Hocevar B A, Burns D J, Fields A P
Department of Pharmacology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106.
J Biol Chem. 1993 Apr 5;268(10):7545-52.
Protein kinase C (PKC) is activated at the nuclear membrane in response to a variety of mitogenic stimuli. In human leukemic cells, the beta II PKC isotype is selectively translocated and activated at the nucleus. We recently identified the nuclear envelope component lamin B1 as a major substrate for nuclear PKC both in whole cells and in vitro. Using highly purified human beta II PKC and isolated nuclear envelopes from the human promyelocytic (HL60) leukemia cell line, we have now determined the major sites for beta II PKC-mediated lamin B phosphorylation. Using a combination of cyanogen bromide cleavage, direct microsequencing, tryptic phosphopeptide, and phosphate release analyses, two major sites of PKC-mediated phosphorylation, Ser395 and Ser405, have been identified. These sites lie within the carboxyl-terminal domain of lamin B immediately adjacent to the central alpha-helical rod domain. Functionally, beta II PKC-mediated phosphorylation of these sites leads to the time-dependent solubilization of lamin B indicative of mitotic nuclear envelope breakdown in vitro. beta II PKC-mediated lamin B phosphorylation is inhibited by 1) a monoclonal antibody directed against the active site of PKC, 2) a PKC pseudosubstrate inhibitor peptide, and 3) a PKC peptide substrate. Two observations indicate that PKC-mediated lamin B phosphorylation and solubilization is due to direct phosphorylation of lamin B by PKC rather than indirect activation of a cdc2 kinase. Neither immunodepletion with p13suc1 Sepharose beads nor the presence of a p34cdc2 kinase peptide substrate had any effect on PKC-mediated lamin B phosphorylation. Therefore, we conclude that beta II PKC represents a physiologically relevant lamin kinase that can directly modulate nuclear lamina structure in vitro. Nuclear beta II PKC, like p34cdc2 kinase, may function to regulate nuclear lamina structural stability during cell cycle.
蛋白激酶C（PKC）在核膜处被激活，以响应多种促有丝分裂刺激。在人类白血病细胞中，βII PKC同工型在细胞核中选择性易位并被激活。我们最近在全细胞和体外实验中均鉴定出核膜成分核纤层蛋白B1是核PKC的主要底物。使用高度纯化的人类βII PKC和从人类早幼粒细胞（HL60）白血病细胞系中分离出的核膜，我们现已确定βII PKC介导的核纤层蛋白B磷酸化的主要位点。通过结合使用溴化氰裂解、直接微量测序、胰蛋白酶磷酸肽和磷酸盐释放分析，已鉴定出PKC介导的磷酸化的两个主要位点，即Ser395和Ser405。这些位点位于核纤层蛋白B紧邻中央α螺旋杆状结构域的羧基末端结构域内。在功能上，βII PKC介导的这些位点的磷酸化导致核纤层蛋白B随时间溶解，这表明在体外有丝分裂时核膜破裂。βII PKC介导的核纤层蛋白B磷酸化受到以下因素的抑制：1）针对PKC活性位点的单克隆抗体；2）PKC假底物抑制剂肽；3）PKC肽底物。两项观察结果表明，PKC介导的核纤层蛋白B磷酸化和溶解是由于PKC直接磷酸化核纤层蛋白B，而不是间接激活cdc2激酶。用p13suc1琼脂糖珠进行免疫去除或存在p34cdc2激酶肽底物均对PKC介导的核纤层蛋白B磷酸化没有任何影响。因此，我们得出结论，βII PKC代表一种生理相关的核纤层蛋白激酶，它可以在体外直接调节核纤层结构。核βII PKC与p34cdc2激酶一样，可能在细胞周期中调节核纤层结构稳定性。
