Dephosphorylation or antibody binding to the carboxy terminus stimulates pp60c-src.

Abstract

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...

Alterations in pp60c-src accompany differentiation of neurons from rat embryo striatum.

“…The protein kinase activity of fibroblast pp6Ocsrc is regulated in a negative fashion by phosphorylation of Tyr-527 on pp60'csrc (20,23). This may explain why pp6Oc-src, which is associated with polyomavirus middle tumor antigen and lacks phosphate on Tyr-527, has increased protein kinase activity (7,10,11,23,25).…”

A mitotic role for a novel fission yeast protein kinase dsk1 with cell cycle stage dependent phosphorylation and localization.

“…The amount of soluble protein used for immunoprecipitation was 6 mg. Immunoprecipitation was done as described in Harlow et al (1988) with modifications appropriate for the extracts of S. pombe (Shiozaki and Yanagida, unpublished observations). To remove phosphate, immunoprecipitates was treated with potato acid phosphatase (PAP; grade II, Boehringer, Mannheim, Germany) with the procedures of Cooper and King (1986). PAP was purified by a MonoQ column (Pharmacia, Uppsala, Sweden) before use.…”

Phosphorylation‐mediated changes in the electrophoretic mobility of CD5 molecules

“…Once washed, immunoprecipitates were digested for 1 h at 37 "C with neuraminidase type VIII (0.2 U/sample) or PAP (30 pg/sample) in the appropriate buffers containing a protease inhibitor (1 mM phenylmethylsulfonylfluoride), as previously described [24,25]. Following incubation, immunoprecipitates were washed again in lysis buffer, eluted and subjected to SDSjPAGE analysis.…”