Detection of myotubularin phosphatases activity on phosphoinositides in vitro and ex vivo.
Phosphoinositides (PPIn) are important regulators of cellular processes like intracellular protein transport, cellular proliferation, apoptosis, and cytoskeletal organization. The amount and localization of these membrane-bound second messengers are regulated through a set of specific phospholipases, lipid kinases, and phosphatases. The elucidation of PPIn-phosphatases and their cellular function has gained much attention because phosphatase dysregulation is often associated with human genetic diseases. Our laboratory has identified the 3'-PPIn-phosphatase myotubularin 1 (MTM1) mutated in X-linked myotubular myopathy (XLMTM). In addition, a whole family of myotubularin-related proteins (MTMR1-MTMR13) has been discovered. Some of them display phosphatase activity, whereas for other family members no enzymatic activity could be detected. Nevertheless, these "dead phosphatases" myotubularins are conserved throughout evolution and probably exert regulatory function by heteromeric interaction with active phosphatase members. It was shown that MTM1 and related phosphatases act on PtdIns3P and PtdIns(3,5)P2; both PPIn species are important regulators of endocytic pathways. We describe two methods to determine phosphatase activity and substrate specificity of myotubularins. One is an immunoprecipitation-phosphatase assay, testing the activity of myotubularin immunoprecipitated from overexpressing cells on artificial PPIn. The other method analyzes phosphatase activity indirectly ex vivo in transiently transfected mammalian cells. The presence and subcellular localization of the myotubularin substrate PtdIns3P were determined using a specific binding domain (2xFYVE) produced recombinantly as a biosensor.
Rohde
Holger Maria
HM
Department of Molecular Pathology, Institut de Génétique et de Biologie Moléculaire et Cellulaire, INSERM U596, CNRS UMR7104, Université Louis Pasteur de Strasbourg, Illkirch.
Tronchère
Hélène
H
Payrastre
Bernard
B
Laporte
Jocelyn
J
D000818:Animals; D015374:Biosensing Techniques; D019921:Biotinylation; D002457:Cell Extracts; D002460:Cell Line; D006801:Humans; D047468:Immunoprecipitation; D018613:Microscopy, Confocal; D010716:Phosphatidylinositols / Q000378:metabolism*; D054558:Protein Tyrosine Phosphatases, Non-Receptor / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D014162:Transfection
D002457:Cell Extracts; D010716:Phosphatidylinositols; D054558:Protein Tyrosine Phosphatases, Non-Receptor; C099588:myotubularin