Identification of myotubularin as the lipid phosphatase catalytic subunit associated with the 3-phosphatase adapter protein, 3-PAP.
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.
Nandurkar
Harshal H
HH
St. Vincent's Hospital, 3065 Melbourne, Australia. Harshal.Nandurkar@svhm.org.au
Layton
Meredith
M
Laporte
Jocelyn
J
Selan
Carly
C
Corcoran
Lisa
L
Caldwell
Kevin K
KK
Mochizuki
Yasuhiro
Y
Majerus
Philip W
PW
Mitchell
Christina A
CA
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D020014:K562 Cells; D008969:Molecular Sequence Data; D009154:Mutation; D020914:Myopathies, Structural, Congenital / Q000235:genetics / Q000378:metabolism; D010641:Phenotype; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D021122:Protein Subunits; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D054558:Protein Tyrosine Phosphatases, Non-Receptor; D011506:Proteins; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
C489234:MTMR12 protein, human; D021122:Protein Subunits; D011506:Proteins; D011993:Recombinant Fusion Proteins; D010744:Phosphoric Monoester Hydrolases; C408986:MTMR2 protein, human; D017027:Protein Tyrosine Phosphatases; D054558:Protein Tyrosine Phosphatases, Non-Receptor; C099588:myotubularin; C061816:phosphatidylinositol-3-phosphatase