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

Abstract

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.

Analysis of phosphoinositide binding domain properties within the myotubularin-related protein MTMR3

“…Alternatively, the phosphoinositide profile of the secretory pathway is richer than previously anticipated, as suggested by recent electron microscopic studies (Watt et al, 2004;Watt et al, 2002). Interestingly, overexpression of MTM1 in muscle cell lines hydrolyses endosomal PtdIns3P to the point at which binding of EEA1 is lost, but the cellular mass of PtdIns3P is little changed (Chaussade et al, 2003). This suggests that the bulk of this lipid may be distributed elsewhere in the cell at concentrations too low to allow effective recruitment of FYVE-domain probes.…”

Oleate-mediated activation of phospholipase D and mammalian target of rapamycin (mTOR) regulates proliferation and rapamycin sensitivity of hepatocarcinoma cells

“…Western blot analysis Cells were scraped and incubated for 20 min in ice-cold lysis buffer [20]. The lysates were centrifuged at 13,000×g for 20 min at 4°C, and protein concentrations were determined by the BCA colorimetric assay.…”

Phosphoinositide 3-kinases as a common platform for multi-hormone signaling

“…Recent experimental evidences suggest that insulin stimulation triggers the production of PtdIns3P, thus possibly involving either class II or III PI3K. For example, overexpression of myotubularins, the PtdIns3P phosphatases, reduces glucose uptake (Chaussade et al 2003). PtdIns3P binds the PX and FYVE domains of proteins often involved in the control of vesicular trafficking.…”