ROLE OF CALMODULIN-DEPENDENT PROTEIN KINASE IN ANERGY

T细胞克隆性无反应性，一种无反应状态，其特征在于关键的 降低T细胞产生白细胞介素-2（IL-2）的能力， 是外周和/或胸腺耐受的一种建议机制。 的 无反应性的生物化学基础是未知的。 我们最近克隆了 多功能钙/钙调蛋白依赖性蛋白激酶γ亚型 (CaM激酶），并已证明其激活后， T细胞受体（TCR）刺激。 淋巴细胞CaM激酶保留 先前在神经元中证实的特征性调节特征 同种型，包括在Thr 286的自磷酸化，将其转化为 部分Ca 2+非依赖性物种。 我们的初步研究表明 在Jurkat细胞中，CaM激酶负调节IL-2的表达， 提示CaM激酶可能作为Ca 2+依赖性效应子发挥作用 介导无反应性的分子。 我们提案的总体目标是 进一步证实了钙调素激酶在无能中的作用， 以下具体目标：（1）通过共表达组成型 （相对于失活的）CAM激酶和IL-2报告基因构建体， 记录了CaM激酶对IL-2转录的负调节;（2）通过 特异性CaM激酶抑制剂，以证明参与 CaM激酶在已建立的模型中，其中克隆的T细胞被赋予 通过在MHC转染的L细胞上呈递抗原而无反应性;（3）通过 组成型CaM激酶的共表达方法，组成型 钙调神经磷酸酶和IL-2报告基因构建体，以确定 CaM激酶的负调节作用存在于上游，下游， 或在钙调神经磷酸酶作用的水平上;（4）确定 共刺激信号通过CD 28受体对Ca 2+信号，钙调素 可用性，以及CaM激酶与 钙调神经磷酸酶

T cell clonal anergy, a nonresponsive state characterized by a critical reduction in the ability of the T cell to produce interleukin-2 (IL-2), is a proposed mechanism for peripheral and/or thymic tolerance. The biochemical basis for anergy is unknown. We have recently cloned the gamma isoform of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM Kinase) from lymphocytes and have demonstrated its activation after T cell receptor (TCR) stimulation. Lymphocyte CaM kinase retains characteristic regulatory features previously demonstrated in neuronal isoforms, including autophosphorylation at Thr286, converting it to a partially Ca2+-independent species. Our preliminary studies indicate that CaM kinase negatively regulates IL-2 expression in Jurkat cells, suggesting that CaM kinase may play a role as the Ca2+ dependent effector molecule mediating anergy. Our overall objective in the proposal is to further substantiate a role for CaM kinase in anergy by addressing the following specific aims: (1) by means of coexpression of constitutive (versus inactive) CAM kinase and IL-2 reporter gene constructs, to document negative regulation of IL-2 transcription by CaM kinase; (2) by means of specific CaM kinase inhibitors, to demonstrate involvement of CaM kinase in an established model in which cloned T cells are rendered anergic by presentation of antigen on MHC transfected L cells; (3) by means of coexpression of constitutive CaM kinase, constitutive calcineurin, and IL-2 reporter gene constructs, to determine if the negative regulatory action of CaM kinase resides upstream, downstream, or at the level of calcineurin action; (4) to determine the effect of the costimulatory signal via the CD28 receptor on the Ca2+ signal, calmodulin availability, and the relative activation of CaM kinase versus calcineurin.