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型蛋白激酶A(PKA-I)同工酶磷酸转移酶活性的严重缺乏，T细胞呈现蛋白激酶A(PKA)催化的蛋白磷酸化受损。最近，我们在SLE T细胞中发现了一种伴随的PKA-II同工酶活性缺陷。PKA-II活性的缺陷与(A)自磷酸化和II型调节亚基(RIIbeta)的β亚基从胞浆到细胞核的异常移位有关；(B)核RIIbeta的积累和保留；以及(C)c-Fos胞浆蛋白的减少或检测不到。因此，我们假设自磷酸化RIIbeta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转录和c-fos蛋白水平降低，(E)AP-1转录因子形成减少，(F)IL-2转录激活受损。这一建议的具体目的是：(L)证明在正常的原代T细胞中，自磷酸化的RIIb-β亚基是一种转录因子，形成RIIb-CREB异构体复合体，并作为CREB介导的c-fos转录的转录抑制因子；(2)确定SLE细胞中RIIbeta-亚基异常移位的机制(S)；(3)确定PKA-II催化的CREB的磷酸化是否受损，并阻碍其与CREB结合蛋白(CBP)的结合和c-Fos启动子的转录激活；以及，(4)确定是否存在AP-1与IL-2启动子/增强子的共识AP-1结合减少，从而导致SLE T细胞产生IL-2减少。本实验的主要目的是建立PKA-II同工酶活性缺陷导致系统性红斑狼疮T细胞c-Fos和IL-2转录激活改变、AP-1丢失和IL-2产生减少的机制(S)。证明T细胞IL-2的产生减少与SLE T细胞中PKA-II同工酶活性低下之间的联系，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的主要认识差距。

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.