Identification of myotubularin as the lipid phosphatase catalytic subunit associated with the 3-phosphatase adapter protein, 3-PAP

• DOI: 10.1073/pnas.1033097100
• Publication Date: 2003-07-22T19:39:06Z
• Authors: Harshal H. Nandurkar; Meredith Layton; Jocelyn Laporte; Carly Selan; Lisa Corcoran; Kevin K. Caldwell; Yasuhiro Mochizuki; Philip W. Majerus; Christina A. Mitc...
• Keywords: 0301 basic medicine; Recombinant Fusion Proteins; Cell Membrane; Molecular Sequence Data; Proteins; In Vitro Techniques; Protein Tyrosine Phosphatases, Non-Receptor; Phosphoric Monoester Hydrolases; Protein Subunits; 03 medical and health sciences; Phenotype; COS Cells; Mutation; Animals; Humans; Amino Acid Sequence; Protein Tyrosine Phosphatases; K562 Cells; Myopathies, Structural, Congenital

Myotubularin is a dual-specific phosphatase that dephosphorylates phosphatidylinositol 3-phosphate and phosphatidylinositol (3,5)-bisphosphate. Mutations in myotubularin result in the human disease X-linked myotubular myopathy, characterized by persistence of muscle fibers that retain an immature phenotype. We have previously reported the identification of the 3-phosphatase adapter protein (3-PAP), a catalytically inactive member of the myotubularin gene family, which coprecipitates lipid phosphatidylinositol 3-phosphate-3-phosphatase activity from lysates of human platelets. We have now identified myotubularin as the catalytically active 3-phosphatase subunit interacting with 3-PAP. A 65-kDa polypeptide, coprecipitating with endogenous 3-PAP, was purified from SDS/PAGE, subjected to trypsin digestion, and analyzed by collision-induced dissociation tandem MS. Three peptides derived from human myotubularin were identified. Association between 3-PAP and myotubularin was confirmed by reciprocal coimmunoprecipitation of both endogenous and recombinant proteins expressed in K562 cells. Recombinant myotubularin localized to the plasma membrane, causing extensive filopodia formation. However, coexpression of 3-PAP with myotubularin led to attenuation of the plasma membrane phenotype, associated with myotubularin relocalization to the cytosol. Collectively these studies indicate 3-PAP functions as an “adapter” for myotubularin, regulating myotubularin intracellular location and thereby altering the phenotype resulting from myotubularin overexpression.