Uemura N, Griffin J D
Department of Adult Oncology, Dana-Farber Cancer Institute, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
J Biol Chem. 1999 Dec 31;274(53):37525-32. doi: 10.1074/jbc.274.53.37525.
Crkl, an SH2-SH3-SH3 adapter protein, is one of the major tyrosine phosphoproteins detected in cells from patients with chronic myelogenous leukemia. Crkl binds to BCR/ABL through its N-terminal SH3 domain and is known to interact with several signaling proteins that have been implicated in integrin signaling, including Cbl, Cas, Hef-1, and paxillin. We have previously shown that overexpression of Crkl enhances adhesion to extracellular matrix proteins through beta(1) integrins. In this study, the effects of Crkl on spontaneous and chemokine-directed migration of the hematopoietic cell line Ba/F3 were examined. Full-length, SH2-, and SH3(N)-domain deletion mutants of Crkl were expressed transiently as fusion proteins with green fluorescent protein. Successfully transfected cells were isolated by fluorescence-activated cell sorting. The ability of these cells to migrate across a fibronectin-coated membrane, either spontaneously or in response to the chemokine stromal-derived factor-1alpha, was determined. Cells expressing green fluorescent protein alone were not distinguishable from untransfected or mock transfected Ba/F3 cells. However, Ba/F3 cells overexpressing full-length Crkl were found to have an increase in spontaneous migration of 2.8 +/- 0.6-fold in seven independent assays. The enhancement of migration required both the SH2 domain and the N-terminal SH3 domain. Migration in response to stromal-derived factor-1alpha was not significantly enhanced by overexpression of Crkl. Overexpression of Crkii also augmented spontaneous migration but to a lesser degree than did Crkl. Because the SH2 domain was required for enhanced migration, we looked for changes in phosphotyrosine containing proteins coprecipitating with Crkl, but not Crkl DeltaSH2, after integrin cross-linking. Full-length Crkl, but not CrklDeltaSH2, coprecipitated with a single major tyrosine phosphoprotein with an M(r) of approximately 120 kDa, identified as Cbl. The major Crkl SH3-binding protein in these cells was found to be the guanine nucleotide exchange factor, C3G. Interestingly, overexpression of C3G also enhanced migration, suggesting that a Cbl-Crkl-C3G complex may be involved in migration signaling in Ba/F3 cells. These data suggest that Crkl is involved in signaling pathways that regulate migration, possibly through a complex with Cbl and C3G.
Crkl是一种含有SH2-SH3-SH3结构域的接头蛋白，是在慢性粒细胞白血病患者细胞中检测到的主要酪氨酸磷酸化蛋白之一。Crkl通过其N端SH3结构域与BCR/ABL结合，并且已知与几种参与整合素信号传导的信号蛋白相互作用，包括Cbl、Cas、Hef-1和桩蛋白。我们之前已经表明，Crkl的过表达通过β1整合素增强对细胞外基质蛋白的黏附。在本研究中，检测了Crkl对造血细胞系Ba/F3自发迁移和趋化因子介导迁移的影响。Crkl的全长、SH2结构域和SH3（N）结构域缺失突变体作为与绿色荧光蛋白的融合蛋白瞬时表达。通过荧光激活细胞分选分离成功转染的细胞。测定这些细胞自发或响应趋化因子基质衍生因子-1α穿过纤连蛋白包被膜迁移的能力。单独表达绿色荧光蛋白的细胞与未转染或mock转染的Ba/F3细胞没有区别。然而，在七次独立试验中发现，过表达全长Crkl的Ba/F3细胞自发迁移增加了2.8±0.6倍。迁移的增强需要SH2结构域和N端SH3结构域。Crkl的过表达并未显著增强对基质衍生因子-1α的迁移反应。Crkii的过表达也增加了自发迁移，但程度低于Crkl。由于迁移增强需要SH2结构域，我们研究了整合素交联后与Crkl（而非Crkl DeltaSH2）共沉淀的含磷酸酪氨酸蛋白的变化。全长Crkl而非CrklDeltaSH2与一种单一的主要酪氨酸磷酸化蛋白共沉淀，其分子量约为120 kDa，鉴定为Cbl。在这些细胞中，主要的Crkl SH3结合蛋白是鸟嘌呤核苷酸交换因子C3G。有趣的是，C3G的过表达也增强了迁移，表明Cbl-Crkl-C3G复合物可能参与Ba/F3细胞的迁移信号传导。这些数据表明，Crkl可能通过与Cbl和C3G形成复合物参与调节迁移的信号通路。
