The sites of phosphorylation by protein kinase C and an intact SH2 domain are required for the enhanced response to beta-adrenergic agonists in cells overexpressing c-src.

Abstract

Previously we demonstrated that C3H1OT½ murine fibroblasts overexpressing avian c-src exhibit elevated levels of cyclic AMP (cAMP) in response to 13-adrenergic believed to be important for signalling processes. In this study we show that the sites of phosphorylation by protein kinase C (PKC) (Ser-12 and Ser-48) as well as the SH2 region of pp60C are required for the enhanced response of c-src overexpressors to 13-agonist stimulation. Mutation at the site of myristylation (Gly-2) results in a decrease in the enhanced response, while mutation at the site of phosphorylation by cAMP-dependent protein kinase (Ser-17) has no effect. Two-dimensional phosphotryptic analyses indicate that phosphorylation on Ser-12 and Ser-48 in unstimulated cells is associated with the ability of overexpressed pp60csrc to potentiate 13-adrenergic signalling. Cells overexpressing wild-type c-src also exhibit enhanced cAMP accumulation upon treatment with cholera toxin, an effect that is abated in cells overexpressing pp60`c<c defective in the kinase or SH2 domains or altered at the sites of phosphorylation by PKC. These studies provide the first evidence for the physiological significance of the pp60C`rC sites of PKC phosphorylation. In addition, they show that the SH2, Ser-12/48, and myristylation regions may be important for efficient interaction of pp60`crc with components of the 13-adrenergic pathway. Our data also support the possibility that the Gs. protein may be an in vivo target for alteration by pp60Oc.

RACK1: a novel substrate for the Src protein-tyrosine kinase

“…We used three Src mutants: (1) Y527F c-Src (constitutively-active) (Cartwright et al, 1987); (2) K297M Src (kinase-inactive) (Snyder et al, 1985); and (3) dl155 Src (contains a three amino acid deletion in the phosphotyrosine binding pocket of the SH2 domain) (Moyers et al, 1993). Previously, we showed that the dl155 Src mutant has reduced binding to RACK1 (Chang et al, 2001).…”

“…Plasmids encoding wild-type (pM5H) or mutant dl155-157 (pM155) chicken c-src were gifts from Sarah Parsons (University of Virginia, Charlottesville, VA, USA; Moyers et al, 1993). pGEM K297M src was a gift from Tony Hunter (The Salk Institute for Biological Studies, La Jolla, CA, USA; Snyder et al, 1985).…”

Reactive Oxygen Species in Inflammation and Tissue Injury

“…The PKC activity is regulated by its translocation to the membrane and phosphorylation and generally denotes enzyme activation (334). PKC is the target for multiple tyrosine phosphorylations by various tyrosine kinases, including Src family kinases (304,396). These modifications further increase kinase activity of PKC (243).…”

Not so strange bedfellows: G-protein-coupled receptors and Src family kinases

“…Early studies with murine fibroblasts stably overexpressing wildtype avian c-Src showed that stimulation of the b adrenergic agonist isoproterenol resulted in a threefold increase in cAMP levels relative to that induced in nontransfected cells or cells expressing catalytically inactive Src (Bushman et al, 1990. The enhanced response to b-agonists requires both an intact Src SH2 domain and the presence of Ser-12 and Ser-48, sites of PKC phosphorylation in the kinase (Moyers et al, 1993). In vitro analysis suggests that Src affects G-protein regulation of adenylyl cyclase at two points, a stimulatory effect at the level of G-protein activation, and a modest inhibitory effect at the level of adenylyl cyclase itself.…”