MTMR9 Increases MTMR6 Enzyme Activity, Stability, and Role in Apoptosis

Myotubularin-related protein 6 (MTMR6) is a catalytically active member of the myotubularin (MTM) family, which is composed of 14 proteins. Catalytically active myotubularins possess 3-phosphatase activity dephosphorylating phosphatidylinositol-3-phoshate and phosphatidylinositol-3,5-bisphosphate, and some members have been shown to form homomers or heteromeric complexes with catalytically inactive myotubularins. We demonstrate that human MTMR6 forms a heteromer with an enzymatically inactive member myotubularin-related protein 9 (MTMR9), both in vitro and in cells. MTMR9 increased the binding of MTMR6 to phospholipids without changing the lipid binding profile. MTMR9 increased the 3-phosphatase activity of MTMR6 up to 6-fold. We determined that MTMR6 is activated up to 28-fold in the presence of phosphatidylserine liposomes. Together, MTMR6 activity in the presence of MTMR9 and assayed in phosphatidylserine liposomes increased 84-fold. Moreover, the formation of this heteromer in cells resulted in increased protein levels of both MTMR6 and MTMR9, probably due to the inhibition of degradation of both proteins. Furthermore, co-expression of MTMR6 and MTMR9 decreased etoposide-induced apoptosis, whereas decreasing both MTMR6 and MTMR9 by RNA interference led to increased cell death in response to etoposide treatment when compared with that seen with RNA interference of MTMR6 alone. Thus, MTMR9 greatly enhances the functions of MTMR6.Myotubularin proteins are a family of 14 proteins with the canonical dual specificity protein tyrosine phosphatase active site CX₅R motif (1–3). Eight members of the myotubularin family possess catalytic activity, dephosphorylating phosphatidylinositol 3-phosphate (PtdIns-3-P)⁴ and phosphatidylinositol 3,5-bisphosphate (PtdIns-3,5-P₂) at the D-3 position, and six members are not catalytically active because they lack the conserved cysteine residue in the protein tyrosine phosphatase motif that is required for activity. Interest in this group of proteins originated from the genetic evidence linking myotubularin, the founding member of this family, to myotubular myopathy, an X-linked disorder characterized by severe hypotonia and generalized muscle weakness (4). Subsequently, mutations in MTMR2 and in its inactive binding partner MTMR13 were linked to a subset of Charcot-Marie-Tooth disease type 4B, a demyelinating neurodegenerative disorder (5, 6).Despite near identical substrate specificity, biochemical and genetic evidence supports the hypothesis that myotubularin proteins are not redundant and have unique functions within cells (2, 7–9). The mechanisms by which loss of function of myotubularin proteins produce diseases are not known. Current evidence supports the hypothesis that each myotubularin protein regulates a specific pool of PtdIns-3-P and/or PtdIns-3,5-P₂, which in turn regulates a variety of cellular functions. Differences in tissue expression and subcellular localization play a role in the specificity of different myotubularins (10–15).The functions of myotubularin proteins are altered by the formation of heteromers between catalytically active and inactive members of the family. The initial biochemical purification of MTM1 demonstrated the presence of MTM1 homodimers and MTM1-3-phosphatase adapter protein (3PAP) heteromers (16), which was later described as MTMR12 (15, 17). MTMR2 was found to form heteromers with MTMR5 (13) and MTMR13 (18), and MTMR7 formed heteromers with MTMR9 (19). In each case, a catalytically active myotubularin protein interacted with an inactive protein. Heteromerization generated two important effects: increased catalytic activity of the active component (13, 15, 19, 20) and targeting of the heteromer to specific subcellular locations (15). Mutations in the inactive member MTMR13 result in a similar phenotype in patients as the mutations in its catalytically active binding partner MTMR2, indicating an indispensable role for the catalytically inactive subunit (21).Myotubularin proteins can be grouped into subfamilies based on homology. Closely related MTMR6, MTMR7, and MTMR8 comprise such a subfamily. We have previously characterized the interaction between mouse MTMR7 and MTMR9 proteins (19). In this report, we characterize the interaction between human MTMR6 and MTMR9. MTMR6 and MTMR9 have been shown to form a heteromeric complex in mouse and Caenorhabditis elegans (19, 22). MTMR6 has been shown to inhibit the activity of a calcium-activated potassium channel (type KCa3.1) (23, 24). Two screening experiments implicate MTMR6 as a regulator of apoptosis. By RNA microarray analysis, increased MTMR6 expression was observed in B cell chronic lymphoid leukemia cells with increased resistance to irradiation-induced apoptosis (25), whereas in an RNA interference screen in HeLa cells, decreased MTMR6 expression promoted apoptosis (26).Here we show that MTMR6 interacts with MTMR9 in vitro and in human cells. This interaction increases the phospholipid binding and enzymatic activity of MTMR6 in vitro. Co-expression of either subunit in cells dramatically increased the protein levels of the individual binding partners, suggesting that heteromer formation increases the stability of the proteins. Finally, MTMR9 was found to potentiate the effects of MTMR6 on apoptosis.