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Expression of Myotubularin by an Adenoviral Vector Demonstrates Its Function as a Phosphatidylinositol 3-Phosphate [PtdIns(3)P] Phosphatase in Muscle Cell Lines: Involvement of PtdIns(3)P in Insulin-Stimulated Glucose Transport

Institut National de la Santé et de la Recherche Médicale Unité 145 (C.C., L.P., S.B., F.P., E.V.O.), Institut Féderatif de Recherche 50, Faculté de Médecine, 06107 Nice Cedex 2, and Unité 326 (H.T., B.P.), Hopital Purpan, 31059 Toulouse, France; Institut de Génétique et de Biologie Moléculaire et Cellulaire (F.B., J.L.), 67404 Illkirch, France; and Institute of Biochemistry (S.B.-V., S.R.), University of Fribourg, CH-1700, Switzerland

Address all correspondence and requests for reprints to: E. Van Obberghen, Institut National de la Santé et de la Recherche Medicale, Unité 145, 28, avenue de Valombrose, 06 107 Nice Cedex 2, France. E-mail: vanobbeg{at}unice.fr.

X-linked myotubular myopathy is a muscle disorder caused by mutations on the myotubular myopathy-1 (MTM-1) gene, coding for myotubularin a 65-kDa polypeptide similar to protein phosphatases. Biochemical and in vivo studies define myotubularin as a phosphatidylinositol 3-phosphate [PtdIns(3)P] phosphatase. To efficiently express myotubularin in muscle cell lines and adipocytes, we used an adenoviral genome recombinogenic to pcDNA3, and to other widely used expression vectors, to produce adenoviruses expressing wild-type (wt), catalytically inactive C375S, and substrate trap D278A myotubularin.

[³²P]Orthophosphate labeling followed by phosphoinositide analysis of differentiated L6 and C2C12 cells expressing myotubularin demonstrated increased PtdIns(3)P levels upon expression of the C375S and D278A mutants. In keeping with its biochemical function, overexpression of wt myotubularin as an enhanced green fluorescent protein fusion disrupted the endosomal punctuated staining of the FYVE (Fab1p/YOTB Vac1p/EEA1)-domain-containing PtdIns(3)P binding protein early endosomal antigen 1 as well as of a gluathione-S-transferase-FYVE probe directed to PtdIns(3)P. Expression of wt myotubularin, although not affecting activation of proximal insulin signal transduction targets such as protein kinase B and MAPK, induced a decrease in insulin-induced glucose uptake, whereas basal glucose uptake was augmented by expression of D278A (DA) and C375S (CS) mutants. Moreover, overexpression of myotubularin in 3T3-L1 adipocytes impaired insulin-induced translocation at the plasma membrane of green fluorescent protein-tagged glucose transporter 4. These data indicate that PtdIns(3)P is required to direct glucose transporter 4 to insulin-responsive compartments and/or to allow the translocation of the latter at the plasma membrane.

We conclude that myotubularin, by modulating the intracellular levels of PtdIns(3)P, plays a role in the control of vesicular traffic related to glucose transport, by counteracting the activities of the PtdIns(3)P-producing phosphatidylinositol 3-kinases.

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