Enzymatic characteristics of pp60^v‐src isolated from vanadium‐treated transformed cells

Abstract

The transforming protein of Rous sarcoma virus (RSV) typically appears as a single phosphorylated polypeptide designated pp60v-src. pp60v-src possesses a protein kinase activity specific for tyrosine residues on select protein substrates. Treatment of RSV-transformed cells with vanadium ions resulted in the appearance of an electrophoretic variant of pp60v-src and was paralleled by a significant increase in the src kinase specific activity in purified enzyme preparations. Both the normal (standard) src kinase and the src kinase preparations obtained from vanadium-treated cells exhibited similar optimal activity profiles for MgCl2, KCl, and pH. Furthermore, their site specificities of phosphorylation of the substrates casein and vinculin were the same. The reaction kinetic profile of the standard src kinase showed a nonlinear pattern, while the vanadium enzyme exhibited conventional linear Michaelis-Menten kinetics. These results are discussed with respect to the possible functional regulation of pp60v-src activity by a vanadium-sensitive protein phosphatase activity.

Vanadium oxoanions and cAMP-dependent protein kinase: an anti-substrate inhibitor

“…Vanadate has been shown to promote the formation of elevated levels of phosphoproteins, specifically those proteins that are phosphorylated on tyrosine residues [1][2][3][4][5][6]. This has been ascribed to activation of protein kinase signal transduction pathways and\or inhibition of phosphotyrosine phosphatases [7].…”

“…Vanadate has been reported to promote the formation of elevated levels of proteins phosphorylated on tyrosine residues [1][2][3][4][5][6]. This may be a consequence of protein phosphatase inhibition [7] and\or protein kinase stimulation [8].…”

Forms of pp60v-src isolated from Rous sarcoma virus-transformed cells

“…Protein kinase reactions. Analytic protein kinase reactions were carried out at 22°C for 30 min in a total volume of 30 ,ul containing 10 mM Tris hydrochloride (pH 8.0), 5 mM MgCl2, 1 ,uM [a-32P]ATP (600 Ci/mmol), and 0.02 to 0.06 pmol of pp6Ov-src polypeptide (7,8). When included, casein (Sigma Chemical Co., St. Louis, Mo.)…”

“…When included, casein (Sigma Chemical Co., St. Louis, Mo.) was present at 300 ,ug/ml, vinculin (purified from chicken gizzards [16]) at 70 p.g/ml supplemented with phosphatidylinositol (Sigma) at 10 jig per reaction mixture (7,8,22), the src kinase cellular protein substrate pp36 (purified as previously described [33] at 70 jig/ml, enolase (Sigma) at 150 p.gIml, and [Val5]-angiotensin II (Sigma) at 600 p.g/ml. Reactions were terminated by addition of 30 p.l of 2x SDS-sample buffer and boiled for 1 min, except for the angiotensin reactions, which were terminated by addition of EDTA to 20 mM.…”

“…In our effort to contribute information toward an understanding of the workings of pp6Ov-src, we have addressed aspects concerning the control and regulation of the function of this transforming protein. Specifically, we have focused on the consequences of tyrosine phosphorylation of the pp60v-src polypeptide on src tyrosyl protein kinase enzymatic activity (7,8,12,33). Indeed, so have numerous other investigators.…”