Phosphorylation of pp60)c-s at Tyr-527, six residues from the carboxy terminus, has been implicated in regulation of the protein-tyrosine kinase activity of pp6OCSF. Here we show that dephosphorylation of pp60C Src by phosphatase treatment in vitro caused a 10-to 20-fold increase in pp60CSc protein-tyrosine kinase activity. Binding of specific antibody to the region of pp60cslc which contains phosphotyrosine-527 also increased kinase activity. Each treatment increased phosphorylation of added substrates and of Tyr-416 within pp6OCSrC by a similar mechanism that involved altered interactions with ATP and increased catalytic rate. We suggest that the phosphorylated carboxy terminus acts as an inhibitor of the protein kinase domain of pp6CSrC, unless its conformation is altered by either dephosphorylation or antibody binding. The antibody additionally stimulated the phosphorylation of forms of pp6OCsrC that had reduced gel mobility, much like those phosphorylated in kinase reactions containing pp6(csC activated by polyomavirus medium tumor antigen. These in vitro experiments provide models for the activation of pp60c c in cells transformed by polyomavirus. We also show that autophosphorylation of pp6()cSC at Tyr-527 occurs only to a very limited extent in vitro, even when Tyr-527 is made available for phosphorylation by treatment with phosphatase. This suggests that other protein-tyrosine kinases may normally phosphorylate Tyr-527 and regulate pp604>5r in the cell.Most vertebrate cells express a number of different protein-tyrosine kinases (26). The activities of some of these, the growth factor receptors, are regulated by binding of messengers from the extracellular environment. The protooncogene product pp6O-src is one of several other proteintyrosine kinases for which regulatory signals have yet to be described. In fibroblasts, pp60-csrc apparently has low activity and contributes little to the aggregate activity of cellular protein-tyrosine kinases (18,27,41). pp60c-src has the potential for much higher specific activity, however. This potential is realized in fibroblasts transformed by polyomavirus. The principal polyomavirus transforming protein, medium T antigen (mT), forms a complex with pp60csrc (16, 17). The specific activity of mT-associated pp6Ocsrc is increased more than 10-fold when assayed in vitro with exogenous substrates (3,14). This activation is probably essential for malignant transformation by polyomavirus, since all mT mutants tested that transform cells also activate pp6c-src.The specific kinase activity of pp6Oc-src may also be increased by mutation. The related viral transforming protein pp60v-src has 20 to 50 times the specific kinase activity of pp60c-src when assayed with model substrates in vitro (18,27). This activity difference is also evident in intact fibroblasts, in which the expression of small quantities of pp60v-src leads to a large increase in the overall level of phosphorylation of cell proteins at tyrosine (41).Studies with mutant v-src's suggest that the high kinase activit...

The lck proto-oncogene encodes a lymphocyte-specific member of the src family of protein tyrosine kinases. Here we demonstrate that pp56kk is phosphorylated in vivo at a carboxy-terminal tyrosine residue analogous to Tyr-527 of pp60`csrc. Substitution of phenylalanine for tyrosine at this position resulted in increased phosphorylation of a second tyrosine residue (Tyr-394) and was associated with an increase in apparent kinase activity. In addition, this single point mutation unmasked the oncogenic potential of pp56kk in NIH 3T3 cell transformation assays. Viewed in the context of similar results obtained with pp60`csrc, it is likely that the enzymatic activity and transforming ability of all src-family protein tyrosine kinases can be regulated by carboxy-terminal tyrosine phosphorylation. We further demonstrate that overexpression of pp56kk in the murine T-cell lymphoma LSTRA as a result of a retroviral insertion event produces a kinase protein that despite wild-type primary structure is nevertheless hypophosphorylated at Tyr-505. Thus, control of normal growth in this lymphoid cell line may have been abrogated through acquistion of a posttranslationally activated version of pps6IckThe src gene family includes seven closely related sequences (fgr, fyn, hck, lck, lyn, src, and yes), all of which diverged from a common evolutionary precursor before the mammalian radiation (25,26,32,34,38,41,43,46,48,49). Each of these genes has the potential to encode a membraneassociated protein tyrosine kinase composed of a specialized amino-terminal domain of about 70 residues that is unique to each src family member joined to a common region containing a prototypical kinase domain. Unlike the related growth factor receptor kinases, which demonstrate ligand-induced increases in kinase activity, physiologic activators of the src-like kinases have not been identified. Thus, the normal function of each of these molecules is obscure. Nevertheless, considerable evidence suggests that the src family kinases can participate in the regulation of cell growth. Three of these kinases (fgr, src, and yes) were originally identified in mutant forms as retroviral oncogenes capable of inducing cell transformation (reviewed in reference 2). Similarly, the Ick gene is rearranged and overexpressed in some murine lymphomas (32,46).Comparison of the deduced structures of the src-like protein tyrosine kinases permits some inferences regarding their physiology. Each of the src family kinases contains an analogous tyrosine residue in a conserved carboxy-terminal sequence context. In pp60c-src this tyrosine (Tyr-527) is phosphorylated in vivo at high stoichiometry (10). Activated forms of pp6Osrc are either hypophosphorylated at Tyr-527, for example, when pp6Oc-src associates with polyomavirus middle T antigen, or lack Tyr-527 altogether, as for pp6v-src (6; reviewed in reference 19). In addition, removal of the phosphate from Tyr-527 in pp60c-rc increases its kinase activity in vitro (12, 13), and mutation of Tyr-527 to phenylalanine increases the k...

