Date of Award

1997

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular and Cellular Biology and Pathobiology

College

College of Graduate Studies

First Advisor

Kathryn E. Meier

Second Advisor

Philippe Arnaud

Third Advisor

Maria G. Buse

Fourth Advisor

Joseph Dolan

Fifth Advisor

John Hildebrant

Sixth Advisor

Mark Willingham

Abstract

The overall goal of this study was to investigate the roles of PLD and neutral SMase in signal transduction in T-lymphocytes. In particular, the abilities of phospholipid metabolites to regulate mitogen-activated protein kinases were explored. The tumor promoting PKC activator, phorbol 12-myristate 13-acetate (PMA), and an antileukemic drug, 1-β-D arabinofuranosylcytosine hydrochloride (Ara-C), were used as model phospholipase activators. The effects of phorbol ester and Ara-C on PLD and SMase activities were examined in EL4, a murine thymoma cell line, and Jurkat, a human leukemia cell line. PLD activity was measured in intact cells using a metabolic labelling assay. PLD and SMase activities were measured in membrane extracts utilizing fluorescent phosphatidylcholine (PLD) and sphingomyelin (SMase) as substrates, respectively. Rapid activation of PLD by PMA was detected in Jurkat cells, but not in EL4 cells. Jurkat, but not EL4 cells, express a 120-kDa protein recognized by an anti-PLD antibody. Ara-C activated neutral SMase within 10 minutes in both Jurkat and EL4. PMA did not activate SMase and Ara-C did not activate PLD. The relationships between activation of PLD, SMase and members of the mitogen-activated protein kinase family, ERKs (extracellular-regulated kinases) and JNKs (c-jun N-terminal kinases), were examined using in vitro phosphorylation assays. PMA induced activation of ERKs in both Jurkat and EL4 cells, while Ara-C or ceramides had no effect. Incubation of EL4 cells with bacterial PLD increased phosphatidic acid levels, but did not activate ERKs. JNK activity was detected within 10 minutes after co-stimulation with PMA and ionomycin in both EL4 and Jurkat cells. Ara-C activated JNKs in these cells only after prolonged incubation (90-120 minutes). These results suggest that activation of PLD is neither necessary nor sufficient for activation of ERKs or JNKs. The delayed effects of Ara-C on JNK activity may be mediated through secondary response pathways. The mechanism by which PMA activates PLD was examined In Jurkat cells and in a prostate cancer cell line. No evidence was obtained for PLD translocation, phosphorylation, or binding to protein kinase Ca. These results are consistent with observations that PLD can be activated by protein-protein and/or protein-lipid interactions. The data obtained in these studies indicate that PLD and neutral SMase are activated via distinct pathways in T-lymphocyte cell lines. While their reaction products do not directly regulate mitogen-activated protein kinases, the activation of these phospholipases by mitogenic and cytotoxic stimuli suggest that further understanding of the regulation of PLD and SMase and their roles in signal transduction may present future avenues for the improved therapy of T-Iymphocyte pathologies.

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