Date of Award

2005

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular and Cellular Biology and Pathobiology

College

College of Graduate Studies

First Advisor

Yusuf A. Hannun

Second Advisor

Maria G. Buse

Third Advisor

Lina M. Obeid

Fourth Advisor

Maurizio Del Poeta

Fifth Advisor

John R. Raymond

Abstract

Members of the protein kinase C (PKC) superfamily transduce a myriad of transmembrane signals initiated by the formation of sn-l ,2-diacylglycerol (DAG). As the primary physiological agonist for C] domain containing PKC isoenzymes, DAG functions to regulate the spatial and temporal parameters of PKC activity. It is now recognized that cellular DAG levels can vary from minutes to hours and even days. These observations have suggested a division of PKC functions that correlate to the duration of the cellular DAG signal. In their classical role, cPKCs respond acutely to the short-lived (1-2 min) DAG generated at the plasma membrane by the phospholipase C-mediated turnover of phosphoinositides. In contrast, a more persistent DAG signal can be produced through a variety of physiologic as well as pathologic mechanisms, and this DAG has also been proposed to regulate PKC activity. Currently, there are conflicting data on this issue and the specific subcellular localization of cPKC during prolonged DAG is unknown. To investigate the target of PKC during sustained activation, green fluorescent protein (GFP) technology was utilized to monitor the subcellular traffic of various PKC isoenzymes in response to treatment with either DAG-mimicking phorbol esters or DAG-generating agonists (e.g. platelet-derived growth factor). In response to a persistent elevation of DAG, cPKC isoenzymes, α and βII, translocated to the plasma membrane and to a juxtanuclear location in a variety of cell lines examined. Characterization of this compartment revealed that it overlapped/co-localized with a rab II-positive subcompartment of recycling endosomes concentrated around the MTOC/centrosome. The cPKC compartment was distinguish from the rab II-positive compartment by several features including a requirement for kinase and phospholipase D activity, an enrichment of lipid raft components, and the independence of microtubules and temperature for maintenance of the structure. Investigation into the significance of this compartment revealed that cPKC translocation coincided with the attendant sequestration of membrane recycling components. Given these distinctions, it was proposed that this novel cPKC-compartment be named the pericentrion in order to distinguish it from the rab 11 subcompartment of recycling endosomes. Subsequent analysis of a disease model wherein cPKC is maintained in a persistently active state, in this case chronic hyperglycemia associated with type I and type II diabetes, revealed a possible role for cPKC in the dysregulation of GLUT4 trafficking in response to insulin. These studies identify a novel site for cPKC translocation and function in response to a physiological and pathological elevation of DAG.

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