蛋白質在細胞中表現的位置對於其具有的功能是很重要的。Eps8 (EGF receptor pathway substrate NO.8) 是EGFR以及v-Src的一個受質，具有97KDa及68KDa兩個isoforms。而在一些人類的癌細胞中也可以看到Eps8有持續性磷酸化的現象。經由蛋白質序列分析可知Eps8有一個分裂的PH domain、一個nuclear localization sequence (NLS)以及一段SH3 domain和四個Proline-rich region。先前的實驗證明C3H10T1/2 Fibroblasts中p97Eps8受到serum的刺激會跑到細胞膜上表現，但是PH-truncated p97Eps8 (261- p97Eps8) 卻無法跑到細胞膜上，因此為了瞭解PH domain對於Eps8在細胞中localization的重要性，我們利用Myc-tagged Eps8及其Myc-tagged mutant proteins包括261-p97Eps8以及具有N-terminal PH domain的Δ424-p97Eps8，以biochemical及immuno-cytometric的方法來研究這些Eps8 protein在細胞中的localization。利用centrifugation fractionation，我們發現在受到serum刺激的細胞中Δ424-p97Eps8主要分布在cytosol (S100)，而利用immunofluorescent microscope觀察Δ424-p97Eps8 expressing cells發現Δ424-p97Eps8會aggregate在cytosol中。因此，我們認為帶有PH domain的Δ424-p97Eps8在細胞內即使在加入serum刺激之後，也無法跑到細胞膜上。

Cellular compartmentalization is important for protein functioning in cells. Eps8 (EGF receptor pathway substrate NO.8), a common substrate of EGF receptor and Src, may exist in two isoforms, i.e. 97- and 68-KDa proteins. In human tumor cell lines, constitutive tyrosine phosphorylation of Eps8 was found. Protein sequence analysis revealed the following domains of p97Eps8 that might contribute to its cellular function: a split PH domain, a putative nuclear targeting sequence (NLS), a SH3 domain, and several proline-rich regions. Our previous studies indicated that p97Eps8, but not PH-truncated p97Eps8 (i.e. 261- p97Eps8) can translocate to plasma membrane following serum stimulation in C3H10T1/2 fibroblasts. In order to further characterize the PH domain of p97Eps8 in cellular localization, we have generated Myc-tagged Eps8 and several Myc-tagged Eps8 mutant proteins including 261-p97Eps8 and the PH domain of p97Eps8 (i.e. Δ424-p97Eps8). Through biochemical and immuno-cytometric methods, we study cellular localization of the Eps8 protein. By centrifugation fractionation, we find that Δ424-p97Eps8 localize predominant in the cytosol portion (S100) of serum-treated cells. And immunofluorescent studies of theΔ424-p97Eps8 expressing cells, we find that Δ424- p97Eps8 aggregates inside the cell. We thus conclude that the PH domain alone can not translocate to plasma membrane in response to serum stimulation.

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