potentiated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. The response occurs in the 0.1–10 nM range of VIP concentrations, half-maximal stimulation being observed with 0.7 nM VIP. The VIP-induced protien kinase activation is thus observed at concentrations similar to those promoting the accumulation of cyclic AMP (11). Secretin also stimulates protien kinase activity but with a 100-times lower potency than VIP, in agreement with the fact that secretin is a VIP agonist of 100-times lower potency with respect to cyclic AMP increase. Prostaglandins E₁ and E₂ (10^?5 M), are also found to increase protein kinase activity.     

Gene regulation by tyrosine kinases: src protein activates various promoters, including c-fos.

A promoter of the nuclear proto-oncogene fos was activated by cotransfection with the viral src gene. Ability to transactivate the c-fos promoter was dependent on tyrosine kinase activity, because (i) src mutants which have reduced tyrosine kinase activity due to mutation of Tyr-416 to Phe showed lower promoter activation, (ii) pp60c-src mutants which have increased tyrosine kinase activity due to mutation of Tyr-527 to Phe also augmented c-fos promoter induction, and (iii) mutation in the ATP-binding site of pp60v-src strongly suppressed c-fos promoter activation. Tyrosine kinase activity alone, however, was not sufficient for promoter activation, because of pp60v-src mutant which lacked its myristylation site and consequently membrane association showed no increased c-fos promoter activation. Both the tyrosine kinase- and membrane-association-defective mutants were also unable to induce transformation. Therefore, phosphorylation of membrane-associated substrates appears to be required for both gene expression and cellular transformation by the src protein. Two regions of the c-fos promoter located between positions -362 and -324 and positions -323 and -294 were responsive to src stimulation. We believe that protein tyrosine phosphorylation represents an important step of signal transduction from the membrane to the nucleus.     

A protein tyrosine kinase involved in regulation of pp60c-src function

We recently identified a novel protein tyrosine kinase that specifically phosphorylates truncated pp60c-src (Mr = 53,000) at a tyrosine residue(s) distinct from its autophosphorylation site. In this study, we examined whether this enzyme phosphorylates intact pp60c-src (Mr = 60,000) and determined its phosphorylation site. Non-neuronal and neuronal forms of intact pp60c-src were separately purified from the membrane fraction of neonatal rat brain by sequential column chromatographies. The novel kinase phosphorylated tyrosine residues of both forms of intact pp60c-src. The phosphorylation occurred in parallel with autophosphorylation of pp60c-src, and in both forms the final stoichiometry estimated was quite similar to that of autophosphorylation (about 5%). The enzyme also phosphorylated pp60c-src in which the kinase activity had been destroyed by an ATP analogue, p-fluorosulfonylbenzoyl 5'-adenosine. The phosphorylation site of the non-neuronal form was analyzed by sequential peptide mapping with tosylphenylalanyl chloromethyl ketone-treated trypsin and alpha-chymotrypsin. Tryptic digestion of the phosphorylated pp60c-src yielded a unique phosphopeptide that cross-reacted with an antibody specific for the carboxyl-terminal sequence of chicken pp60c-src. Digestion of the phosphopeptide with chymotrypsin yielded a product that comigrated with a synthetic phosphopeptide corresponding to the carboxyl-terminal 15 residues of chicken pp60c-src. These results clearly indicate that the carboxyl-terminal sequence of rat pp60c-src is identical to that of chicken pp60c-src, and a tyrosine residue corresponding to chicken Tyr527 is the phosphorylation site. This phosphorylation resulted in a decrease in the enolase phosphorylating activity of pp60c-src. Kinetic experiments indicated that this decrease in activity was due to a decrease in the Vmax value of pp60c-src. These findings support our previous proposal that the novel tyrosine kinase acts as a specific regulator of pp60c-src in cells.     

Protein kinase C promotes the phosphorylation of immunoprecipitated middle T antigen from polyoma-transformed cells

Stimulation of protein kinase C in polyoma virus-transformed cells increased the phosphorylation of tyrosine residues of the viral middle T (mT) antigen in mT:pp60c-src complexes precipitated by anti-mT antibodies. This increase might have been due to a stimulation of the complex's pp60c-src tyrosine kinase activity or to an increased ability of the mT protein to be phosphorylated by pp60c-src. These observations suggest that cellular protein kinase C might control the ability of polyoma virus to transform its host cell.     

Tyrosine phosphorylation of a 50K cellular polypeptide associated with the Rous sarcoma virus transforming protein pp60src.

We have examined the phosphorylation of a 50,000-dalton cellular polypeptide associated with the Rous sarcoma virus (FSV) transforming protein pp60-src. It has been shown that pp60src forms a complex with two cellular polypeptides, an 89,000-dalton heat-shock protein (89K) and a 50,000-dalton phosphoprotein (50K). The pp60src-associated protein kinase activity phosphorylates at tyrosine residues, and the 50K polypeptide present in the complex contains phosphotyrosine and phosphoserine. These observations suggest that the 50K polypeptide may be a substrate for the protein kinase activity of pp60src. To examine this possibility, we isolated the 50K polypeptide by two-dimensional polyacrylamide gel electrophoresis from lysates of uninfected or virally infected cells. Tryptic phosphopeptide analysis indicated that the 50K polypeptide isolated by this method was the same polypeptide as that complexed to pp60src. In uninfected cells or cells infected by a transformation-defective mutant, the 50K polypeptide contained phosphoserine but little or no phosphotyrosine. In cells infected by Schmidt-Ruppin or Prague RSV, there was a 40- to 50-fold increase in the quantity of phosphotyrosine in the 50K protein. Thus, the phosphorylation of the 50K polypeptide at tyrosine is dependent on the presence of pp60src. However, the 50K polypeptide isolated from cells infected by temperature-sensitive mutants of RSV was found to be phosphorylated at tyrosine at both permissive and nonpermissive temperatures; this behavior is different from that of other substrates or putative substrates of the pp60src kinase activity. It is possible that the 50K polypeptide is a high-affinity substrate of pp60src.     

Abscisic Acid Induces Mitogen-Activated Protein Kinase Activation in Barley Aleurone Protoplasts

Abscisic acid (ABA) induces a rapid and transient mitogen-activated protein (MAP) kinase activation in barley aleurone protoplasts. MAP kinase activity, measured as myelin basic protein phosphorylation by MAP kinase immunoprecipitates, increased after 1 min, peaked after 3 min, and decreased to basal levels after ~5 min of ABA treatment in vivo. Antibodies recognizing phosphorylated tyrosine residues precipitate with myelin basic protein kinase activity that has identical ABA activation characteristics and demonstrate that tyrosine phosphorylation of MAP kinase occurs during activation. The half-maximal concentration of ABA required for MAP kinase activation, 3 x 10-7 M, is very similar to that required for ABA-induced rab16 gene expression. The tyrosine phosphatase inhibitor phenylarsine oxide can completely block ABA-induced MAP kinase activation and rab16 gene expression. These results lead us to conclude that ABA activates MAP kinase via a tyrosine phosphatase and that these steps are a prerequisite for ABA induction of rab16 gene expression.     

Enzymatically inactive p60c-src mutant with altered ATP-binding site is fully phosphorylated in its carboxy-terminal regulatory region

Cellular src protein, p60c-src, is phosphorylated on tyrosine 527 in chicken embryo fibroblasts, and this phosphorylation is implicated in suppressing the protein-tyrosine kinase activity and transforming potential of p60c-src. To determine whether tyrosine 527 phosphorylation is dependent on p60c-src kinase activity, the ATP-binding site of chicken p60c-src was destroyed by substitution of lysine 295 with methionine. The resultant protein, p60c-src(M295), expressed either in chicken cells or in yeast, lacked detectable kinase activity. Nevertheless, tyrosine and serine phosphorylation of p60c-src(M295) overproduced in chicken cells were indistinguishable from that of authentic p60c-src. By contrast, p60c-src(M295) was not phosphorylated on tyrosine in yeast. These results suggest that a protein kinase present in chicken cells but not in yeast phosphorylates tyrosine 527 in trans, and are consistent with the possibility that this kinase is distinct from p60c-src.