IDENTIFICATION OF NOVEL PHOSPHORYLATION SITES IN MURINE A-TYPE LAMINS
IDENTIFICATION OF NOVEL PHOSPHORYLATION SITES IN MURINE A-TYPE LAMINS
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DOI：
10.1111/j.1432-1033.1993.tb17806.x
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发表时间：
1993-04-15
期刊：
影响因子：
--
通讯作者：
JOST, E
中科院分区：
文献类型：
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作者：
EGGERT, M;RADOMSKI, N;JOST, E
我们报告的分布在小鼠核纤层蛋白A和C（A型核纤层蛋白）在体外和体内的磷酸化位点，然后通过反相高效液相色谱和微测序的肽跨越几乎完整的核纤层蛋白序列。我们发现，两种不同的蛋白激酶，细胞分裂周期-2激酶（cdc2激酶）和蛋白激酶C（PKC），磷酸化小鼠A型核纤层蛋白在非α-螺旋氨基和羧基末端结构域在体外和体内。Cdc2激酶，但不是PKC，能够诱导解聚的透化细胞的核纤层。因此，提出层蛋白是cdc2激酶和PKC的直接体内底物，对层动力学具有不同的影响。原始A型核纤层蛋白的分析揭示了残基Ser5和Ser392的磷酸化。Ser392残基亚化学计量磷酸化的底物和cdc2激酶在体外。PKC磷酸化肽，其激酶特异性基序围绕Ser5，Thr199，Thr416，Thr480和Ser625。在体内，一个有丝分裂特异性磷酸化的cdc2激酶特异性磷酸化受体位点Ser392和N-末端肽被确定。确定了与PKC共有序列相匹配的Ser525处的间期特异性磷酸化以及由未知激酶磷酸化的肽的磷酸化。结果使我们提出，不同的细胞周期蛋白依赖性激酶的活动作为核纤层蛋白激酶在有丝分裂和间期。其他激酶可能与cdc2激酶在有丝分裂可逆拆卸过程中合作，并可能调节核纤层蛋白丝的超分子组装。
We report the distribution of phosphorylation sites in murine lamins A and C (A-type lamins) in vitro and in vivo followed by reverse-phase high-performance liquid chromatography and micro-sequencing of peptides spanning the almost complete lamin sequence. We show that two distinct protein kinases, cell-division-cycle-2 kinase (cdc2 kinase) and protein kinase C (PKC), phosphorylate murine A-type lamins at the non-alpha-helical amino- and carboxy-terminal domains in vitro and in vivo. Cdc2 kinase, but not PKC, is capable of inducing depolymerization of the nuclear lamina in permeabilized cells. Accordingly, lamins were proposed to be direct in vivo substrates of cdc2 kinase and PKC with different effects on the lamina dynamics. Analysis of the original A-type lamins revealed phosphorylation of residues Ser5 and Ser392. Residue Ser392 was substoichiometrically phosphorylated in the substrate and by cdc2 kinase in vitro. PKC phosphorylated peptides with its kinase-specific motifs surrounding Ser5, Thr199, Thr416, Thr480 and Ser625. In vivo, a mitosis-specific phosphorylation at the cdc2-kinase-specific phosphoacceptor site Ser392 and of the N-terminal peptide was identified. An interphase-specific phosphorylation at Ser525 matching the PKC consensus sequence and of peptides phosphorylated by unknown kinases was determined. The results lead us to propose that different cyclin-dependent kinase activities act as lamin kinases in mitosis and in interphase. Other kinases may cooperate with cdc2 kinase during reversible disassembly in mitosis and may modulate the supramolecular assembly of lamin filaments.