Chew C S, Parente J A, Zhou C, Baranco E, Chen X
Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia 30912, USA.
Am J Physiol. 1998 Jul;275(1):C56-67. doi: 10.1152/ajpcell.1998.275.1.C56.
Activation of the cAMP signaling pathway is correlated with increased secretory-related events in a wide variety of cell types including the gastric parietal cell. Within this pathway, as well as in other intracellular signaling pathways, protein phosphorylation serves as a major downstream regulatory mechanism. However, although agonist and cAMP-dependent activation of cAMP-dependent protein kinase (PKA) has been demonstrated, little is currently known about the downstream in vivo phosphoprotein substrates of this enzyme. Here we report the isolation, microsequencing, and cloning of a LIM and SH3 domain-containing, cAMP-responsive, 40-kDa phosphoprotein (pp40) from rabbit gastric parietal cells. The deduced amino acid sequence for pp40 is 93.5%, homologous with the putative protein product of the human gene lasp-1, which was recently identified based on its overexpression in some breast carcinomas. In addition to LIM and SH3 domains, the rabbit homolog contains two highly conserved PKA consensus sequences as well as two conserved SH2 binding motifs and several other putative protein kinase phosphorylation sites, including two for tyrosine kinase(s). Combined Northern and Western blot analyses indicate that pp40/lasp-1 is widely expressed (through a single 3.3-kb message) not only in epithelial tissues but also in muscle and brain. Furthermore, stimulation of isolated parietal cells, distal colonic crypts, and pancreatic cells with the adenylyl cyclase activator forskolin leads to the appearance of a higher molecular weight form of pp40/lasp-1, a finding which is consistent with an increase in protein phosphorylation. Thus pp40/lasp-1 appears to be regulated within the cAMP signaling pathway in a wide range of epithelial cell types. Because the cAMP-dependent increase in pp40 phosphorylation is correlated with secretory responses in the parietal cell and because pp40 appears to be widely distributed among various secretory tissues, this newly defined signaling protein may play an important role in modulating ionic transport or other secretory-related activities in many different cell types.
环磷酸腺苷（cAMP）信号通路的激活与多种细胞类型（包括胃壁细胞）中与分泌相关事件的增加有关。在该信号通路以及其他细胞内信号通路中，蛋白质磷酸化是主要的下游调节机制。然而，尽管已经证实了激动剂和cAMP依赖性激活环磷酸腺苷依赖性蛋白激酶（PKA），但目前对该酶在体内的下游磷蛋白底物知之甚少。在此，我们报告了从兔胃壁细胞中分离、微量测序和克隆一种含有LIM和SH3结构域、对cAMP有反应的40 kDa磷蛋白（pp40）。pp40推导的氨基酸序列与人类基因l asp-1的推定蛋白质产物有93.5%的同源性，l asp-1最近因其在某些乳腺癌中的过表达而被鉴定。除了LIM和SH3结构域，兔同源物还包含两个高度保守的PKA共有序列以及两个保守的SH2结合基序和其他几个推定的蛋白激酶磷酸化位点，包括两个酪氨酸激酶的位点。Northern印迹和Western印迹联合分析表明，pp40/l asp-1不仅在上皮组织中广泛表达（通过单一3.3 kb的信使RNA），而且在肌肉和大脑中也广泛表达。此外，用腺苷酸环化酶激活剂福斯高林刺激分离的壁细胞、远端结肠隐窝和胰腺细胞会导致出现更高分子量形式的pp40/l asp-1，这一发现与蛋白质磷酸化增加一致。因此，pp40/l asp-1似乎在广泛的上皮细胞类型的cAMP信号通路中受到调节。由于壁细胞中pp40磷酸化的cAMP依赖性增加与分泌反应相关，并且由于pp40似乎广泛分布于各种分泌组织中，这种新定义的信号蛋白可能在调节许多不同细胞类型中的离子转运或其他与分泌相关的活动中发挥重要作用。
