Aarts L H, Schrama L H, Hage W J, Bos J L, Gispen W H, Schotman P
Department of Physiological Chemistry, Utrecht University, 3584 CG, Utrecht, The Netherlands.
Mol Biol Cell. 1998 Jun;9(6):1279-92. doi: 10.1091/mbc.9.6.1279.
In the present study we show that expression of the neural PKC-substrate B-50 (growth-associated protein [GAP-43]) in Rat-1 fibroblasts induced the formation of filopodial extensions during spreading. This morphological change was accompanied by an enhanced formation of peripheral actin filaments and by accumulation of vinculin immunoreactivity in filopodial focal adhesions, colocalizing with B-50. In time lapse experiments, the B-50-induced filopodial extensions were shown to stay in close contact with the substratum and appeared remarkably stable, resulting in a delayed lamellar spreading of the fibroblasts. The morphogenetic effects of the B-50 protein were entirely dependent on the integrity of the two N-terminal cysteines involved in membrane association (C3C4), but were not significantly affected by mutations of the PKC-phosphorylation site (S41) or deletion of the C terminus (177-226). Cotransfection of B-50 with dominant negative Cdc42 or Rac did not prevent B-50-induced formation of filopodial cells, whereas this process could be completely blocked by cotransfection with dominant negative Rho or Clostridium botulinum C3-transferase. Conversely, constitutively active Rho induced a similar filopodial phenotype as B-50. We therefore propose that the induction of surface extensions by B-50 in spreading Rat-1 fibroblasts depends on Rho-guanosine triphosphatase function.
在本研究中,我们发现,大鼠-1成纤维细胞中神经型蛋白激酶C底物B-50(生长相关蛋白[GAP-43])的表达在铺展过程中诱导丝状伪足延伸的形成。这种形态变化伴随着外周肌动蛋白丝的形成增加以及丝状伪足黏着斑中纽蛋白免疫反应性的积累,且与B-50共定位。在延时实验中,B-50诱导的丝状伪足延伸与基质保持紧密接触,并且显得非常稳定,导致成纤维细胞的片状铺展延迟。B-50蛋白的形态发生效应完全依赖于参与膜结合的两个N端半胱氨酸(C3C4)的完整性,但不受蛋白激酶C磷酸化位点(S41)突变或C端缺失(177 - 226)的显著影响。将B-50与显性负性Cdc42或Rac共转染并不能阻止B-50诱导丝状伪足细胞的形成,而与显性负性Rho或肉毒杆菌C3转移酶共转染则可完全阻断这一过程。相反,组成型激活的Rho诱导出与B-50类似的丝状伪足表型。因此,我们提出,在铺展的大鼠-1成纤维细胞中,B-50诱导表面延伸依赖于Rho-鸟苷三磷酸酶的功能。