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J Biol Chem. 2001 Jan 19;276(3):1911-5. Epub 2000 Oct 19.

Phosphorylation of GRK2 by protein kinase C abolishes its inhibition by calmodulin.

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  • 1Institut für Pharmakologie und Toxikologie, Versbacher Str. 9, D-97078 Würzburg, Germany. krasel@wpxx02.toxi.uni-weirzburg.de

Abstract

G-protein-coupled receptor kinases (GRKs) are important regulators of G-protein-coupled receptor function. Two members of this family L, GRK2 and GRK5 L, have been shown to be substrates for protein kinase C (PKC). Whereas PKC-mediated phosphorylation results in inhibition of GRK5, it increases the activity of GRK2 toward its substrates probably through increased affinity for receptor-containing membranes. We show here that this increase in activity may be caused by relieving a tonic inhibition of GRK2 by calmodulin. In vitro, GRK2 was preferentially phosphorylated by PKC isoforms alpha, gamma, and delta. Two-dimensional peptide mapping of PKCalpha-phosphorylated GRK2 showed a single site of phosphorylation, which was identified as serine 29 by HPLC-MS. A S29A mutant of GRK2 was not phosphorylated by PKC in vitro and showed no phorbol ester-stimulated phosphorylation when transfected into human embryonic kidney (HEK)293 cells. Serine 29 is located in the calmodulin-binding region of GRK2, and binding of calmodulin to GRK2 results in inhibition of kinase activity. This inhibition was almost completely abolished in vitro when GRK2 was phosphorylated by PKC. These data suggest that calmodulin may be an inhibitor of GRK2 whose effects can be abolished with PKC-mediated phosphorylation of GRK2.

PMID:
11042191
[PubMed - indexed for MEDLINE]
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