Phosphoinositides control many different processes required for normal cellular 
function. Myotubularins are a family of Phosphatidylinositol 3-phosphate 
(PtdIns3P) phosphatases identified by the positional cloning of the MTM1 gene in 
patients suffering from X-linked myotubular myopathy and the MTMR2 gene in 
patients suffering from the demyelinating neuropathy Charcot-Marie-Tooth disease 
type 4B. MTM1 is a phosphatidylinositol phosphatase with reported specificity 
toward PtdIns3P, while the related proteins MTMR2 and MTMR3 hydrolyze both 
PtdIns3P and PtdIns(3,5)P2. We have investigated MTM1 and MTMR6 and find that 
they use PtdIns(3,5)P2 in addition to PtdIns3P as a substrate in vitro. The 
product of PtdIns(3,5)P2 hydrolysis, PtdIns5P, causes MTM1 to form a heptameric 
ring that is 12.5 nm in diameter, and it is a specific allosteric activator of 
MTM1, MTMR3, and MTMR6. A disease-causing mutation at arginine 69 of MTM1 
falling within a putative pleckstrin homology domain reduces the ability of the 
enzyme to respond to PtdIns5P. We propose that the myotubularin family of 
enzymes utilize both PtdIns3P and PtdIns(3,5)P2 as substrates, and that PtdIns5P 
functions in a positive feedback loop controlling their activity. These findings 
highlight the importance of regulated phosphatase activity for the control of 
phosphoinositide metabolism.