LPS-MEDIATED REGULATION OF PROTEIN KINASE C DURING MACROPHAGE ACTIVATION

拟议研究的总体目标是阐明 LPS引发的细胞内信号转导中的生化事件 巨噬细胞的杀肿瘤活性。 根据初步结果，我们 我建议检查一个假设，即蛋白激酶C（PKC），它起着重要的作用。 在杀肿瘤活化中的关键作用，在LPS-巨噬细胞 通过两种不同的途径相互作用。 在LPS激活和 非活化巨噬细胞系，LPS激活PIP 2特异性 磷脂酶C，导致产生的第二信使，DAG 和Ca 2+通过IP 3的作用从细胞内储存位点释放， 然后激活PKC 在LPS激活的细胞中，LPS可能与 Ca 2+依赖性中性蛋白酶calpain的30 kDa亚基， 激活钙蛋白酶的80 kDa亚基，然后在V3切割PKC 区域以产生表示为PKM的催化结构域片段。 非 可激活的细胞可能缺乏第二种PKC激活机制， PKC的V3区的结构改变或定性和/或 钙蛋白酶/钙蛋白酶抑制素系统的定量缺陷。 的假设 将通过追求四个具体目标进行测试：1）调查是否 或不LPS处理LPS可激活的细胞将导致钙蛋白酶- 催化PKC裂解成调节和催化结构域片段; 2)为了确定脂多糖非激活细胞中的PKC是否 与可活化细胞中的结构不同; 3）表征 LPS介导的小鼠巨噬细胞中钙蛋白酶的调节;和4） 与莫里森博士（项目1负责人）合作， 无论80 kDa LPS结合蛋白（LPS受体）和80 kDa LPS结合蛋白（LPS受体）是否 钙蛋白酶kDa亚基之间存在相关性。 我们的方法将是：1）亲和力 用放射性标记PKC的调节和催化结构域 对每个结构域特异的配体; 2）多克隆抗体的产生 通过使用合成肽作为抗原针对每个结构域; 3） PKC及其结构域的分离和鉴定; 4）PKC及其结构域的分离和鉴定， LPC激活型和非激活型PKC β基因序列分析 细胞; 5）钙蛋白酶和钙蛋白酶抑制素的表征; 6） LPS与分离的两种钙蛋白酶亚基之间的相互作用 7）LPS结合蛋白和80 kDa巨噬细胞的比较 钙蛋白酶亚基。 其中许多研究将在 与莫里森和罗素博士的合作。 一些生物化学 这些发现也应该适用于Parmely博士对TGF-β的研究。 所获得的结果将是重要的，因为它们有望阐明 巨噬细胞激活肿瘤细胞的早期信号传导事件 杀伤，PKC的结构和功能，相互作用的性质 PKC与calpain/calpastatin的调节机制，LPS- 诱导激活钙蛋白酶，和一些生化基础， 异常的巨噬细胞反应

The overall objective of the proposed research is to elucidate the biochemical events involved in signal transduction during LPS-triggered tumoricidal activation of macrophages. Based on our initial results, we propose to examine a hypothesis that protein kinase C (PKC), which plays a key role in tumoricidal activation, is activated, following LPS-macrophage interaction, through two different pathways. In both LPS-activatable and nonactivatable macrophage cell lines, LPS activates PIP2-specific phospholipase C, leading to the generation of the second messengers, DAG and Ca2+ released from intracellular storage site by the action of IP3, which then activate PKC. In LPS-activatable cells, LPS may interact with the 30kDa subunit of the Ca2+-dependent neutral protease, calpain, to activate the 80kDa subunit of calpain, which then cleaves PKC at the V3 region to generate the catalytic domain fragment denoted as PKM. Non- activatable cells may lack the second PKC activation mechanism due to either structural alteration of the V3 region of PKC or qualitative and/or quantitative defects in the calpain/calpastatin system. The hypothesis will be tested by pursuing four specific aims: 1) to investigate whether or not LPS treatment of LPS-activatable cells will result in the calpain- catalyzed cleavage of PKC into regulatory and catalytic domain fragments; 2) to determine whether or not PKC in LPS-non-activatable cells id different in structure from that in activatable cells; 3) to characterize the LPS-mediated regulation of calpain in mouse macrophages; and 4) to investigate, in collaboration with Dr. Morrison (project #1 leader), whether or not the 80 kDa LPS-binding protein (an LPS receptor) and the 80 kDa subunit of calpain are related. Our approaches will be: 1) affinity labeling of regulatory and catalytic domains of PKC with the radioactive ligands specific for each domain; 2) production of polyclonal antibodies directed against each domain by using synthetic peptides as antigens; 3) isolation and characterization of PKC and its domains; 4) isolation and sequence analysis of PKCbeta gene of LPC-activatable and non-activatable cells; 5) characterization of calpain and calpastatin; 6) studies of the interaction between LPS and two subunits of calpain isolated from macrophages; and 7) comparison of the LPS-binding protein and the 80kDa subunit of calpain. Many of these studies will be carried out in collaboration with Drs. Morrison and Russell. Some of the biochemical findings should also be applicable to Dr. Parmely's studies of TGF-beta. Results obtained will be important because they are expected to illuminate early signal transduction events in macrophage activation for tumor cell killing, the structure and function of PKC, the nature of the interaction between PKC and calpain/calpastatin, the regulatory mechanism of LPS- induced activation of calpain, and some of the biochemical bases of aberrant macrophage responses.