Loo L W, Berestecky J M, Kanemitsu M Y, Lau A F
Molecular Carcinogenesis Program, Cancer Research Center of Hawaii, Honolulu, USA.
J Biol Chem. 1995 May 26;270(21):12751-61. doi: 10.1074/jbc.270.21.12751.
Several laboratories have demonstrated a decrease in gap junctional communication in cells transformed by the src oncogene of the Rous sarcoma virus. The decrease in gap junctional communication was associated with tyrosine phosphorylation of the gap junction protein, connexin 43 (Cx43). This study was initiated to determine if the phosphorylation of Cx43 is the result of a direct kinase-substrate interaction between the highly active tyrosine kinase, pp60v-src, and Cx43. Previous biochemical studies have been limited by the low levels of Cx43 protein in fibroblast cell lines. To obtain larger quantities of Cx43, we constructed a recombinant baculovirus expressing Cx43 in Spodoptera frugiperda (Sf-9) cells and subsequently purified the expressed Cx43 by immunoaffinity chromatography. We observed that this partially purified Cx43 was phosphorylated on tyrosine in vitro in the presence of kinase-active pp60src. Phosphotryptic peptide mapping indicated that the in vitro phosphorylated Cx43 contained phosphopeptides which comigrated with a subset of tryptic peptides prepared from Cx43 phosphorylated in vivo. Furthermore, coinfection of Sf-9 cells with recombinant baculoviruses encoding pp60v-src and Cx43 resulted in the accumulation of phosphotyrosine in Cx43. Taken together, the evidence presented in this paper demonstrates that kinase active pp60c-src is capable of phosphorylating Cx43 in a direct manner. Since the presence of phosphotyrosine on Cx43 is correlated with the down-regulation of gap-junctional communication, these results suggest that pp60v-src regulates gap junctional gating activity via tyrosine phosphorylation of Cx43.
几个实验室已证明，感染劳氏肉瘤病毒src癌基因后发生转化的细胞中，间隙连接通讯减少。间隙连接通讯的减少与间隙连接蛋白连接蛋白43（Cx43）的酪氨酸磷酸化有关。开展本研究是为了确定Cx43的磷酸化是否是高活性酪氨酸激酶pp60v-src与Cx43之间直接激酶-底物相互作用的结果。以往的生化研究因成纤维细胞系中Cx43蛋白水平较低而受到限制。为了获得更多的Cx43，我们构建了一种重组杆状病毒，该病毒在草地贪夜蛾（Sf-9）细胞中表达Cx43，随后通过免疫亲和层析法纯化表达的Cx43。我们观察到，在激酶活性pp60src存在的情况下，这种部分纯化的Cx43在体外发生了酪氨酸磷酸化。磷酸化胰蛋白酶肽图谱分析表明，体外磷酸化的Cx43所含的磷酸肽与体内磷酸化的Cx43制备的一组胰蛋白酶肽迁移情况相同。此外，将编码pp60v-src和Cx43的重组杆状病毒共感染Sf-9细胞，导致Cx43中磷酸酪氨酸的积累。综上所述，本文提供的证据表明，激酶活性pp60c-src能够直接使Cx43磷酸化。由于Cx43上磷酸酪氨酸的存在与间隙连接通讯的下调相关，这些结果表明pp60v-src通过Cx43的酪氨酸磷酸化调节间隙连接门控活性。