Infection of A431 cells with vaccinia virus, or exposure to a mitogenic polypeptide secreted by vaccinia virus-infected cells, induces tyrosine phosphorylation of epidermal growth factor receptors.

K-252a, a protein kinase inhibitor isolated from the culture broth of Nocardiopsis sp., inhibits the nerve growth factor (NGF)-stimulated phosphorylation of microtubule-associated protein 2 (MAP2) and Kemptide (synthetic Leu-Arg-Arg-Ala-Ser-Leu-Gly) by blocking the activation of two independent kinases in PC12 cells: MAP2/pp250 kinase and Kemptide kinase. The NGF-stimulated activation of these kinases is inhibited in a dose-dependent manner following treatment of the cells with K-252a. Although these kinases also are activated by epidermal growth factor (EGF) and 12-O-tetradecanoyl-phorbol 13-acetate, K-252a has no inhibitory effect when these agents are used. Half-maximal inhibition of the activation of both kinases was observed at 10-30 nM K-252a. K-252a was shown to directly inhibit the activity of MAP2/pp250 kinase and Kemptide kinase when added to the phosphorylation reaction mixture in vitro; however, half-maximal inhibition under these conditions was observed at greater than or equal to 50 nM K-252a. These data suggest that K-252a exerts its effects at a step early in the cascade of events following NGF binding. The effects of K-252a are similar to those reported for 5'-S-methyladenosine (MTA) and other methyltransferase inhibitors. Treatment of PC12 cells with MTA inhibited NGF-, but not EGF-mediated activation of MAP2/pp250-kinase (Ki greater than 500 microM). MTA, when added to the phosphorylation reaction mixture in vitro, directly inhibited kinase activity (Ki = 50 microM), suggesting that the effects of MTA may be the result of its action on protein kinases rather than methyltransferases.

p56kk and p60c-src are closely related protein-tyrosine kinases that are activated by similar oncogenic mutations. We have used fibroblast cell lines that express p56ick from introduced DNA molecules to compare the subcellular localizations of p60c-src and p561ck and their abilities to bind polyomavirus middle T antigen (mT). p561ck is associated with the detergent-insoluble matrix, as defined by extraction with solutions containing nonionic detergents, whereas p60csrc is soluble under these conditions. p56lck is also associated with detergent-insoluble structures in a lymphoid cell line, LSTRA. p60csrC binds to mT, but P56lck does not bind detectably. In terms of both solubility and mT interactions, the nononcogenic p56lck more closely resembles oncogenically activated p60csrc mutants than it resembles p60c-src. Because published results show that an intact carboxy terminus is required for p60CsrC to bind mT and be soluble, we tested whether the different localization and mT binding properties of p561ck and p6C-srC were dictated by their different carboxy termini. A protein consisting largely of p6oc-srC sequences but carrying a p561ck carboxy terminus was soluble and bound to mT. We suggest that both the solubility and mT-binding properties of p60'src not only require sequences common to the carboxy termini of p60csrc and p56 ck, but also require sequences unique to the body of p60csrc. p561ck and p60c-src are homologous membrane-associated protein-tyrosine kinases (J. A. Cooper, in B. Kemp and P. F. Alewood, ed., Peptides and Protein Phosphorylation, in press). Both proteins have tyrosines near their carboxy termini that are normally phosphorylated (12, 37). These phosphorylations correlate with suppression of the kinase activities and transforming abilities of p60csrc and p56Ick (13,