Pawelczyk T, Matecki A, Dettlaff A
Department of Clinical Biochemistry, Medical University of Gdansk, Poland.
FEBS Lett. 1998 Feb 13;423(1):31-4. doi: 10.1016/s0014-5793(98)00054-4.
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.
大鼠脑PKC-γ调节区C1（92-173）的第二个富含半胱氨酸（Cys-2）结构域在大肠杆菌细胞中表达并纯化。对Cys-2蛋白的核磁共振研究确定了该分子内佛波醇和其他磷脂的结合位点（Xu, R.X., Pawelczyk, T., Xia, T-H.和Brown, S.T.（1997年）《生物化学》37卷，第10709-10717页）。在此，我们测试了该结构域结合其他蛋白质的能力。通过覆盖分析，我们发现Cys-2结构域能结合非洲爪蟾卵母细胞可溶性组分中的其他蛋白质。与激酶活性不同，在不添加磷脂的情况下也能检测到Cys-2与其他蛋白质的结合。将Cys-2蛋白显微注射到非洲爪蟾卵母细胞中可抑制胰岛素诱导的成熟，但不影响孕酮诱导的成熟。增强胰岛素诱导成熟的最小剂量是注射0.45×10⁻¹²摩尔的Cys-2。这些结果表明，PKC-γ的Cys-2结构域除了是佛波酯/二酰甘油和磷脂酰丝氨酸的结合位点外，还能结合其他蛋白质。与PKC的Cys-2结构域相互作用的蛋白质对于卵母细胞中胰岛素诱导的成熟程序至关重要。
