Recombinant protein kinase C-gamma phorbol binding domain upon microinjection blocked insulin-induced maturation of Xenopus laevis oocytes

Abstract

The second cysteine-rich (Cys-2) domain of rat brain PKC-gamma regulatory region C1 (92-173) was expressed in Escherichia coli cells and purified. NMR studies of Cys-2 protein identified the phorbol and other phospholipid binding sites within this molecule (Xu, R.X., Pawelczyk, T., Xia, T-H. and Brown, S.T. (1997) Biochemistry 37, 10709-10717). Here, we tested the ability of this domain to bind other proteins. Using an overlay assay we show that the Cys-2 domain binds other proteins in Xenopus oocyte soluble fraction. Unlike the kinase activity, binding of Cys-2 to other proteins was detected in the absence of added phospholipids. Microinjection of Cys-2 protein into Xenopus leavis oocytes inhibited insulin-induced but not progesterone-induced maturation. The smallest dose that enhanced insulin-induced maturation was 0.45 x 10(-12) mol injected Cys-2. These results demonstrate that the PKC-gamma Cys-2 domain beside being the binding site for phorbol ester/DAG and phosphatidylserine binds also other proteins. The proteins that interact with Cys-2 domain of PKC are essential for insulin-induced maturation program in oocytes.