Abstract 1305 Understanding the mechanism of LCMT-1 methylation of PP2A

Protein phosphatase 2A (PP2A) is a ubiquitously expressed heterotrimeric serine/threonine phosphatase that has broad substrate specificity and acts as a potent tumor suppressor. PP2A plays a role in critical processes including cell proliferation, signal transduction, and apoptosis. The PP2A holoenzyme complex exists as a core dimer, composed of the scaffold A and catalytic C subunits, bound to one of over 40 regulatory B subunits that determine the cellular localization and substrate specificity of the enzyme. Post-translation modifications (PTMs) play critical roles in regulating the recruitment of B subunits, thereby regulating the populations of specific PP2A holoenzymes in the cell and overall PP2A activity. One such PTM is the methylation of the carboxyl-terminal tail of the C subunit. Methylation is catalyzed by PP2A-specific methyltransferase: leucine carboxyl methyltransferase (LCMT-1). Our cryo-electron microscopy single particle analysis revealed large conformational dynamics at the N-terminus of the PP2A A subunit in the biochemically trapped enzyme-substrate (ES) complex. To elucidate the contributions of both the A-subunit and LCMT-1 N-terminus in regulating the C-tail methylation, we have generated mutant constructs of LCMT-1 with N-terminal deletions, which were subsequently expressed and purified. These mutants were then used to assess the impact of N-terminal deletions on A-C dimer methylation by Western Blot where we measured a methylation reaction byproduct, SAH to characterize how much methylation happened. We saw that qualitatively there were major differences in the amount of methylation occurring between the mutants and the WT LCMT-1. Based upon these results, we performed a methylation assay to quantitatively demonstrate that an N-terminal deletion partially decreased the rate of methylation and increased the Km. This supports our hypothesis that both the flexible region of the A subunit as well as the N-terminus of LCMT-1 play a role in the methylation mechanism. Through a deeper understanding of the LCMT-1-PP2A mechanism, this research aims to provide insights into PP2A methylation, its regulation, and potential implications in diseases associated with PP2A dysregulation. The funding for this research has been provided by the Taylor Lab and in part by the Undergraduate Research Office of Case Western Reserve University. The funding for this research has been provided by the Taylor Lab in the Case Western Reserve University School of Medicine, Department of Pharmacology and in-part by the Undergraduate Research Office of Case Western Reserve University.