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）已用作脂质调节蛋白的原型，在磷脂酰肌醇（PI）循环的情况下得到了最好的说明，其中PI衍生的二酰基甘油（DAG）用作PKC的直接激活剂。鉴于PI周期的快速周转（在受体接合的90秒内），PKC激活的这种范例必然集中在PKC调节的非常急性期。重要的是，这种模式没有解决DAG生成的其他机制，最值得注意的是通过磷脂酶D（PLD），它没有区分调节特定同工酶激活的机制。广泛的初步研究结果产生了令人兴奋的新的PKC机制和功能的信息。简而言之，我们发现i）持续激活PKC（30-60分钟）诱导PKC α和β II易位（但不是密切相关的PKC β I）到一个新的细胞核/中心周围区室; ii）这个区室具有以前确定的特征，iii）PLD是该易位所需的;和iv）PKC易位到该非核区室伴随着一些质膜蛋白（例如转铁蛋白受体和GM 4）和脂质（例如GM 1）重新定位/隔离到该区室。基于这些观察结果，我们推测PLD的刺激导致PKC α和β II的持续激活及其转运到中心粒周围区室。这种机制可能在质膜的再循环组分的螯合和可用性中起关键作用。这一假说将通过以下具体目标进行研究：1）定义和建立一种新的PKC易位; 2）定义非核PKC的调节机制和PLD的作用; 3）定义PKC在质膜和/或再循环成分的隔离中的作用。如果正确的话，这种新型的PKC易位和功能可能参与多种生理和病理生理过程，这些过程取决于PM处关键蛋白和/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.