Structure and function of a mitochondrial PP2A holoenzyme that regulates neuronal survival

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 Bβ regulatory gene termed Bβ2. I found that the divergent N-terminus of Bβ2 does not affect phosphatase activity or holoenzyme association but encodes a mitochondrial targeting signal. Moreover, transient and stable expression of wild-type Bβ2 but not Bβ1, Bβ2 mutants defective in mitochondrial targeting or a monomeric mutant unable to associate with the holoenzyme, promotes apoptosis in neurons while knock-down of endogenous Bβ2 is neuroprotective. Furthermore, I identified the mechanisms by which Bβ2 incorporates the PP2A holoenzyme. By performing charge reversal mutagenesis in Bγ 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 β-propeller. To identify residues in Bβ2 important for mitochondrial association, I performed mutagenesis of the divergent N-terminus of Bβ2 and identified basic and hydrophobic residues that are critical for mitochondrial association. The variable N-terminal tail of Bβ2 is a cryptic mitochondrial import sequence that promotes import of GFP, but not full-length Bβ2, because its β-propeller domain resists the partial unfolding step necessary for translocation. Lastly, I addressed the mechanism by which Bβ2 promotes apoptosis in neurons. I found that overexpressing Bβ2 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)