Multinucleation induced by Chk overexpression in hematopoietic cells

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家族激酶的活性受到酪氨酸磷酸化和去磷酸化事件的严格调节。广泛表达的C-末端src激酶（Csk）已显示磷酸化Src家族激酶的C-末端负调节酪氨酸残基并抑制其激酶活性。在造血细胞中表达的Csk家族的第二个成员最近被鉴定为Csk同源激酶（Chk）。与Csk类似，Chk通过磷酸化Src家族激酶的C-末端负调控酪氨酸残基来抑制Src家族激酶的催化活性。Chk在COS-1细胞中的异位和瞬时表达显示Chk的核定位和生长抑制。为了进一步探索Chk在细胞生长中的作用，我们在人未成熟髓系KMT-2细胞中过表达Chk。Chk过度表达导致生长迟缓和染色体异常运动，导致多核化，这些事件伴随着有丝分裂纺锤体形成不足。体外激酶试验表明，Chk过表达抑制酪氨酸激酶活性的林恩，Src家族的成员，免疫沉淀的Triton X-100裂解物。亚细胞分级分离研究表明，一部分Chk和林恩，耐Triton X-100增溶，与有丝分裂染色体支架和纺锤体。Chk过表达诱导林恩的自磷酸化减少，并伴随着与两种组分相关的蛋白质酪氨酸磷酸化水平的变化。这些结果表明，Chk，林恩和酪氨酸磷酸化蛋白定位于有丝分裂染色体和纺锤体，这表明Chk依赖的酪氨酸磷酸化可能通过林恩可能参与染色体动力学。

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.