Posttranslational regulation of protein function and stability at the mitochondria and beyond

The goal of this dissertation research is to determine how post translational modifications of Mfn2, Drp1 and PP2A B'beta regulate protein function and stability. Mitofusin 2 (Mfn2) and Dynamin related protein 1 (Drp1) work in opposing manners to balance mitochondrial morphology and maintain organelle function. Loss of balanced fission and fusion results in mitochondrial dysfunction, a major contributor to the pathology of many neurodegenerative diseases and cancer. Regulation of fission is mediated through the reversible posttranslational modifications of Drp1, which has been shown to be (de)phosphorylated on multiple residues, (de)sumoylated, nitrosylated and is believed to be ubiquitinated. Yet there are currently no known post-translational modifications of Mfn2 that regulate fusion. The first two experimental chapters of this thesis focus on the regulation of PKA induced phosphorylation of mitochondrial fission and fusion proteins. In Chapter 2 I utilize a Mfn2 phospho Ser442 specific antibody to determine the phosphorylation state of both over-expressed and endogenous neuronal Mfn2. Using this technique I found that under the conditions studied, Mfn2 is not PKA phosphorylated on Ser442. To look at the requirement of Mfn2 Ser442 in promoting mitochondrial elongation and protecting hippocampal neurons from excitotoxic cell death, Hela cells and primary hippocampal neurons (PHN) were evaluated by immunofluorescence. Results from these studies suggest Mfn2's neuroprotective effects require Ser442, and are separate from its function to promote mitochondrial fusion, and independent of PKA phosphorylation. Chapter 3 utilizes biochemical and immunofluorescence techniques to determine whether the PKA induced phosphorylation of Drp1 on Ser656 and the nitrosylation of Drp1 on Cys663 are mutually exclusive events. While previous work demonstrated the requirement of Drp1 Cys663 in mediating nitric oxide (NO) induced mitochondrial fragmentation and cell death, results presented herein demonstrate Cys663 and Ser656 act independently to blunt, but not inhibit, mitochondrial fragmentation following NO treatment. Additional work demonstrated that blocking Drp1 nitrosylation with a conserved Cys663Val mutation enhanced the PKA mediated phosphorylation of Ser656 both biochemically and morphologically, while direct Drp1 nitrosylation had no effect. These results suggest Drp1 nitrosylation has no functional consequence, while Cys663 appears to be structurally important residue.

The final chapter characterizes the formation of a novel E3 ubiquitin ligase complex, composed of KLHL15 and Cul3, that mediates the specific degradation of protein phosphatase 2A (PP2A) regulatory subunit B'beta. PP2A B'beta is expressed in neurons where it functions to regulate catecholamine synthesis through the dephosphorylation and inactivation of Tyrosine hydroxylase (TH). Here I establish the role of a novel kelch domain containing protein, KLHL15, which serves as an adaptor molecule, bridging the E3 ubiquitin ligase Cul3, though the N-terminal BTB domain, with the substrate B'beta through the C-terminal kelch domain. Additionally, the requirement of B'beta Tyr52 for its interaction with KLHL15 suggests MAPK phosphorylation of this residue might regulate the inclusion of PP2A B'beta in the complex, ultimately regulating downstream dopamine synthesis.