Myristylation is required for Tyr-527 dephosphorylation and activation of pp60c-src in mitosis.

Abstract

The chicken proto-oncoprotein c-Src is phosphorylated by p34cdc2 during mitosis concomitant with increased c-Src tyrosine kinase activity. On the basis of indirect evidence, we previously suggested that this is caused by partial dephosphorylation at Tyr-527, the phosphorylation of which suppresses c-Src kinase activity. In support of this hypothesis, we now show that treatment of cells with a protein tyrosine phosphatase inhibitor, sodium vanadate, blocks the mitotic increase in Src kinase activity. Also, we show that an amino-terminal mutation that prevents myristylation (and membrane localization) of c-Src does not interfere with the p34cdc2-mediated phosphorylations but blocks both mitotic dephosphorylation of Tyr-527 (in kinase-defective Src) and stimulation of c-Src kinase activity. Furthermore, in unsynchronized cells, the kinase activity of nonmyristylated c-Src is suppressed by 60% relative to wild-type c-Src, presumably because of increased Tyr-527 phosphorylation. Consistent with this, the Tyr-527 dephosphorylation rate measured in cell homogenates is much higher for wild-type, myristylated c-Src than for nonmyristylated c-Src. Tyr-527 phosphatase activity was primarily associated with the nonsoluble subcellular fraction. These findings suggest that the phosphatase(s) that acts on Tyr-527 is membrane bound and indicate that membrane localization of c-Src is necessary for its mitotic activation by dephosphorylation of Tyr-527.Chicken c-src is the cellular homolog of the Rous sarcoma virus transforming gene v-src. Both genes encode membrane-associated 60-kDa phosphoproteins with tyrosine kinase activity (25). Association of v-Src and c-Src with cellular membranes requires covalent attachment of myristate to Gly at position 2, and mutation of this residue prevents myristylation and membrane binding (3, 12,31,33,34). Other amino-terminal sequence determinants are also involved in membrane association (18), and at least for v-Src, association may involve a 32-kDa Src receptor protein (29, 30). Myristylation is not required for the mitogenic effect of v-Src (4) but is required for transformation of chicken embryo fibroblasts by v-Src (10, 17) and activated c-Src (31, 41) (although our preliminary results suggest that nonmyristylated, activated c-Src is weakly transforming in mouse fibroblasts).During mitosis, c-Src is phosphorylated by p34cdc2 or a related kinase at three amino-terminal serine and threonine residues, and c-Src kinase activity is increased (6,22,35,38,40). These mitosis-specific phosphorylations (MSPs) retard c-Src electrophoretic mobility but, by themselves, do not stimulate its kinase activity (22, 38). C-Src kinase activity can be negatively regulated by phosphorylation of its C-terminal Tyr-527 residue (5, 20, 28), and we (1, 37) and others (16) have proposed that a 10 to 20% decrease in Tyr-527 phosphorylation is the direct cause of its mitotic activation. Although we cannot reliably directly detect such a small decrease, the kinase activities of c-Src mutants lacking Tyr-527 are not fu...

Protein myristoylation in health and disease

“…Src is activated by autophosphorylation at Tyr416 and by dephosphorylation of Tyr527, which when phosphorylated causes an inter-domain interaction that folds the protein into a closed, inactive state [127]. A non-myristoylated mutant has a decreased rate of phosphorylation and suppressed kinase activity, and so is not as efficient at inducing events in proliferation [128]. The accessibility of Src to regulatory phosphatases and kinases may depend on subcellular localisation and hence on myristoylation.…”

Selected glimpses into the activation and function of Src kinase

“…During mitosis, phosphorylations within the unique domain of c-Src at Thr34, Thr46, and Ser72 (Shenoy et al, 1989) appear to destabilize the SH2/Tyr(527/530) interactions, thereby sensitizing c-Src to the action of a PTP capable of dephosphorylating Tyr(527/530) (Bagrodia et al, 1991(Bagrodia et al, , 1994Shenoy et al, 1992;Stover et al, 1994), which leads to c-Src activation. c-Src is also a target of the activated PDGF receptor and becomes phosphorylated within its SH3 domain at Y138 (Broome and Hunter, 1997) and within its SH2 domain at Y213 (Stover et al, 1996).…”

Adhesion signaling by a novel mitotic substrate of src kinases

“…Src kinase activity is increased during mitosis in association with amino-terminal serine and threonine phosphorylations as well as partial dephosphorylation of tyrosine 527 by RPTPalpha (Chackalaparampil and Shalloway, 1988;Bagrodia et al, 1991;Park and Cartwright, 1995;Mustelin and Hunter, 2002). Dephosphorylation of tyrosine 527 leads to the release of intramolecular interactions and increased accessibility of the SH2 and SH3 domains for interaction with proteins specifically during mitosis (Bagrodia et al, 1994). Only one mitotic substrate of src, Sam68, has been identified so far (Fumagalli et al, 1994;Taylor et al, 1995).…”