Graduation Year

2006

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Chemistry

Major Professor

Robert Potter, Ph.D.

Keywords

Translocation, Isoforms, Astroglial cells, Phosphorylation, Apoptosis

Abstract

O-linked N-acetylglucosamine (O-GlcNAc) is an abundant and ubiquitous post-translational modification that has been shown to play a role in regulating a variety of intracellular processes. The pathway responsible for generating the O-GlcNAc modification, the hexosamine biosynthetic pathway (HBP), has also been shown to affect the activity and translocation of certain protein kinase C (PKC) isoforms. To investigate if the effects of HBP flux on PKC translocation observed by others is related to the O-GlcNAc modification, O-GlcNAc levels in human astroglial cells were elevated using four separate O-GlcNAc modulating agents followed by analysis of cytosol and membrane concentrations of PKC-epsilon, -alpha, -betaII, and -iota. Of the four PKC isoforms analyzed, PKC-epsilon showed a significant reduction in its membrane associated levels in response to all agents tested whereas PKC-alpha showed reductions in response to only two agents.

Investigation of the mechanism for the reductions in membrane associated PKC-epsilon and -alpha indicate that the increased O-GlcNAc levels did not disrupt the activation of these isoforms or their ability to translocate to the plasma membrane. Furthermore, results indicate that these reductions are not due to a disruption in the Hsp70 mediated recycling of the isoforms. It was found; however, that increased O-GlcNAc levels resulted in increased degradation of PKC-epsilon suggesting that the decreases in membrane associated PKC-epsilon may be a result of increased phosphatase or protease activity.

Additional studies revealed that decreases in membrane bound PKC-epsilon and PKC-alpha, both of which act as anti-apoptotic enzymes, correlated with an increase in poly-(ADP-ribose) polymerase (PARP) cleavage -- a well characterized hallmark of apoptosis. In addition to PKC, the effects of increased O-GlcNAc levels on a related kinase, Akt, were also examined. Initial investigation of the effects of increased O-GlcNAc modification of Akt activation using glucosamine or streptozotocin revealed a relatively large, short-term increase in Akt phosphorylation in response to these treatments. However, further analysis with other O-GlcNAc modulators indicated that this activation was not related to O-GlcNAc protein modification. Furthermore, this activation does not appear to be related to any hyperosmotic effects associated with the treatment conditions, nor does it appear to be related to oxidative stress. Therefore, further investigation is needed to characterize the novel pathway responsible for Akt activation following glucosamine or streptozotocin treatment.

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