Deficiency of MIP phosphatase induces a muscle disorder by disrupting Ca 2+ homeostasis Jinhua Shen 1,6 , Wen-Mei Yu 1,6 , Marco Brotto 2,6 , Joseph A. Scherman 3 , Caiying Guo 4 , Christopher Stoddard 4 , Thomas M. Nosek 5 , Héctor H. Valdivia 3 , and Cheng-Kui Qu 1,7 1 Department of Medicine, Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, Ohio 44106 2 Schools of Nursing and Medicine, University of Missouri, Kansas City, Missouri 64108 3 Department of Physiology, University of Wisconsin Medical School, Madison, Wisconsin 53706 4 Gene Targeting and Transgenic Facility, University of Connecticut Health Center, Farmington, Connecticut 06030 5 Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106 Abstract The intracellular Ca 2+ ([Ca 2+ ] i ) level of skeletal muscles must be rapidly regulated during the excitation-contraction-relaxation process 1 . However, the signaling components involved in such rapid Ca 2+ movement are not fully understood. Here, we report that mice deficient in the novel phosphatidylinositol phosphate (PIP) phosphatase MIP displayed muscle weakness and fatigue. Muscles isolated from MIP −/− mice produced less contractile force, markedly prolonged relaxation, and exhibited exacerbated fatigue. Further analyses revealed that MIP deficiency resulted in spontaneous Ca 2+ leak from the internal store — the sarcoplasmic reticulum (SR). This was attributed to the decreased metabolism/dephosphorylation and the subsequent accumulation of MIP substrates, especially PI(3,5)P 2 and PI(3,4)P 2 . Furthermore, we found that PI(3,5)P 2 and PI(3,4)P 2 bound to and directly activated the Ca 2+ release channel/ryanodine receptor (RyR1) of the SR. These studies provide the first evidence that finely controlled PIP levels in muscle cells are essential for maintaining Ca 2+ homeostasis and muscle performance. During our systematic genome-wide survey for tyrosine/dual specificity phosphatases (unpublished work), we discovered a novel phosphatase by hidden Markov database mining using the conserved catalytic motif ([V/I][V/I]HCXXGXXR[T/S]) as the bait sequence. Both human (BC035690) and mouse (BC018294) homologies were identified. They share 90% identity in amino acid sequences (Supplementary Information, Fig. S1). Northern blotting analyses illustrated that this phosphatase was predominantly expressed in skeletal muscle and heart (Fig. 1a). Immunostaining indicates that it is primarily localized in the cytoplasm (data not shown). To verify its phosphatase property, we generated a GST fusion protein and tested its catalytic activity using pNPP (p-Nitrophenyl Phosphate), a widely used non-specific 7Correspondence should be addressed to: C.K.Q. (e-mail: E-mail: cxq6@case.edu). 6 These authors contributed equally to this work. AUTHOR CONTRIBUTIONS J.S., W.M. Y., M.B., J.A.S., and C.S. conducted the research and summarized the data. C.K.Q., M.B., H.H.V., T.M.N., and C.G. designed the experiments and wrote the manuscript. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. NIH Public Access Author Manuscript Nat Cell Biol. Author manuscript; available in PMC 2009 December 1. Published in final edited form as: Nat Cell Biol. 2009 June ; 11(6): 769–776. doi:10.1038/ncb1884. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript