Platelet-derived growth factor induces multisite phosphorylation of pp60c-src and increases its protein-tyrosine kinase activity.

Abstract

We have shown previously that pp69>-sc is a substrate for protein kinase C in vitro and in vivo and that the target of protein kinase C phosphorylation in mammalian pp6OcSrc is serine 12. We now demonstrate that in addition to tumor promoters, all activators of phosphatidylinositol turnover that we have tested in fibroblasts (platelet-derived growth factor, fibroblast growth factor, serum, vasopressin, sodium orthovanadate, and prostaglandin F2.) lead to the phosphorylation of pp60c-sr at serine 12. In addition to stimulating serine 12 phosphorylation in pp6Oc-"s, platelet-derived growth factor treatment of quiescent fibroblasts induces phosphorylation of one or two additional serine residues and one tyrosine residue within the N-terminal 16 kilodaltons of the enzyme and activates its immune complex protein-tyrosine kinase activity.pp6f-src is a membrane-associated phosphoprotein with intrinsic protein-tyrosine kinase activity (for a review, see reference 32). It can be activated to transform cells by C-terminal truncation or various point mutations (6,35,36,40,49,56). pp60c,src is expressed ubiquitously in animal cells, in which its transforming potential is normally suppressed and its specific tyrosine protein kinase activity is lower than that of its transforming counterparts (24,34,45). The highest levels of pp6csrc have been found in the brain (21), platelets (28), peripheral blood lymphocytes (28), and adrenal medullary chromaffin cells (46). Although this distribution has suggested that pp60c-src functions in development, maintenance of a differentiated state, or secretion, its role in normal cellular physiology and its mode of regulation are not understood.We are interested in the role of phosphorylation in the regulation of pp6c-src function. Under most conditions, serine 17 (S17) is the major site of serine phosphorylation in pp60c-src (48, 61). pp60csrc is also phosphorylated in cells to near stoichiometry on a tyrosine near its C terminus, tyrosine 527 (Y527) in chicken pp60csrc (16). Phosphorylation of Y527 constrains the protein kinase activity of the molecule (18,22), and deletion or mutation of this residue results in a protein with transforming ability and higher protein kinase activity (6,36,49,56). Another tyrosine residue within pp60c-src, tyrosine 416 (Y416), is the major site of autophosphorylation in vitro (47, 61). In vivo, Y416 is phosphorylated when Y527 has been mutated (as in Y527 to phenylalanine 527) or deleted (as in pp60vsrc) (6,36,49,56). If it is assumed that pp60c-src is active as a protein kinase under certain circumstances in normal cells, a mechanism(s) must exist for activating and subsequently inhibiting its activity. Such regulation could be achieved by the transient dephosphorylation of Y527, but to date, there have been no reports that this occurs in nontransformed cells.We (29) and others (26, 62) have shown that pp60c src is phosphorylated rapidly and stoichiometrically at a normally unphosphorylated serine(s) when cells are treated with tumor promoters that bind and ...

Expression and regulation of osteopontin in type 1 diabetes

“…Pancreata or islets were lysed in modified RIPA lysis buffer, 37 and the protein concentrations in the supernatant were determined using the BCA protein assay reagent (Pierce; Rockford, IL). Equal protein concentrations (50 µg) were denatured in a gel loading buffer at 85 o C for 5 minutes and then loaded onto 10 % SDS-polyacrylamide slab gels and transferred to polyvinylidene difluoride membranes and incubated at 4 o C overnight with primary mouse monoclonal osteopontin antibody, 1:150 diluted in PBST (Santa Cruz Biotechnology, Santa Cruz, CA).…”

Mutation of amino acids in pp60c-src that are phosphorylated by protein kinases C and A.

“…Since phosphorylation of Thr-654 by PKC suppresses epidermal growth factor receptor activity (8,16), it is plausible that Ser-12 phosphorylation regulates pp60c-'r( activity. Biochemical studies have detected no change in pp60c"`in vitro kinase activity after either in vitro or in vivo phosphorylation at Ser-12 (10)(11)(12)29); however, these assays would not be sensitive to in vivo effects that involve interactions between pp6Oc"rc and other proteins. Genetic studies to detect such effects have not yet been reported.…”

“…An attractive hypothesis is that the multiple phosphorylation sites in the amino region may govern pp60 C-N activity as part of an interrelated phosphorylation-dephosphorylation network that regulates cellular growth, differentiation, or both. For example, novel mitosis-specific phosphorylations at amino-region threonine and serine residues (4) and at an amino-region tyrosine after treatment with platelet-derived growth factor (11,30) are separately correlated with increased pp60c-'' specific kinase activity. pp60c%I( is also phosphorylated in the amino region by cyclic AMP-dependent protein kinase A (PKA) (6,26) and by the calciumphospholipid-dependent protein kinase, protein kinase C (PKC) (10)(11)(12)39).…”

“…For example, novel mitosis-specific phosphorylations at amino-region threonine and serine residues (4) and at an amino-region tyrosine after treatment with platelet-derived growth factor (11,30) are separately correlated with increased pp60c-'' specific kinase activity. pp60c%I( is also phosphorylated in the amino region by cyclic AMP-dependent protein kinase A (PKA) (6,26) and by the calciumphospholipid-dependent protein kinase, protein kinase C (PKC) (10)(11)(12)39). Both kinases participate in intracellular signaling (reviewed in references 9 and 24), but it is not known whether their phosphorylations of pp60O'-N are functionally important.…”

“…PKC phosphorylates Ser-12 and Ser-48 in chicken pp6O`cs" (whether expressed in chicken or mouse fibroblasts) and Ser-12 in human platelet pp6Oc%-r after stimulation with 12-O-tetradecanoyl phorbol acetate (TPA) (10)(11)(12). The neighboring basic amino acids, which are thought to be important for PKC substrate specificity (40), and the spatial relationship of Ser-12 to the plasma membrane are similar to those of Thr-654 in the epidermal growth factor receptor (12).…”