Recombinant protein kinase C‐γ phorbol binding domain upon microinjection blocked insulin‐induced maturation of Xenopus leavis oocytes

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Abstract
The second cysteine‐rich (Cys‐2) domain of rat brain PKC‐γ 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 0.45×10−12 mol injected Cys‐2. These results demonstrate that the PKC‐γ 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.