Role and Regulation of Protein Kinase C Isoenzymes

描述（由申请人提供）：从概念上讲，蛋白激酶C （PKC）作为脂质调节蛋白的原型，在磷脂酰肌醇（PI）循环中得到了最好的说明，其中PI衍生的二酰基甘油（DAG）作为PKC的直接激活剂。考虑到PI周期的快速周转（受体参与后90秒内），PKC激活的这种范式必然集中在PKC调控的非常急性阶段。重要的是，这种模式并没有解决DAG产生的其他机制，最明显的是通过磷脂酶D (PLD)，也没有区分调节特定同工酶激活的机制。广泛的初步结果为PKC的新机制和功能提供了令人兴奋的信息。简而言之，我们发现i) PKC的持续激活（30-60分钟）诱导PKC α和β i（但不包括密切相关的PKC β i）易位到一个新的核旁/中心周围室；Ii)这个隔室具有先前确定的（但不被充分认识的）循环核内体成分的特征；iii)此易位需要PLD；iv) PKC易位到核旁室时，一些质膜蛋白（如转铁蛋白受体和glut4）和脂质（如GM1）被重新定位/隔离到核旁室。基于这些观察结果，我们假设PLD的刺激导致PKC α和β i的持续激活，并将它们转移到中心周围室。这一机制可能在质膜的隔离和回收组分的可用性中起关键作用。这一假设将通过以下具体目标进行调查：1)定义并建立一种新的PKC易位；2)明确核旁PKC的调控机制及PLD的作用；3)确定PKC在质膜固存和/或回收组分中的作用。如果正确的话，这种新的PKC易位和功能可能涉及多种生理和病理生理过程，这些过程取决于PM中关键蛋白质和/或脂质的可用性，包括糖尿病和癌症。

DESCRIPTION (provided by applicant): Conceptually, Protein kinase C (PKC) has served as the prototype of lipid-regulated proteins, best illustrated in the case of the phosphatidylinositol (PI) cycle whereby PI-derived diacylglycerol (DAG) serves as the direct activator of PKC. Given the rapid turnover of the PI cycle (within 90 seconds of receptor engagement), this paradigm of PKC activation has by necessity focused on the very acute phase of PKC regulation. Importantly, this paradigm does not address other mechanisms of DAG generation, most notably through phospholipase D (PLD), and it does not distinguish mechanisms for regulating activation of specific isoenzymes. Extensive preliminary results have generated exciting information on novel mechanisms and functions of PKC. Briefly, we find that i) sustained activation of PKC (30-60 min) induces the translocation of PKC alpha and betaII (but not the closely related PKC betaI) to a novel juxtanuclear/pericentriolar compartment; ii) this compartment has features of a previously-identified (but not well-appreciated) component of recycling endosomes; iii) PLD is required for this translocation; and iv) translocation of PKC to this juxtanuclear compartment is accompanied by re-localization/sequestration of some plasma membrane proteins (e.g. transferrin receptor and glut4) and lipids (e.g. GM1) to this compartment. Based on these observations, we hypothesize that stimulation of PLD results in sustained activation of PKC alpha and betaII and their translocation to a pericentriolar compartment. This mechanism may play a key role in the sequestration and availability of recycling components of the plasma membrane. This hypothesis will be investigated by pursuing the following specific aims 1) Define and establish a novel translocation of PKC; 2) Define mechanisms of regulation of juxtanuclear PKC and role of PLD; 3) Define role of PKC in sequestration of plasma membrane and/or recycling components. If correct, this novel translocation and function of PKC may be involved in multiple physiologic and pathophysiologic processes that depend on availability of key proteins and/or lipids at the PM, including diabetes, and cancer.