Document Type

PhD diss.

Date of Degree

2006

Degree Name

PhD (Doctor of Philosophy)

Department

Pharmacology

First Advisor

Stefan Strack

Abstract

Serine/threonine phosphatase 2A (PP2A) consists of an AC core dimer composed of catalytic (C), structural (A) subunits complexed to a variable regulatory subunit derived from three gene families (B, B', B"). My dissertation work characterized the structure and function of a neuron-specific splice variant of the Bbeta regulatory gene termed Bbeta2. I found that the divergent N-terminus of Bbeta2 does not affect phosphatase activity or holoenzyme association but encodes a mitochondrial targeting signal. Moreover, transient and stable expression of wild-type Bbeta2 but not Bbeta1, Bbeta2 mutants defective in mitochondrial targeting or a monomeric mutant unable to associate with the holoenzyme, promotes apoptosis in neurons while knock-down of endogenous Bbeta2 is neuroprotective. Furthermore, I identified the mechanisms by which Bbeta2 incorporates the PP2A holoenzyme. By performing charge reversal mutagenesis in Bgamma as a model for B family regulatory subunits, I found that holoenzyme association requires multiple electrostatic charges clustered in WD repeats 3 and 4 of the beta-propeller. To identify residues in Bbeta2 important for mitochondrial association, I performed mutagenesis of the divergent N-terminus of Bbeta2 and identified basic and hydrophobic residues that are critical for mitochondrial association. The variable N-terminal tail of Bbeta2 is a cryptic mitochondrial import sequence that promotes import of GFP, but not full-length Bbeta2, because its beta-propeller domain resists the partial unfolding step necessary for translocation. Lastly, I addressed the mechanism by which Bbeta2 promotes apoptosis in neurons. I found that overexpressing Bbeta2 fragments mitochondria while RNAi of the endogenous protein promotes mitochondrial fusion in neurons. Conversely, targeting PKA, a well characterized prosurvival kinase, to the OMM by overexpressing A kinase anchoring protein 121 (AKAP121) opposes the effects of the phosphatase by elongating mitochondria. Furthermore, downregulating the endogenous AKAP121 by RNAi, or inhibiting PKA at the OMM by overexpressing an inhibitor of PKA (OMM-PKI) fragments mitochondria. The effects of OMM-targeted PP2A or PKA on survival require remodeling of mitochondria, since blocking mitochondrial fission reversed the proapoptotic effects of Bbeta2 and OMM-PKI. My dissertation provides a novel mechanism by which kinase/phosphatase signaling determines neuronal survival.

Pages

xvii, 192

Bibliography

173-191

Copyright

Copyright 2006 Ruben Karim Dagda

Included in

Pharmacology Commons

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