Myotubularin is the archetype of a family of highly conserved protein-tyrosine 
phosphatase-like enzymes. The myotubularin gene, MTM1, is mutated in the genetic 
disorder, X-linked myotubular myopathy. We and others have previously shown that 
myotubularin utilizes the lipid second messenger, phosphatidylinositol 
3-phosphate (PI(3)P), as a physiologic substrate. We demonstrate here that the 
myotubularin-related protein MTMR2, which is mutated in the neurodegenerative 
disorder, type 4B Charcot-Marie-Tooth disease, is also highly specific for 
PI(3)P as a substrate. Furthermore, the MTM-related phosphatases MTMR1, MTMR3, 
and MTMR6 also dephosphorylate PI(3)P, suggesting that activity toward this 
substrate is common to all myotubularin family enzymes. A direct comparison of 
the lipid phosphatase activities of recombinant myotubularin and MTMR2 
demonstrates that their enzymatic properties are indistinguishable, indicating 
that the lack of functional redundancy between these proteins is likely to be 
due to factors other than the utilization of different physiologic substrates. 
To this end, we have analyzed myotubularin and MTMR2 transcripts during induced 
differentiation of cultured murine C2C12 myoblasts and find that their 
expression is divergently regulated. In addition, myotubularin and MTMR2 
enhanced green fluorescent protein fusion proteins exhibit overlapping but 
distinct patterns of subcellular localization. Finally, we provide evidence that 
myotubularin, but not MTMR2, can modulate the levels of endosomal PI(3)P. From 
these data, we conclude that the developmental expression and subcellular 
localization of myotubularin and MTMR2 are differentially regulated, resulting 
in their utilization of specific cellular pools of PI(3)P.