Identification of protein kinase C (PKC) phosphorylation sites on human lamin B. Potential role of PKC in nuclear lamina structural dynamics.
Identification of protein kinase C (PKC) phosphorylation sites on human lamin B. Potential role of PKC in nuclear lamina structural dynamics.
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DOI：
10.1016/s0021-9258(18)53210-5
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发表时间：
1993-04
期刊：
影响因子：
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通讯作者：
B. A. Hocevar;D. Burns;A. Fields
中科院分区：
文献类型：
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作者：
B. A. Hocevar;D. Burns;A. Fields
蛋白激酶C（PKC）在核膜上被激活以响应各种促有丝分裂刺激。在人类白血病细胞中，β II PKC同种型选择性地易位并在细胞核中活化。最近，我们确定了核被膜组件核纤层蛋白B1作为一个主要的底物，无论是在整个细胞和体外的核蛋白激酶C。使用高度纯化的人β II PKC和分离的人早幼粒细胞（HL 60）白血病细胞系的核膜，我们现在已经确定了β II PKC介导的核纤层蛋白B磷酸化的主要位点。使用溴化氰切割，直接微测序，胰蛋白酶磷酸肽，磷酸盐释放分析的组合，PKC介导的磷酸化，Ser 395和Ser 405的两个主要网站，已被确定。这些位点位于核纤层蛋白B的羧基末端结构域内，紧邻中心α-螺旋杆结构域。在功能上，β II PKC介导的这些位点的磷酸化导致核纤层蛋白B的时间依赖性溶解，指示体外有丝分裂核膜的破坏。β II PKC介导的核纤层蛋白B磷酸化被1）针对PKC活性位点的单克隆抗体，2）PKC假底物抑制剂肽，和3）PKC肽底物抑制。两个观察结果表明，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，而不是间接激活的cdc 2激酶。p13 suc 1琼脂糖凝胶珠的免疫耗竭和p34 cdc 2激酶肽底物的存在对PKC介导的核纤层蛋白B磷酸化都没有任何影响。因此，我们得出结论，β II PKC代表一个生理相关的核纤层蛋白激酶，可以直接调节核纤层结构在体外。细胞核β-Ⅱ型蛋白激酶C与p34 cdc 2激酶一样，在细胞周期中可能起着调节核纤层结构稳定性的作用。
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.