PROTEOLYTIC REGULATION & NEURAL FUNCTION OF ATYPICAL PKC

蛋白水解调节

• 批准号: 2675632
• 负责人: TODD C SACKTOR
• 金额: $ 21.4万
• 依托单位: SUNY DOWNSTATE MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-04-01 至 2001-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2675632
• 关键词: enzyme activity; hippocampus; laboratory rat; long term potentiation; neural inhibition; neural transmission; phosphorylation; protein kinase C; proteolysis

DESCRIPTION (Adapted from applicant's abstract): PKC has been implicated in both short-term and long-term forms of synaptic plasticity which are believed to contribute to the physiological mechanisms of memory, and thus to behavioral and mental states. One widely held hypothesis for the persistence of changes in synaptic strength is that enzymes, such as PKC, which can enhance synaptic transmission in the short-term, become persistently active in the long-term. We have examined the regulation of PKC following a high-frequency tetanus of afferents in the CA1 region of the hippocampus that produces a long-term synaptic enhancement, LTP. We have observed that during LTP, PKC becomes persistently active through the proteolytic removal of the enzyme's autoinhibitory regulatory domain, increasing the level of the free, independently active catalytic fragment, termed PKM. This proteolytic activation, however, is specifically directed towards the ayptical zeta isoform of the enzyme, only one of the 10 isozymes of PKC. In addition, we have observed that low-frequency stimulation of afferents that produces LTD decreases the level of PKMzeta by proteolytic downregulation. The overall goal of this grant is to investigate how the unique properties of a typical PKCzeta, working in combination with other signal transduction pathways, regulate proteolytic processing to produce increases or decreases in PKMzeta during LTP and LTD, respectively. We will use a combination of biochemical and physiological techniques, both in vitro and in the hippocampal slice preparation, to address the following 3 Specific Aims: (1) to develop an assay to study the proteolytic processing of zeta. Preliminary results in vitro indicate that the calcium-dependent protease, calpain, can either form or degrade PKMzeta, depending upon the site of proteolytic cleavage on zeta. (2) To characterize the mechanisms that modulate this proteolytic processing. We have observed that the atypical isoform zeta, but not the conventional isoforms of PKC, has 2 potential CaM-kinase II phosphorylation sites in its catalytic domain. We will examine whether these, or other forms of post-translational modification of zeta, regulate the accessibility of zeta's cleavage sites to determine the direction of the change in PKMzeta. (3) To determine the functional consequences of the proteolytic activation of zeta on the long-term modulation of synaptic transmission. We have found that zeta phosphorylation can be isolated by the application of specific combinations of pharmacological agents. We will use these combinations in hippocampal slices to isolate the role of zeta phosphorylation during the maintenance of long-term synaptic plasticity. These specific aims will help to achieve a detailed molecular and functional analysis of the proteolytic regulation of PKMzeta that causes both increases and decreases of constitutively active PKC during bidirectional synaptic plasticity.

描述（改编自申请人的摘要）：PKC已与 短期和长期突触可塑性的形式 被认为有助于记忆的生理机制，因此 行为和精神状态。 一个广泛持有的假设 突触强度变化的持久性是酶，例如PKC， 可以在短期内增强突触传播 从长远来看持续活跃。 我们已经检查了 PKC之后，在CA1区域的传入量高频破伤力之后 海马产生长期突触增强，LTP。 我们有 观察到在LTP期间，PKC通过 蛋白水解去除酶的自身抑制性调节结构域， 提高自由，独立活性催化片段的水平， 称为PKM。 但是，这种蛋白水解活化是专门定向的 朝向酶的杀伤性Zeta同工型，只有10个同工酶中的一个 PKC。 此外，我们已经观察到低频刺激 产生LTD的传入会降低蛋白水解的pkmzeta水平 下调。 这笔赠款的总体目标是调查如何 典型的pkczeta的独特特性，与其他合并 信号转导途径，调节蛋白水解处理以产生 在LTP和LTD期间，PKMZETA的增加或减少。 我们将 使用生化技术和生理技术的结合，都在体外 在海马切片准备中，以解决以下3 具体目的：（1）开发研究蛋白水解处理的测定 Zeta。 初步结果体外表明钙依赖性 蛋白酶，钙蛋白酶可以形成或降解pkmzeta，具体取决于 Zeta上的蛋白水解切割部位。 （2）表征机制 调节这种蛋白水解处理。 我们已经观察到 非典型同工型Zeta，但没有PKC的常规同工型，有2个 潜在的CAM-激酶II磷酸化位点其催化结构域中。 我们 将检查这些或其他形式的翻译后 修改Zeta，调节Zeta的裂解位点的可访问性 确定PKMZETA变化的方向。 （3）确定 Zeta蛋白水解激活在Zeta上的功能后果 长期调节突触传播。 我们发现Zeta 可以通过应用特定组合来隔离磷酸化 药理学剂。 我们将在海马中使用这些组合 切片以隔离Zeta磷酸化在维持过程中的作用 长期突触可塑性。 这些具体目标将有助于实现 蛋白水解调节的详细分子和功能分析 pKmzeta会导致组成性活跃的增加和减少 双向突触可塑性期间的PKC。