Xiaohong Yan

Date of Award

6-27-2005

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Bryon D. Grove

Abstract

Gravin, an A Kinase Anchoring Protein, has been shown through biochemical studies to bind to the regulatory subunit of type II PKA and to PKCα. Preliminary studies also show that gravin localizes to the cell periphery but redistributes to an intracellular vesicular compartment in response to PKC activation using PMA. Although the intracellular targeting mechanisms of some AKAPs have been explored, the targeting domains and possible targeting mechanisms of gravin remain unknown. In addition, the capacity for gravin to affect the subcellular distribution of PKA has not been investigated in vivo. To identify the domains critical in determining the subcellular distribution of gravin, a full-length gravin-EGFP construct and a series of gravin-EGFP mutant constructs were generated and transfected into ANKA cells. Fluorescence micrographs and quantitative data showed that mutant gravin-EGFP lacking either the myristoylation site or the polybasic domains had a peripheral distribution similar to that of full-length gravin, but mutant gravin-EGFP lacking both the myristoylation site and the three polybasic domains had a cytoplasmic distribution. Gravin-EGFP mutants lacking the myristoylation site but containing each of the individual polybasic domains showed a partial peripheral distribution. To study the influence of gravin expression on the distribution of PKA RII, PKA RII-ECFP was coexpressed with either the full-length gravin-EGFP or its mutants in AN3CA and HEC-1-A cells. Microscopic localization studies revealed that PKA RII-ECFP localized to the cell periphery in cells coexpressing full-length gravin-EGFP, but was diffusely distributed in cells coexpressing mutant gravin-EGFP lacking either the membrane targeting domains or the PKA binding domain. PKA RII-ECFP was also diffusely distributed in cells transfected with the PKA RII-ECFP construct alone. Treatment of cells coexpressing the full-length gravin-EGFP and PKA RII-ECFP with PMA resulted in gravin-EGFP and PKA RII-ECFP relocalizing to a perinuclear vesicular. These results demonstrate that a putative myristoylation site and three polybasic regions participate in directing gravin to the cell periphery. These results further show that gravin targets PKA RII to the cell periphery and to a perinuclear vesicular compartment when PKC is activated. It is postulated that gravin may participate in crosstalk between PKA and PKC signaling pathways.

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