Role of Hic-5, a protein localized at focal adhesion, in promoting cellular senescence in the stress-responses by cells.

Hic-5定位于成纤维细胞的局灶性粘连。这种定位表明，Hic-5可能介导细胞信号转导的整合素和生长因子受体的刺激。也有报道Hic-5易位到细胞核中，这些研究暗示Hic-5在细胞衰老和应激反应过程中的功能。因此，为了研究Hic-5的功能，我们研究了Hic-5在细胞内的定位是如何调节的。首先，我们分析了刺激细胞中Hic-5的酪氨酸磷酸化，以研究Hic-5在信号转导中的可能参与。我们的前期实验表明，当细胞暴露于高渗应激时，Hic-5被酪氨酸磷酸化。在本研究中，我们将Hic-5 cDNA导入不内源性表达Hic-5的COS-7细胞中。我们没有观察到酪氨酸磷酸化的Hic-5，即使在这些COS-7细胞暴露于渗透胁迫。然而，Hic-5表达在细胞内， ...更多信息 当Fyn和CAK β与Hic-5一起表达时，COS-7细胞被磷酸化，但FAK不表达。在COS-7细胞中的条件下，Hic-5在酪氨酸60上被磷酸化。结果表明，酪氨酸60的磷酸化是Hic-5与Csk的SH 2结构域特异性结合所必需的。这一结果表明，Hic-5和SH 2蛋白通过激活CAKβ和Fyn而发生特异性的蛋白-蛋白相互作用，导致下游信号传导。据报道，桩蛋白的酪氨酸磷酸化与细胞丝状伪足结构的突出和粘着斑数量的增加相关。我们通过使用重组腺病毒在成纤维细胞中过表达Hic-5，试图用Hic-5替代粘着斑处的桩蛋白。我们发现细胞突起确实受到Hic-5表达的影响。因为Hic-5缺乏桩蛋白中特有的SH 2和SH 3结合基序，我们的研究结果表明，这些基序可能在组织新的局灶性粘连中很重要。因此，与桩蛋白不同，Hic-5的酪氨酸60的磷酸化不能诱导粘着斑的形成，但可能激活一些其他信号通路。少

Hic-5 is localized at focal adhesions of fibroblast. This localization suggests a possibility that Hic-5 may mediate cellular signaling in response to the stimulation of integrins and growth factor receptors. It was also reported that Hic-5 translocated into nucleus : these studies implicated a function of Hic-5 in the process of cellular senescence and stress response. We therefore investigated, in order to study function of Hic-5, how the localization of Hic-5 within cells is regulated.First, we analyzed tyrosine phosphorylation of Hic-5 in stimulated cells to study possible involvement of Hic-5 in signal transduction. Our previous experiments showed that Hic-5 was tyrosine phosphorylated when cells were exposed to hyperosmotic stress. In the present study, we introduced Hic-5 cDNA into COS-7 cells which do not express Hic-5 endogenously. We did not observe tyrosine phoshporylation of Hic-5 even after the exposure of these COS-7 cells to osmotic stress. However, the Hic-5 expressed i … More n COS-7 cells was phosphorylated upon osmotic stress when Fyn and CAK β, but not FAK, was expressed together with Hic-5. Under the conditions in COS-7 cells, Hic-5 was phosphorylated on tyrosine 60. It was shown that the phosphorylation of tyrosine 60 was necessary for the specific binding of Hic-5 to the SH2 domain of Csk. This result implies specific protein-protein interactions of Hic-5 and SH2 proteins, caused by activation of CAKβ and Fyn, leading to downstream signalings.It was reported that tyrosine phosphorylation of paxillin correlates with protrusion of filopodial structures and an increase in the number of focal adhesions of the cell. We overexpressed Hic-5 in fibroblasts by the use of recombinant adenovirus in an attempt to replace paxillin at focal adhesions with Hic-5. We found that the cell protrusion was indeed affected by Hic-5 expression. Because Hic-5 lacks the SH2 and SH3 binding motifs characteristically found in paxillin, our results suggest that these motifs is probably important in organizing new focal adhesions. Thus, unlike paxillin, phosphorylation of tyrosine 60 of Hic-5 cannot induce the formation of focal adhesion but may activate some other signaling pathway. Less