Protein Kinase A-II in the Pathogenesis of Lupus

系统性红斑狼疮（SLE）以T淋巴细胞信号转导紊乱为特征。由于I型PKA（PKA-I）同工酶磷酸转移酶活性的严重缺乏，T细胞表现出受损的蛋白激酶A（PKA）催化的蛋白磷酸化。最近，我们确定了PKA-II同工酶活性在SLE T细胞的伴随缺陷。PKA-II活性缺陷与（a）自磷酸化和II型调节亚基β亚型（RII β）从胞质溶胶到细胞核的异常易位有关;（B）细胞核RII β的蓄积和保留;以及（c）c-Fos胞质溶胶蛋白减少或检测不到。因此，我们假设自磷酸化RII β的异常核转位导致（a）PKA-II活性缺陷，（B）核cAMP反应元件结合蛋白（CREB）转录因子磷酸化不足，（c）c-fos转录激活受损，（d）c-fos转录物和c-Fos蛋白水平降低，（e）AP-1转录因子形成减少，和（f）IL-2转录激活受损。本研究的具体目的是：（1）证明自身磷酸化的RII β亚基是一种转录因子，它能形成RIIb-CREB异聚体复合物，并在正常原代T细胞中作为CREB介导的c-fos转录的转录抑制因子;（2）鉴定导致SLE T细胞中RII β亚基从胞浆到核的异常易位的机制;（3）确定PKA-II催化的CREB磷酸化是否受损并阻碍其与CREB结合蛋白（CBP）的结合和SLE T细胞中c-Fos启动子的转录激活;和（4）确定AP-1与IL-2启动子/增强子的共有AP-1位点的结合是否减少，这导致SLE T细胞中IL-2产生减少。所提出的实验的主要目标是建立PKA-II同工酶活性缺陷导致改变的c-Fos和IL-2转录激活、AP-1丧失和SLE T细胞产生减少的IL-2的机制。证明SLE T细胞中T细胞IL-2产生减少和PKA-II同工酶活性缺乏之间的联系将解决我们对SLE T细胞免疫功能障碍的分子和细胞病理生理学理解中的主要空白。

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.