Zhao J, Zheng C, Guan J
Cancer Biology Laboratories, Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA.
J Cell Sci. 2000 Sep;113 ( Pt 17):3063-72. doi: 10.1242/jcs.113.17.3063.
We have previously identified FAK and its associated signaling pathways as a mediator of cell cycle progression by integrins. In this report, we have analyzed the potential role and mechanism of Pyk2, a tyrosine kinase closely related to FAK, in cell cycle regulation by using tetracycline-regulated expression system as well as chimeric molecules. We have found that induction of Pyk2 inhibited G(1) to S phase transition whereas comparable induction of FAK expression accelerated it. Furthermore, expression of a chimeric protein containing Pyk2 N-terminal and kinase domain and FAK C-terminal domain (PFhy1) increased cell cycle progression as FAK. Conversely, the complementary chimeric molecule containing FAK N-terminal and kinase domain and Pyk2 C-terminal domain (FPhy2) inhibited cell cycle progression to an even greater extent than Pyk2. Biochemical analyses indicated that Pyk2 and FPhy2 stimulated JNK activation whereas FAK or PFhy1 had little effect on it, suggesting that differential activation of JNK by Pyk2 may contribute to its inhibition of cell cycle progression. In addition, Pyk2 and FPhy2 to a greater extent also inhibited Erk activation in cell adhesion whereas FAK and PFhy1 stimulated it, suggesting a role for Erk activation in mediating differential regulation of cell cycle by Pyk2 and FAK. A role for Erk and JNK pathways in mediating the cell cycle regulation by FAK and Pyk2 was also confirmed by using chemical inhibitors for these pathways. Finally, we showed that while FAK and PFhy1 were present in focal contacts, Pyk2 and FPhy2 were localized in the cytoplasm. Interestingly, both Pyk2 and FPhy2 (to a greater extent) were tyrosine phosphorylated and associated with Src and Fyn. This suggested that they may inhibit Erk activation in an analogous manner as the mislocalized FAK mutant (&Dgr;)C14 described previously by competing with endogenous FAK for binding signaling molecules such as Src and Fyn. This model is further supported by an inhibition of endogenous FAK association with active Src by Pyk2 and FPhy2 and a partial rescue by FAK of Pyk2-mediated cell cycle inhibition.
我们之前已确定黏着斑激酶（FAK）及其相关信号通路是整合素介导细胞周期进程的介质。在本报告中，我们通过使用四环素调控表达系统以及嵌合分子，分析了与FAK密切相关的酪氨酸激酶Pyk2在细胞周期调控中的潜在作用及机制。我们发现，诱导Pyk2表达会抑制G1期到S期的转变，而诱导等量的FAK表达则会加速这一转变。此外，包含Pyk2 N端和激酶结构域以及FAK C端结构域的嵌合蛋白（PFhy1）的表达会像FAK一样促进细胞周期进程。相反，包含FAK N端和激酶结构域以及Pyk2 C端结构域的互补嵌合分子（FPhy2）对细胞周期进程的抑制作用比Pyk2更大。生化分析表明，Pyk2和FPhy2会刺激JNK激活，而FAK或PFhy1对其影响很小，这表明Pyk2对JNK的差异性激活可能导致其对细胞周期进程的抑制作用。此外，Pyk2和FPhy2在细胞黏附中也更大程度地抑制了Erk激活，而FAK和PFhy1则刺激了Erk激活，这表明Erk激活在介导Pyk2和FAK对细胞周期的差异性调控中发挥作用。使用这些信号通路的化学抑制剂也证实了Erk和JNK信号通路在介导FAK和Pyk2对细胞周期的调控中所起的作用。最后，我们发现虽然FAK和PFhy1存在于黏着斑中，但Pyk2和FPhy2定位于细胞质中。有趣的是，Pyk2和FPhy2（更大程度上）都发生了酪氨酸磷酸化，并与Src和Fyn相关联。这表明它们可能通过与内源性FAK竞争结合Src和Fyn等信号分子，以类似于之前描述的错误定位的FAK突变体（Δ）C14的方式抑制Erk激活。Pyk2和FPhy2对内源性FAK与活性Src结合的抑制以及FAK对Pyk2介导的细胞周期抑制的部分挽救进一步支持了这一模型。
