Biochemical characterisation of the interaction of the myosin light chain phosphatase with the regulatory subunits of protein kinase A in cells of the circulatory system

Saldanha, Paulo André Cardoso Bicho da Silva

Abstract

Regulation of actomyosin-driven contraction is important for numerous cardiovascular cell functions, including smooth muscle tone, endothelial cell permeability and platelet shape change and spreading. These processes are primarily regulated by the phosphorylation and dephosphorylation of the myosin light chain, catalysed by myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP), respectively. MLCP is composed of a 38-kDa protein phosphatase catalytic subunit (PP1c) and a 130-kDa myosin phosphatase targeting subunit 1 (MYPT1). MYPT1 serves as a scaffold that positions PP1c in proximity to its substrates. The activity, protein-protein interactions and localisation of MLCP are fine-tuned by the phosphorylation status of MYPT1, which is targeted by multiple protein kinases. Notably, phosphorylation by protein kinase A (PKA) can counteract the inhibition of MLCP elicited by ROCK phosphorylation.
Several isoforms of MYPT1 have been identified, resulting from cassette-type alternative splicing of exons E13, E14, E22, and E24 of the PPP1R12A gene. An analysis of public databases reveals a greater transcriptional complexity of this gene than previously appreciated. Many studies on PPP1R12A expression and transcription variants have focused on a limited number of organs and tissues, often composed of mixed cell types. There is a scarcity of studies on specific cell lines or individual cell types. To address this gap, a comprehensive examination of the transcriptional landscape of the PPP1R12A gene, emphasising protein-coding transcripts was conducted. This in silico analysis of public EST and mRNA databases revealed that PPP1R12A comprises 32 exons, 29 of which are used in 11 protein-coding transcripts. Using publicly available RNAseq data and reverse transcription PCR, the relative abundance of each transcript in human umbilical vein endothelial cells (HUVEC), human saphenous vein smooth muscle cells (HSVSMC), and platelets were determined. HUVECs and HSVSMCs were found to predominantly express the full-length variant (58.3% and 64.3%, respectively), with the E13-skipping variant being the second most common (33.7% and 23.1%, respectively). Variants including E24 account for 5.4% of transcripts in platelets but are rare in HUVECs and HSVSMCs. These insights set the stage for future studies on the specific roles of MYPT1 isoforms in physiology and pathology.
MYPT1 is part of a complex signalling node involving PP1c, kinases, and other enzymes, and previous studies have unravelled a direct interaction between MYPT1 and the regulatory subunits of PKA. The hypothesis in this study was that MYPT1 and PKA subunits interact directly in vascular cells, potentially functioning as an A-kinase anchoring protein (AKAP) to target PKA to the MLCP signalling node. Using immunoprecipitation, affinity pulldown, and in situ proximity ligation assays in human platelets and endothelial cells, it was demonstrated that MYPT1 directly interacts with all four PKA regulatory subunit variants. Peptide array overlays identified K595, E676, and the PKA/ROCK kinase substrate motif R693/R694/S695/T696 as critical for this interaction. Substitution of S695, T696, or both with aspartic acid or the corresponding phosphorylated residues abolished binding. A model is proposed where MYPT1 functions as a non-canonical AKAP, anchoring PKA near non-phosphorylated S695/T696 to prevent PKA catalytic subunits and potentially ROCK from phosphorylating MYPT1. Collectively, these findings provide valuable insights into the anchoring role of MYPT1 and lay the groundwork for future research into the specific roles of MYPT1 isoforms in cardiovascular function.

Citation

Saldanha, P. A. C. B. D. S. Biochemical characterisation of the interaction of the myosin light chain phosphatase with the regulatory subunits of protein kinase A in cells of the circulatory system. (Thesis). Hull York Medical School. https://hull-repository.worktribe.com/output/4790706

• Thesis Type: Thesis
• Deposit Date: Aug 22, 2024
• Publicly Available Date: Aug 22, 2024
• Keywords: Medical sciences
• Public URL: https://hull-repository.worktribe.com/output/4790706
• Additional Information: Hull York Medical School; University of Hull and University of York
• Award Date: Jul 1, 2023