Endothelin Signaling and Regulation of Protein Kinases

描述（由申请方提供）：内皮素1（ET-1）对肾血管系统和肾小球系膜细胞（GMC）发挥血管收缩、肥大和促有丝分裂作用。ET-1依赖的GMC收缩可调节肾小球超滤系数，ET-1诱导的肾小球肥大和增生可能参与多种类型的增生性和硬化性肾小球疾病。用ET-1刺激GMC引起各种各样的信号传导事件;然而，ET-1诱导的细胞增殖和肥大主要通过激活细胞外信号调节激酶（ERK）发生。GMC的收缩反应依赖于p38 MAPK的激活。我们推测，蛋白质-蛋白质相互作用是内皮素诱导的细胞内信号通路中的关键组成部分，导致ET-1的短期和长期效应，并且通常调节这些相互作用的蛋白质酪氨酸磷酸化可能是肾脏疾病演变的主要因素。具体目标1将评估酪氨酸激酶Pyk 2在ET-1介导的GMC收缩性中的假定参与。我们将用腺病毒介导的方法将显性干扰突变体和野生型Pyk 2基因导入大鼠和人GMC，并分析Pyk 2信号转导抑制对ET-1介导的系膜细胞收缩、调节p38 MAPK的小GTP酶激活、MAP激酶激活的蛋白激酶2/3激活的影响。（p38 MAPK的下游底物）、HSP 25的磷酸化和肌动蛋白丝动力学的调节。具体目标2将评估鸟嘌呤核苷酸交换因子Pix介导内皮素对GMC中小GTdc 42的刺激的假设。我们将研究a）Pix与参与系膜细胞中ET-1信号传导的异源三聚体G蛋白的直接相互作用; B）Pix的显性干扰突变体抑制ET-1依赖性cdc 42活化的能力; c）Pix的显性干扰突变体抑制MAPK的ET-1依赖性活化的能力。具体目标3将评估的假设，内皮素信号转导的细胞内MAPK级联的水平上的双特异性磷酸酶的负调节是一个重要组成部分的正常信号转导的ET-1。我们假设MKP-3抑制ET-1介导的ERK激活，而MKP-1减弱p38 MAPK，MKP-1和MKP-3对MAPK信号的严格调节控制GMC肥大关键基因的表达。

DESCRIPTION (provided by applicant): Endothelin 1 (ET-1) exerts vasoconstrictor, hypertrophic and mitogenic actions on the renal vasculature and glomerular mesangial cells (GMC). ET-1-dependent contraction of GMC can regulate the glomerular ultrafiltration coefficient and ET-1 induced glomerular hypertrophy and proliferation may contribute to diverse types of proliferative and sclerotic glomerular diseases. Stimulation of GMC with ET-1 evokes a wide variety of signaling events; however, ET-1-induced cell proliferation and hypertrophy occurs primarily via activation of the extracellular signal-regulated kinase (ERK). Contractile responsiveness of GMC was shown to depend on activation of the p38 MAPK. We hypothesize that protein-protein interactions are among crucial components in the endothelin-induced intracellular signaling pathways leading to short- and long-term effects of ET-1 and that protein tyrosine phosphorylation, which often regulates these interactions, may be a principal factor in evolution of renal diseases. Specific Aim 1 will evaluate the putative involvement of tyrosine kinase Pyk2 in ET-1-mediated GMC contractility. We will carry out adenoviral mediated transfer of genes encoding dominant interfering mutant and wild type Pyk2 into rat and human GMC and analyze the effect of inhibition of signaling via Pyk2 upon ET-1 mediated mesangial cell contraction, activation of small GTPases regulating p38 MAPK, activation of MAP kinase-activated protein kinase 2/3 (downstream substrate of p38 MAPK), phosphorylation of HSP 25 and regulation of actin filament dynamics. Specific Aim 2 will evaluate the hypothesis that guanine nucleotide exchange factor Pix mediates stimulation of small GTPase cdc42 by endothelin in GMC. We will study a) the direct interaction of Pix with heterotrimeric G proteins, involved in ET-1 signaling in mesangial cells; b) ability of dominant interfering mutants of Pix to inhibit ET-l-dependent activation of cdc42; c) ability of dominant interfering mutants of Pix to inhibit ET-1- dependent activation of MAPKs. Specific Aim 3 will evaluate the hypothesis that negative regulation of endothelin signaling on the level of intracellular MAPK cascades by dual specificity phosphatases is an essential component of the normal signal transduction by ET-1. We hypothesize that MKP-3 inhibits ET-1 mediated ERK activation, whereas MKP-1 attenuates p38 MAPK and tight regulation of MAPK signaling by MKP-1 and MKP-3 controls expression of genes critical for GMC hypertrophy.