Phosphorylation of GΖα by protein kinase C blocks interaction with the βγ complex

Abstract

GΖα is a G protein α subunit with biochemical properties that distinguish it from other members of the G protein α subunit family. One such property is its ability to be stoichiometrically phosphorylated by protein kinase C (PKC), both in vitro and in intact cells. The site of this phosphorylation has been mapped to a region near the N terminus of GΖα, but no functional significance of the modification has been established. To investigate this question, we have developed a baculovirus/Sf9 cell expression system to produce GΖα. The protein purified from Sf9 cells is functional as assessed by its ability both to bind guanine nucleotide in a Mg2+-sensitive fashion and to serve as a substrate for phosphorylation by PKC. Furthermore, addition of the G protein βγ complex purified from bovine brain inhibits phosphorylation of GΖα in a dose-dependent manner. Conversely, phosphorylation of GΖα inhibits its ability to interact with βγ subunits. These results establish a functional consequence for PKC-catalyzed phosphorylation of GΖα and suggest a mechanism for regulation of signaling through GΖ by preventing reassociation of its subunits.