Scaife R M, Langdon W Y
Department of Pathology, University of Western Australia, QE II Medical Centre, Nedlands WA 6907, Australia.
J Cell Sci. 2000 Jan;113 Pt 2:215-26. doi: 10.1242/jcs.113.2.215.
Adhesive and locomotive properties of cells have key roles in normal physiology and disease. Cell motility and adhesion require the assembly and organization of actin microfilaments into stress fibers, lamellipodia and filopodia, and the formation of these structures is mediated by signalling through Rho; GTPases. Here we identify c-Cbl (a multi-adaptor proto-oncogene product involved in protein tyrosine kinase signalling) as an important regulator of the actin cytoskeleton. By immunofluorescence microscopy we have determined that c-Cbl co-localizes with the adaptor protein Crk to submembranous actin lamellae in NIH 3T3 fibroblasts and that c-Cbl's actin localization requires specific SH3-binding sequences. Further, we have found that truncation of this SH3-binding domain in c-Cbl profoundly alters the morphology of NIH 3T3 fibroblasts by inhibiting the formation of actin lamellae, lamellipodia and membrane ruffles. The induction of lamellipodia and membrane ruffles are also inhibited during cell spreading and migration, conditions when these structures are normally most prominent. The inhibitory effect of truncated c-Cbl expression on lamellipodia formation can be reversed by mutational inactivation of its divergent SH2 domain, by the co-expression of constitutively active Rac or by the overexpression of c-Cbl. This study therefore identifies a cytoskeletal role for c-Cbl which may involve the regulation of Crk and Rac, and which is dependent on targeting of c-Cbl to actin lamellae and the ability to recruit signalling protein(s) associated with its divergent SH2 domain.
细胞的黏附与运动特性在正常生理和疾病过程中发挥着关键作用。细胞运动和黏附需要将肌动蛋白微丝组装并组织成应力纤维、片状伪足和丝状伪足，而这些结构的形成是由Rho；GTPases信号传导介导的。在此，我们确定c-Cbl（一种参与蛋白酪氨酸激酶信号传导的多衔接子原癌基因产物）是肌动蛋白细胞骨架的重要调节因子。通过免疫荧光显微镜观察，我们确定c-Cbl在NIH 3T3成纤维细胞中与衔接蛋白Crk共定位于膜下肌动蛋白薄片，并且c-Cbl的肌动蛋白定位需要特定的SH3结合序列。此外，我们发现c-Cbl中该SH3结合结构域的截短通过抑制肌动蛋白薄片、片状伪足和膜皱褶的形成，深刻改变了NIH 3T3成纤维细胞的形态。在细胞铺展和迁移过程中，即这些结构通常最为突出的情况下，片状伪足和膜皱褶的诱导也受到抑制。截短的c-Cbl表达对片状伪足形成的抑制作用可通过其不同的SH2结构域的突变失活、组成型活性Rac的共表达或c-Cbl的过表达来逆转。因此，本研究确定了c-Cbl在细胞骨架中的作用，这可能涉及对Crk和Rac的调节，并且依赖于将c-Cbl靶向至肌动蛋白薄片以及募集与其不同的SH2结构域相关的信号蛋白的能力。
