Arai A, Nosaka Y, Kohsaka H, Miyasaka N, Miura O
First Department of Internal Medicine, Tokyo Medical and Dental University, Tokyo, Japan.
Blood. 1999 Jun 1;93(11):3713-22.
CrkL is a member of the Crk family of adapter proteins consisting mostly of SH2 and SH3 domains. CrkL is most abundantly expressed in hematopoietic cells and has been implicated in pathogenesis of chronic myelogenous leukemia. However, its function has not been precisely defined. Here, we show that overexpression of CrkL enhances the adhesion of hematopoietic 32D cells to fibronectin. The CrkL-induced increase in cell adhesion was blocked by antibodies against VLA-4 (alpha4beta1) and VLA-5 (alpha5beta1) but was observed without changes in surface expression levels of these integrins. Studies using CrkL mutants demonstrated that the SH2 domain is partially required for enhancing cell adhesion, whereas the C-terminal SH3 domain as well as the tyrosine phosphorylation site (Y207) is dispensable. In contrast, the N-terminal SH3 domain, involved in binding C3G and other signaling molecules, was showed to play a crucial role, because a mutant defective of this domain showed an inhibitory effect on the cell adhesion to fibronectin. Furthermore, overexpression of C3G also increased the adhesion of hematopoietic cells to fibronectin, whereas a C3G mutant lacking the guanine nucleotide exchange domain abrogated the CrkL-induced increase in cell adhesion. On the other hand, a dominant negative mutant of H-Ras or that of Raf-1 enhanced the basal and CrkL-induced cell adhesion and that of R-Ras modestly decreased the adhesion. Taken together, these results indicate that the CrkL-C3G complex activates VLA-4 and VLA-5 in hematopoietic cells, possibly by activating the small GTP binding proteins, including R-Ras, through the guanine nucleotide exchange activity of C3G.
CrkL是衔接蛋白Crk家族的成员，主要由SH2和SH3结构域组成。CrkL在造血细胞中表达最为丰富，并与慢性粒细胞白血病的发病机制有关。然而，其功能尚未得到精确界定。在此，我们表明CrkL的过表达增强了造血32D细胞与纤连蛋白的黏附。CrkL诱导的细胞黏附增加被抗VLA-4（α4β1）和VLA-5（α5β1）的抗体阻断，但在这些整合素的表面表达水平未发生变化的情况下也观察到了这种增加。使用CrkL突变体的研究表明，SH2结构域对于增强细胞黏附部分必需，而C末端SH3结构域以及酪氨酸磷酸化位点（Y207）则是可有可无的。相反，参与结合C3G和其他信号分子的N末端SH3结构域被证明起着关键作用，因为该结构域缺陷的突变体对细胞与纤连蛋白的黏附具有抑制作用。此外，C3G的过表达也增加了造血细胞与纤连蛋白的黏附，而缺乏鸟嘌呤核苷酸交换结构域的C3G突变体则消除了CrkL诱导的细胞黏附增加。另一方面，H-Ras或Raf-1的显性负性突变体增强了基础和CrkL诱导的细胞黏附，而R-Ras的显性负性突变体则适度降低了黏附。综上所述，这些结果表明CrkL-C3G复合物可能通过C3G的鸟嘌呤核苷酸交换活性激活包括R-Ras在内的小GTP结合蛋白，从而激活造血细胞中的VLA-4和VLA-5。
