Focal adhesion kinase (FAK)-dependent regulation of S-phase kinase-associated protein-2 (Skp-2) stability - A novel mechanism regulating smooth muscle cell proliferation
Focal adhesion kinase (FAK)-dependent regulation of S-phase kinase-associated protein-2 (Skp-2) stability - A novel mechanism regulating smooth muscle cell proliferation
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DOI：
10.1074/jbc.m404307200
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发表时间：
2004-09-03
影响因子：
4.8
通讯作者：
Newby, AC
中科院分区：
文献类型：
--
作者：
Bond, M;Sala-Newby, GB;Newby, AC
平滑肌细胞（SMC）增殖在完整血管中受到抑制，但在动脉粥样硬化、血管成形术后再狭窄和静脉移植物疾病中受到刺激。细胞周期蛋白依赖性激酶抑制剂p27（Kip 1）在维持SMC静止中起重要作用。p27（Kip 1）的水平取决于细胞外基质（ECM）的附着和组成。在这里，我们试图阐明潜在的ECM依赖性调节p27（Kip 1），因此，SMC增殖的机制。血清刺激降低p27（Kip 1）在离体SMC的水平，但在大鼠主动脉。这种作用是翻译后的，由蛋白酶体降解介导。我们研究了S期相关激酶蛋白2（Skp 2），一种参与泛素化和蛋白酶体介导的降解的F-box蛋白。Skp-2蛋白在分离的SMC中被血清强烈诱导，从检测不到的水平，但在主动脉中仍然检测不到; Skp-2 mRNA在主动脉中也较低。野生型Skp-2在SMC中的过表达降低了p27（Kip 1）水平，而显性负性F-box缺失突变体（DeltaF-Skp-2）Skp-2增加了p27（Kip 1）水平。此外，视网膜母细胞瘤蛋白的过度磷酸化和SMC增殖也受到野生型和显性负性Skp-2的显著影响。Skp-2的表达是绝对依赖于细胞附着的ECM和抑制层粘连蛋白和1型纤维胶原蛋白，但增加纤连蛋白。Skp-2蛋白的表达，而不是mRNA，与粘着斑激酶（FAK）的活性和抑制过表达的FAK相关的非激酶和显性负FAK（Y397 F）突变体。此外，显性负性FAK对Skp-2表达的抑制可被蛋白酶体抑制剂MG-132逆转。总之，这些数据表明，血管ECM通过Skp-2蛋白稳定性的FAK依赖性调节来控制SMC增殖。
Smooth muscle cell (SMC) proliferation is suppressed in intact blood vessels but stimulated in atherosclerosis, restenosis after angioplasty, and vein graft disease. The cyclin-dependent kinase inhibitors, including p27(Kip1), play important roles in maintaining SMC quiescence. Levels of p27(Kip1) are dependent on attachment to and the composition of the extracellular matrix (ECM). Here we sought to elucidate mechanisms underlying the ECM-dependent regulation of p27(Kip1) and hence, SMC proliferation. Serum stimulation decreased p27(Kip1) levels in isolated SMC but not in rat aorta. The effect was posttranslational and mediated by proteasomal degradation. We studied the S-phase-associated kinase protein-2 (Skp2), an F-box protein involved in ubiquitination and proteasome-mediated degradation. Skp-2 protein is strongly induced by serum from undetectable levels in isolated SMCs but remains undetectable in aorta; Skp-2 mRNA is also lower in aorta. Overexpression of wild-type Skp-2 in SMCs decreased p27(Kip1) levels, whereas dominant negative F-box deleted mutant (DeltaF-Skp-2) Skp-2 increased p27(Kip1) levels. Furthermore, hyperphosphorylation of retinoblastoma protein and SMC proliferation were also reciprocally affected by wild-type and dominant negative Skp-2. Skp-2 expression was absolutely dependent on cell attachment to the ECM and was inhibited by laminin and type-1 fibrillar collagen but increased by fibronectin. Expression of Skp-2 protein, but not mRNA, was associated with focal adhesion kinase (FAK) activity and inhibited by overexpression of FAK-related non-kinase and a dominant negative FAK(Y397F) mutant. Furthermore, the inhibition of Skp-2 expression by dominant negative FAK was reversed by the proteasome inhibitor MG-132. Taken together, these data demonstrate that the vascular ECM controls SMC proliferation via FAK-dependent regulation of Skp-2 protein stability.