Protein Kinase A-II in the Pathogenesis of Lupus

狼疮发病机制中的蛋白激酶 A-II

• 批准号: 6327266
• 负责人: GARY M KAMMER
• 金额: $ 25.32万
• 依托单位: WAKE FOREST UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-03-01 至 2006-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6327266
• 关键词: AP1 protein; cAMP response element binding protein; clinical research; enzyme activity; enzyme deficiency; genetic regulatory element; human subject; interleukin 2; isozymes; molecular pathology; phlebotomy; protein kinase A; protooncogene; systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is characterized by disordered T lymphocyte signal transduction. T cells exhibit impaired protein kinase A (PKA)-catalyzed protein phosphorylation due to a profound deficiency of type I PKA (PKA-I) isozyme phosphotransferase activity. Recently, we identified a concomitant deficiency of PKA-II isozyme activity in SLE T cells. Deficient PKA-II activity is associated with (a) autophosphorylation and aberrant translocation of the beta isoform of the type II regulatory subunit (RIIbeta) from the cytosol to the nucleus; (b) accumulation and retention of nuclear RIIbeta; and, (c) reduced or undetectable c-Fos cytosolic protein. Therefore, we hypothesize that aberrant nuclear translocation of autophosphorylated RIIbeta results in (a) deficient PKA-II activity, (b) under-phosphorylation of nuclear cAMP response element binding protein (CREB) transcription factor, (c) impaired c-fos transcriptional activation, (d) reduced levels of c-fos transcript and c-Fos protein, (e) decreased formation of AP-1 transcription factor, and (f) impaired IL-2 transcriptional activation. The specific aims of this proposal are: (l) To demonstrate that autophosphorylated RIIbeta-subunit is a transcription factor that forms a RIIb-CREB heteromeric complex and acts as a transcriptional repressor of CREB-mediated c-fos transcription in normal primary T cells; (2) To identify the mechanism(s) leading to aberrant translocation of the RIIbeta-subunit from the cytosol to the nucleus in SLE T cells; (3) To determine if PKA-II-catalyzed phosphorylation of CREB is impaired and hinders its binding to CREB binding protein (CBP) and transcriptional activation of the c-Fos promoter in SLE T cells; and, (4) To determine if there is diminished AP-1 binding to consensus AP-1 sites of the IL-2 promoter/enhancer that results in reduced IL-2 production in SLE T cells. The principal goal of the proposed experiments is to establish the mechanism(s) by which deficient PKA-II isozyme activity contributes to altered c-Fos and IL-2 transcriptional activation, loss of AP-1, and diminished IL-2 production by SLE T cells. Demonstrating a connection between reduced T cell IL-2 production and deficient PKA-II isozyme activity in SLE T cells will address a principal gap in our understanding of the molecular and cellular pathophysiology of T cell immunodysfunctions in SLE.

全身性红斑狼疮（SLE）的特征是T淋巴细胞信号转导。 T细胞表现出受损的蛋白激酶A（PKA）催化的蛋白磷酸化，这是由于I型PKA（PKA-I）同工酶磷酸转移酶活性的严重缺陷。最近，我们确定了SLE T细胞中PKA-II同工酶活性的伴随缺乏。缺乏PKA-II活性与（a）II型调节亚基（Riibeta）从细胞质到细胞核的II型调节亚基（Riibeta）的β同工型的自磷酸化和异常易位有关； （b）核riibeta的积累和保留； （c）降低或无法检测到的C-FOS胞质蛋白。因此，我们假设自磷酸化的riibeta的异常核易位导致（a）核营地响应蛋白（CREB）转录因子（CREB）转录因子（C）C-FOS转录率的降低，（D）降低了C-FOS的pROS-FOS蛋白质（e-FOS）的水平，（e （f）IL-2转录激活受损。该提案的具体目的是：（l）证明自磷酸化的riibeta-subunit是一种转录因子，形成了RIIB-CREB异质体复合物，并充当正常主要T细胞中CREB介导的C-FOS转录的转录抑制剂； （2）确定导致riibeta-subunit异常易位的机制，从细胞质到SLE T细胞中的细胞核； （3）确定CREB的PKA-II催化磷酸化是否受到损害，并阻碍其与CREB结合蛋白（CBP）的结合以及C-FOS启动子在SLE T细胞中的转录激活； （4）确定与IL-2启动子/增强子的共有AP-1位点的AP-1结合减少，从而导致SLE T细胞中IL-2产生降低。提出的实验的主要目标是建立缺乏PKA-II同工酶活性有助于改变C-FOS和IL-2转录激活，AP-1的丢失以及SLE T细胞产生IL-2的机制。证明SLE T细胞中T细胞IL-2的产生减少与缺乏PKA-II同工酶活性之间的联系将解决我们对SLE中T细胞免疫功能的分子和细胞病理生理学的理解。