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灶性粘连。这种定位表明，Hic-5可能介导了细胞信号对整合素和生长因子受体的刺激。也有报道Hic-5移位到细胞核内：这些研究表明Hic-5在细胞衰老和应激反应过程中发挥作用。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可能参与的信号转导。我们先前的实验表明，当细胞暴露在高渗应激下时，Hic-5被酪氨酸磷酸化。在本研究中，我们将Hic-5基因导入不表达Hic-5的COS-7细胞。即使在这些COS-7细胞暴露于渗透胁迫后，我们也没有观察到Hic-5的酪氨酸磷酸化。然而，HIC-5表达了I…当Fyn和Cakβ与Hic-5一起表达时，在渗透胁迫下，nCOS7细胞被更多的磷酸化，而不是FAK。在COS-7细胞中，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SH2结构域特异性结合所必需的。这一结果暗示了Hic-5和SH2蛋白之间的特异性蛋白质相互作用，这种相互作用是由Cakβ和Fyn激活引起的，从而导致下游信号的产生。据报道，巴西林的酪氨酸磷酸化与丝孔结构的突起和细胞局部粘连数量的增加有关。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取代局部粘连处的帕西林。我们发现细胞突起确实受到Hic-5表达的影响。由于Hic-5缺乏典型的帕克西林中的SH2和SH3结合基序，我们的结果表明这些基序可能在组织新的局灶性粘连中起重要作用。因此，与巴西林不同，Hic-5酪氨酸60的磷酸化不能诱导局部黏附的形成，但可能激活其他一些信号通路。较少

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