Multinucleation induced by Chk overexpression in hematopoietic cells

造血细胞中 Chk 过表达诱导的多核

基本信息

批准号：
11470212
负责人：
YAMAGUCHI Naoto
金额：
$ 7.3万
依托单位：
Chiba University (2000)University of Shizuoka (1999)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11470212/
关键词：
Cell growth Chromosome Csk Multinucleation Intracellular localization CSk homologous kinase (Chk)Src family tyrosine kinase Tyrosine phosphorylation Csk homologous kinase (Chk)Csk homolosous kinase(Chk)

项目摘要

Src family protein-tyrosine kinases play crucial roles in regulating proliferation and differentiation of multiple cell types including hematopoietic cells. The activity of Src family kinases is tightly regulated by tyrosine phosphorylation and dephosphorylation events. The C-terminal src kinase (Csk), which is expressed ubiquitously, has been shown to phosphorylate the C-terminal negative regulatory tyrosine residue of Src family kinases and suppress their kinase activity. A second member of the Csk family expressed in hematopoietic cells was recently identified as the Csk homologous kinase (Chk). Like Csk, Chk suppresses the catalytic activity of Src family kinases by phosphorylating their C-terminal negative regulatory tyrosine residues. Ectopic and transient expression of Chk in COS-1 cells showed nuclear localization of Chk and growth inhibition. To further explore the role of Chk in cell growth, we overexpressed Chk in human immature myeloid KMT-2 cells. Chk overexpression brought about growth retardation and aberrant chromosome movement leading to multinucleation, and these events were accompanied by insufficient formation of mitotic spindles. In vitro kinase assays showed that Chk overexpression suppressed the tyrosine kinase activity of Lyn, a member of the Src family, immunoprecipitated from Triton X-100 lysates. Subcellular fractionation studies revealed that a fraction of Chk and Lyn, resistant to Triton X-100 solubilization, are associated with mitotic chromosome scaffolds and spindles. Chk overexpression induced a decrease in autophosphorylation of Lyn and concomitant changes in levels of tyrosine phosphorylation of proteins associated with both fractions. These results indicate that Chk, Lyn and the tyrosine-phosphorylated proteins localize to mitotic chromosomes and spindles, suggesting that Chk-dependent tyrosine phosphorylation presumably through Lyn may be involved in chromosome dynamics.

SRC家族蛋白 - 酪氨酸激酶在调节包括造血细胞在内的多种细胞类型的增殖和分化中起着至关重要的作用。 SRC家族激酶的活性受酪氨酸磷酸化和去磷酸化事件的严格调节。普遍表达的C端SRC激酶（CSK）已被证明可以磷酸化SRC家族激酶的C末端负调控酪氨酸残基并抑制其激酶活性。最近将在造血细胞中表达的CSK家族的第二个成员被确定为CSK同源激酶（CHK）。像CSK一样，CHK通过磷酸化其C末端负调控酪氨酸残基来抑制SRC家族激酶的催化活性。 COS-1细胞中CHK的异位和瞬态表达显示CHK的核定位和生长抑制作用。为了进一步探索CHK在细胞生长中的作用，我们在人类不成熟的髓样KMT-2细胞中过表达CHK。 CHK过表达导致生长迟钝和异常的染色体运动导致多核，这些事件伴随着有丝分裂纺锤体的形成不足。体外激酶分析表明，CHK过表达抑制了SRC家族成员Lyn的酪氨酸激酶活性，并从Triton X-100裂解物中免疫沉淀。亚细胞分馏研究表明，抗Triton X-100溶解化的CHK和Lyn的一部分与有丝分裂染色体支架和纺锤体有关。 CHK的过表达诱导LYN的自磷酸化降低，以及与两种级分有关的蛋白质酪氨酸磷酸化水平的随之变化。这些结果表明，CHK，Lyn和酪氨酸磷酸化的蛋白质定位于有丝分裂染色体和纺锤体，这表明可能通过Lyn可能与CHK依赖性酪氨酸磷酸化可能有关染色体动力学。