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TRPM7 regulates myosin IIA filament stability and protein localization by heavy chain phosphorylation |
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| Authors: | Clark Kristopher Middelbeek Jeroen Lasonder Edwin Dulyaninova Natalya G Morrice Nick A Ryazanov Alexey G Bresnick Anne R Figdor Carl G van Leeuwen Frank N |
| Affiliation: | 1 Department of Tumor Immunology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands 2 Laboratory of Pediatric Oncology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands 3 Centre for Molecular and Biomolecular Informatics, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands 4 Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA 5 MRC Protein Phosphorylation Unit, James Black Centre, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, UK 6 Department of Pharmacology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854, USA |
| Abstract: | Deregulation of myosin II-based contractility contributes to the pathogenesis of human diseases, such as cancer, which underscores the necessity for tight spatial and temporal control of myosin II activity. Recently, we demonstrated that activation of the mammalian α-kinase TRPM7 inhibits myosin II-based contractility in a Ca2+- and kinase-dependent manner. However, the molecular mechanism is poorly defined. Here, we demonstrate that TRPM7 phosphorylates the COOH-termini of both mouse and human myosin IIA heavy chains—the COOH-terminus being a region that is critical for filament stability. Phosphorylated residues were mapped to Thr1800, Ser1803 and Ser1808. Mutation of these residues to alanine and that to aspartic acid lead to an increase and a decrease, respectively, in myosin IIA incorporation into the actomyosin cytoskeleton and accordingly affect subcellular localization. In conclusion, our data demonstrate that TRPM7 regulates myosin IIA filament stability and localization by phosphorylating a short stretch of amino acids within the α-helical tail of the myosin IIA heavy chain. |
| Keywords: | MHCII, myosin II heavy chain GST, glutathione S-transferase WT, wild type KD, kinase dead MALDI-TOF, matrix-assisted laser desorption/ionization time of flight MS, mass spectrometry LTQ-FT, linear ion trap quadrupole-Fourier transform LC-MS/MS, nano-liquid chromatography tandem mass spectrometry YFP, yellow fluorescent protein F-actin, filamentous actin PKC, protein kinase C CKII, casein kinase II HA, hemagglutinin GFP, green fluorescent protein PBS, phosphate buffered saline |
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