The Sites of Phosphorylation by Protein Kinase C and an Intact SH2 Domain Are Required for the Enhanced Response to β-Adrenergic Agonists in Cells Overexpressing c-src

Abstract

Previously we demonstrated that C3H10T½ murine fibroblasts overexpressing avian c-src exhibit elevated levels of cyclic AMP (cAMP) in response to β-adrenergic agonists compared with that in control cells and that this enhanced response requires c-src kinase activity (W. A. Bushman, L. K. Wilson, D. K. Luttrell, J. S. Moyers, and S. J. Parsons, Proc. Natl. Acad. Sci. USA 87:7462-7466, 1990). However, it is not yet known which components of the β-adrenergic receptor pathway, if any, interact with pp60^c-src. It has recently been shown that immune complexes of pp60^c-src phosphorylate recombinant G_α proteins in vitro to stoichiometric levels, resulting in alterations of GTP binding and GTPase activity (W. P. Hausdorff, J. A. Pitcher, D. K. Luttrell, M. E. Linder, H. Kurose, S. J. Parsons, M. G. Caron, and R. J. Lefkowitz, Proc. Natl. Acad. Sci. USA 89:5720-5724, 1992), raising the possibility that the G_sα protein may be an in vivo target for the interaction with pp60^c-src. To further characterize the involvement of pp60^c-src in the β-adrenergic signalling pathway, we have overexpressed, in 10T½ cells, pp60^c-src containing mutations in several domains which are 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 pp60^c-src are required for the enhanced response of c-src overexpressors to β-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 pp60^c-src to potentiate β-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-src 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 pp60^c-src 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^c-src with components of the β-adrenergic pathway. Our data also support the possibility that the G_sα protein may be an in vivo target for alteration by pp60^c-src.