Date on Master's Thesis/Doctoral Dissertation

8-2018

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Biochemistry and Molecular Biology

Degree Program

Biochemistry and Molecular Biology, PhD

Committee Chair

Schaner Tooley, Christine

Committee Co-Chair (if applicable)

Klinge, Carolyn

Committee Member

Klinge, Carolyn

Committee Member

Clark, Barbara

Committee Member

Cheng, Alan

Committee Member

Ceresa, Brian

Author's Keywords

alpha-amino PTM; protein methylation; acetylation; regulatory light chain; non-muscle myosin; compartmentalization

Abstract

Dysregulation of alpha-amino post-translational modifications (Nα-PTMs) is found in multiple cancers and developmental disorders. However, the exact roles Nα-PTMs play in regulating protein function remain poorly understood. I sought to clarify the role of Nα-methylation and Nα-acetylation in the regulation of Myosin Regulatory Light Chain 9 (MYL9). MYL9 is a key cytoskeletal regulator and transcription factor and is the first protein confirmed to undergo both Nα-methylation and Nα-acetylation. Through this work I revealed novel regulatory features of MYL9, while also presenting a framework by which to understand the coordinated regulation of proteins by Nα-methylation and Nα-acetylation. Nα-PTM selective mutants of MYL9 were generated by modifying the consensus sequence targeted by Nα-methyltransferases and Nα-acetyltransferases. These mutants were assayed alongside wildtype (WT) protein for effects on MYL9 stability and function in mammalian cell lines. Offering a counterpoint to the dogmatic perspective of Nα-PTMs as modifiers of protein stability, loss of either Nα-methylation or Nα-acetylation did not alter MYL9 half-life. Instead, distinct functionality of Nα-methylated and Nα-acetylated MYL9 was observed. MYL9 is known primarily as a regulator of cytoskeletal dynamics through its regulation of non-muscle myosin II (NMII) activity. A specialized nuclear role for MYL9 has been described in which it binds at select gene promoters and activates transcription. My results show that Nα-methylation promotes this nuclear function of MYL9. The Nα-methylation selective mutant of MYL9 increased TNFα stimulated transcription of intercellular adhesion molecule 1 (ICAM1) as compared to all other variants of MYL9. In addition, this MYL9 mutant showed increased binding at the ICAM1 promoter. Nα-methylation has been shown to increase protein-DNA interactions, implicating this mechanism in the regulation of MYL9 nuclear function. Phosphorylation of MYL9 at serine 19 (pS19) is required for NMII activity. Expression of a Nα-methylation deficient mutant of MYL9 increased the occurrence of pS19 after stimulation as compared to all other variants of MYL9. This mutant of MYL9 also displayed increased cell migration and cell spreading on fibronectin, measures of NMII activity. As such, I propose a model in which Nα-methylation of MYL9 favors nuclear activity while blocking cytoplasmic activity.

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