James G, Olson E
Department of Biochemistry and Molecular Biology, University of Texas M.D. Anderson Cancer Center, Houston 77030.
J Cell Biol. 1992 Feb;116(4):863-74. doi: 10.1083/jcb.116.4.863.
Members of the protein kinase C (PKC) family are characterized by an NH2-terminal regulatory domain containing binding sites for calcium, phosphatidylserine, and diacylglycerol (or tumor-promoting phorbol esters), a small central hinge region and a COOH-terminal catalytic domain. We have constructed fusion proteins in which the regulatory domain of PKC alpha was removed and replaced by a 19-amino acid leader sequence containing a myristoylation consensus or by the same sequence in which the amino-terminal glycine was changed to alanine to prevent myristoylation. The goal was to generate constitutively active mutants of PKC that were either membrane bound, due to their myristoylation, or cytoplasmic. Western blotting of fractions from COS cells transfected with plasmids encoding wild-type and mutant proteins revealed that PKC alpha resided entirely in a Triton X-100 soluble (TS) fraction, whereas both the myristoylated and nonmyristoylated mutants were associated primarily with the nuclear envelope fraction. A similar mutant that lacked the 19 amino acid leader sequence was also found almost entirely in the nuclear envelope, as was a truncation mutant containing only the regulatory domain, hinge region, and a small portion of the catalytic domain. However, an additional truncation mutant consisting of only the regulatory domain plus the first one-third of the hinge region was almost entirely in the TS fraction. A nonmyristoylated fusion protein containing only the catalytic domain was also found in the nuclear envelope. Immunostaining of cells transfected with these constructs revealed that both the myristoylated and nonmyristoylated mutants were localized in nuclei, whereas wild-type PKC alpha was primarily cytoplasmic and perinuclear. Phorbol dibutyrate treatment of PKC alpha-transfected cells resulted in increased perinuclear and nuclear staining. The results are consistent with a model in which activation of PKC, by phorbol esters or by deletion of the regulatory domain, exposes regions in the hinge and catalytic domains that interact with a PKC "receptor" present in the nuclear envelope, and may explain the ability of wild-type PKC to be translocated to the nucleus under certain conditions.
蛋白激酶C（PKC）家族成员的特征是具有一个NH2末端调节结构域，该结构域包含钙、磷脂酰丝氨酸和二酰基甘油（或促肿瘤佛波酯）的结合位点，一个小的中央铰链区和一个COOH末端催化结构域。我们构建了融合蛋白，其中PKCα的调节结构域被去除，并用一个包含豆蔻酰化共有序列的19个氨基酸的前导序列取代，或者用相同序列，其中氨基末端甘氨酸被改为丙氨酸以防止豆蔻酰化。目的是产生组成型活性的PKC突变体，这些突变体要么由于豆蔻酰化而与膜结合，要么存在于细胞质中。对用编码野生型和突变蛋白的质粒转染的COS细胞的各部分进行蛋白质印迹分析表明，PKCα完全存在于Triton X-100可溶性（TS）部分，而豆蔻酰化和未豆蔻酰化的突变体主要与核膜部分相关。一个缺乏19个氨基酸前导序列的类似突变体也几乎完全存在于核膜中，一个仅包含调节结构域、铰链区和一小部分催化结构域的截短突变体也是如此。然而，另一个仅由调节结构域加上铰链区的前三分之一组成的截短突变体几乎完全存在于TS部分。一个仅包含催化结构域的未豆蔻酰化融合蛋白也存在于核膜中。对用这些构建体转染的细胞进行免疫染色表明，豆蔻酰化和未豆蔻酰化的突变体都定位于细胞核中，而野生型PKCα主要存在于细胞质和核周区域。用佛波二丁酸处理PKCα转染的细胞导致核周和核染色增加。这些结果与一个模型一致，在该模型中，佛波酯或调节结构域的缺失激活PKC，暴露出铰链区和催化结构域中与核膜中存在的PKC“受体”相互作用的区域，这可能解释了野生型PKC在某些条件下转移到细胞核的能力。
