Аннотация
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.
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