Specificity profiling of Pak kinases allows identification of novel phosphorylation sites
Specificity profiling of Pak kinases allows identification of novel phosphorylation sites
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DOI：
10.1074/jbc.m700253200
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发表时间：
2007-05-25
影响因子：
4.8
通讯作者：
Peterson, Jeffrey R.
中科院分区：
文献类型：
--
作者：
Rennefahrt, Ulrike E. E.;Deacon, Sean W.;Peterson, Jeffrey R.
p21激活的激酶（Paks）作为Rho家族GTP酶Rac和Cdc 42的效应子。基于序列相似性将六种人Pak分为两组。组I Pak（Pak 1至Pak-3）磷酸化将该组连接至细胞骨架的调节以及增殖和抗凋亡信号传导的许多底物。II组Pak（Pak 4至Pak-6）被认为发挥不同的功能作用，然而它们的少数已知底物也被I组Pak靶向。为了确定这两个组是否识别不同的靶序列，我们使用简并肽文库方法来全面表征组I和II Paks的共有磷酸化基序。我们发现，Pak 1和Pak 2表现出几乎相同的底物特异性，这是从Pak 4不同。基于结构比较和诱变，我们确定了两个关键的氨基酸残基介导的不同特异性的组I和II Paks，并建议这些差异的结构基础。这些结果暗示，第一次，从底物选择性的激酶的小叶残基。最后，我们利用Pak 1的共识基序预测一个新的Pak 1磷酸化位点的Pix（帕克互动交换因子），并证明Pak 1磷酸化这个网站在体外和培养的细胞。总的来说，这些结果阐明了Pak激酶的特异性，并说明了一个通用的方法，用于识别新的Paks磷酸化位点。
The p21-activated kinases (Paks) serve as effectors of the Rho family GTPases Rac and Cdc42. The six human Paks are divided into two groups based on sequence similarity. Group I Paks (Pak1 to -3) phosphorylate a number of substrates linking this group to regulation of the cytoskeleton and both proliferative and anti-apoptotic signaling. Group II Paks (Pak4 to -6) are thought to play distinct functional roles, yet their few known substrates are also targeted by Group I Paks. To determine if the two groups recognize distinct target sequences, we used a degenerate peptide library method to comprehensively characterize the consensus phosphorylation motifs of Group I and II Paks. Wefind that Pak1 and Pak2 exhibit virtually identical substrate specificity that is distinct from that of Pak4. Based on structural comparisons and mutagenesis, we identified two key amino acid residues that mediate the distinct specificities of Group I and II Paks and suggest a structural basis for these differences. These results implicate, for the first time, residues from the small lobe of a kinase in substrate selectivity. Finally, we utilized the Pak1 consensus motif to predict a novel Pak1 phosphorylation site in Pix (Pak-interactive exchange factor) and demonstrate that Pak1 phosphorylates this site both in vitro and in cultured cells. Collectively, these results elucidate the specificity of Pak kinases and illustrate a general method for the identification of novel sites phosphorylated by Paks.