Ishizaki T, Maekawa M, Fujisawa K, Okawa K, Iwamatsu A, Fujita A, Watanabe N, Saito Y, Kakizuka A, Morii N, Narumiya S
Department of Pharmacology, Kyoto University Faculty of Medicine, Japan.
EMBO J. 1996 Apr 15;15(8):1885-93.
The small GTP-binding protein Rho functions as a molecular switch in the formation of focal adhesions and stress fibers, cytokinesis and transcriptional activation. The biochemical mechanism underlying these actions remains unknown. Using a ligand overlay assay, we purified a 160 kDa platelet protein that bound specifically to GTP-bound Rho. This protein, p160, underwent autophosphorylation at its serine and threonine residues and showed the kinase activity to exogenous substrates. Both activities were enhanced by the addition of GTP-bound Rho. A cDNA encoding p160 coded for a 1354 amino acid protein. This protein has a Ser/Thr kinase domain in its N-terminus, followed by a coiled-coil structure approximately 600 amino acids long, and a cysteine-rich zinc finger-like motif and a pleckstrin homology region in the C-terminus. The N-terminus region including a kinase domain and a part of coiled-coil structure showed strong homology to myotonic dystrophy kinase over 500 residues. When co-expressed with RhoA in COS cells, p160 was co-precipitated with the expressed Rho and its kinase activity was activated, indicating that p160 can associate physically and functionally with Rho both in vitro and in vivo.
小GTP结合蛋白Rho在粘着斑和应力纤维的形成、胞质分裂及转录激活过程中起分子开关的作用。这些作用背后的生化机制尚不清楚。利用配体覆盖分析，我们纯化了一种160 kDa的血小板蛋白，它能特异性结合GTP结合形式的Rho。这种蛋白，即p160，在其丝氨酸和苏氨酸残基处发生自磷酸化，并对外源底物表现出激酶活性。加入GTP结合形式的Rho后，这两种活性均增强。编码p160的cDNA编码一个由1354个氨基酸组成的蛋白。该蛋白在其N端有一个丝氨酸/苏氨酸激酶结构域，其后是一个约600个氨基酸长的卷曲螺旋结构，在C端有一个富含半胱氨酸的锌指样基序和一个普列克底物蛋白同源区域。包括激酶结构域和部分卷曲螺旋结构的N端区域与强直性肌营养不良激酶在超过500个残基上有很强的同源性。当在COS细胞中与RhoA共表达时，p160与表达的Rho共沉淀，其激酶活性被激活，这表明p160在体外和体内均可与Rho发生物理和功能上的联系。
