Recombinant protein kinase C-gamma phorbol binding domain upon microinjection blocked insulin-induced maturation of Xenopus laevis oocytes.
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.
Pawelczyk
T
T
Department of Clinical Biochemistry, Medical University of Gdansk, Poland. tkpaw@amedec.amg.gda.pl
Matecki
A
A
Dettlaff
A
A
D000818:Animals; D001665:Binding Sites; D007328:Insulin / Q000494:pharmacology*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D008845:Microinjections; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D010703:Phorbol Esters / Q000378:metabolism*; D011374:Progesterone / Q000494:pharmacology; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D014982:Xenopus laevis
D007328:Insulin; D007527:Isoenzymes; D010703:Phorbol Esters; D011993:Recombinant Fusion Proteins; D011374:Progesterone; C087857:protein kinase C gamma; D011493:Protein Kinase C