PHOSPHORYLATION OF CONNEXIN43 GAP JUNCTION PROTEIN IN UNINFECTED AND ROUS-SARCOMA VIRUS-TRANSFORMED MAMMALIAN FIBROBLASTS
PHOSPHORYLATION OF CONNEXIN43 GAP JUNCTION PROTEIN IN UNINFECTED AND ROUS-SARCOMA VIRUS-TRANSFORMED MAMMALIAN FIBROBLASTS
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DOI：
10.1128/mcb.10.4.1754
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发表时间：
1990-04-01
影响因子：
5.3
通讯作者：
LAU, AF
中科院分区：
文献类型：
--
作者：
CROW, DS;BEYER, EC;LAU, AF
间隙连接是允许离子和其他低分子量分子在相邻细胞之间交换的膜通道。劳斯肉瘤病毒（RSV）诱导的转化的标志是早期和深刻的中断缝隙连接通信，这表明这些膜结构可能作为网站的pp 60 v-src的行动。我们已经开始了这种可能性的调查，通过确定和表征假定的蛋白质在成纤维细胞，目前用于研究RSV诱导的转化的主要细胞类型的交界处通信。我们发现，未感染的哺乳动物成纤维细胞似乎不包含RNA或蛋白质相关的连接蛋白32，主要大鼠肝脏间隙连接蛋白。相比之下，田鼠和小鼠成纤维细胞含有一个同源的3.0-腺苷酸酶RNA，其大小与编码差距连接蛋白（connexin 43）的心脏组织RNA相似。抗连接蛋白43肽抗血清特异性地与来自通信成纤维细胞的约43、45和47千道尔顿（kDa）的三种蛋白质反应。心脏细胞的缝隙连接主要包含45-和47-kDa的物种类似于在成纤维细胞中发现的。未感染的成纤维细胞45-和47-kDa蛋白磷酸化丝氨酸残基。45-和47-kDa的蛋白质和脉冲追踪标记的研究表明，这些蛋白质的磷酸化形式的43-kDa的蛋白质的磷酸化酶diglase。连接蛋白的磷酸化似乎在合成后不久发生，随后同样快速去磷酸化。与这些结果相比，RSV转化的成纤维细胞中的连接蛋白43蛋白含有磷酸酪氨酸和磷酸丝氨酸。因此，连接蛋白43中磷酸酪氨酸的存在与RSV转化成纤维细胞中观察到的缝隙连接通讯的丧失相关。
Gap junctions are membrane channels that permit the interchange of ions and other low-molecular-weight molecules between adjacent cells. Rous sarcoma virus (RSV)-induced transformation is marked by an early and profound disruption of gap-junctional communication, suggesting that these membrane structures may serve as sites of pp60v-src action. We have begun an investigation of this possibility by identifying and characterizing putative proteins involved in junctional communication in fibroblasts, the major cell type currently used to study RSV-induced transformation. We found that uninfected mammalian fibroblasts do not appear to contain RNA or protein related to connexin32, the major rat liver gap junction protein. In contrast, vole and mouse fibroblasts contained a homologous 3.0-kilobase RNA similar in size to the heart tissue RNA encoding the gap junction protein, connexin43. Anti-connexin43 peptide antisera specifically reacted with three proteins of approximately 43, 45 and 47 kilodaltons (kDa) from communicating fibroblasts. Gap junctions of heart cells contained predominantly 45- and 47-kDa species similar to those found in fibroblasts. Uninfected fibroblast 45- and 47-kDa proteins were phosphorylated on serine residues. Phosphatase digestions of 45- and 47-kDa proteins and pulse-chase labeling studies indicated that these proteins represented phosphorylated forms of the 43-kDa protein. Phosphorylation of connexin protein appeared to occur shortly after synthesis, followed by an equally rapid dephosphorylation. In comparison with these results, connexin43 protein in RSV-transformed fibroblasts contained both phosphotyrosine and phosphoserine. Thus, the presence of phosphotyrosine in connexin43 correlates with the loss of gap-junctional communication observed in RSV-transformed fibroblasts.