The Phosphoinositide-3-phosphatase MTMR2 Associates with MTMR13, a Membrane-associated Pseudophosphatase Also Mutated in Type 4B Charcot-Marie-Tooth Disease

Abstract

Charcot-Marie-Tooth disease type 4B (CMT4B) is a severe, demyelinating peripheral neuropathy characterized by distinctive, focally folded myelin sheaths. CMT4B is caused by recessively inherited mutations in either myotubularin-related 2 (MTMR2) or MTMR13 (also called SET-binding factor 2). MTMR2 encodes a member of the myotubularin family of phosphoinositide-3-phosphatases, which dephosphorylate phosphatidylinositol 3-phosphate (PI(3)P) and bisphosphate PI(3,5)P 2 . MTMR13 encodes a large, uncharacterized member of the myotubularin family. The MTMR13 phosphatase domain is catalytically inactive because the essential Cys and Arg residues are absent. Given the genetic association of both MTMR2 and MTMR13 with CMT4B, we investigated the biochemical relationship between these two proteins. We found that the endogenous MTMR2 and MTMR13 proteins are associated in human embryonic kidney 293 cells. MTMR2-MTMR13 association is mediated by coiled-coil sequences present in each protein. We also examined the cellular localization of MTMR2 and MTMR13 using fluorescence microscopy and subcellular fractionation. We found that (i) MTMR13 is a predominantly membrane-associated protein; (ii) MTMR2 and MTMR13 cofractionate in both a light membrane fraction and a cytosolic fraction; and (iii) MTMR13 membrane association is mediated by the segment of the protein which contains the pseudophosphatase domain. This work, which describes the first cellular or biochemical investigation of the MTMR13 pseudophosphatase protein, suggests that MTMR13 functions in association with MTMR2. Loss of MTMR13 function in CMT4B2 patients may lead to alterations in MTMR2 function and subsequent alterations in 3-phosphoinositide signaling. Such a mechanism would explain the strikingly similar phenotypes of patients with recessive mutations in either MTMR2 or MTMR13.

Systematic analysis of myotubularins: heteromeric interactions, subcellular localisation and endosomerelated functions

“…2A shows a representative experiment of this nature, showing specific interaction between MTMR3 and MTMR4. Previous studies have ascribed a role of the coiled-coil domain in MTM interactions (Berger et al, 2003;Kim et al, 2003;Robinson and Dixon, 2005). Surprisingly, both the MTMR3-MTMR4 and the MTMR3 homomeric interaction is retained following deletion of the C-terminal section of MTMR3 containing both FYVE and coiled-coil domains [MTMR3 (1-821), Fig.…”

“…Various reports have demonstrated heteromeric and homomeric interactions between myotubularin family members (Berger et al, 2003;Dang et al, 2003;Kim et al, 2003;Mochizuki and Majerus, 2003;Nandurkar et al, 2003;Robinson and Dixon, 2005;Schaletzky et al, 2003). Several lines of evidence suggest that these associations are functionally significant: (1) increased enzyme activity through association with an inactive partner (Kim et al, 2003;Mochizuki and Majerus, 2003;Schaletzky et al, 2003); (2) altered subcellular distribution through expression of binding partners (Kim et al, 2003); (3) defects in either MTMR2 and MTMR13/Sbf2 lead to a clinically indistinguishable form of Charcot-Marie-Tooth syndrome (Robinson and Dixon, 2005).…”

“…Several lines of evidence suggest that these associations are functionally significant: (1) increased enzyme activity through association with an inactive partner (Kim et al, 2003;Mochizuki and Majerus, 2003;Schaletzky et al, 2003); (2) altered subcellular distribution through expression of binding partners (Kim et al, 2003); (3) defects in either MTMR2 and MTMR13/Sbf2 lead to a clinically indistinguishable form of Charcot-Marie-Tooth syndrome (Robinson and Dixon, 2005). To date heteromeric interactions have been detected biochemically through coimmunoprecipitation experiments of both endogenous and overexpressed proteins.…”

“…Thus MTM1 and MTMR2 interact with MTMR12/3-PAP (Nandurkar et al, 2003), MTMR6 and MTMR7 interact with MTMR9 , and MTMR2 interacts with both MTMR5/Sbf1 and MTMR13/Sbf2 (Kim et al, 2003;Robinson and Dixon, 2005). Furthermore, MTMR2 has been shown to dimerise through its coiled-coil domain (Berger et al, 2003;Kim et al, 2003) and MTM1 can form heptameric ring stuctures (Schaletzky et al, 2003).…”

The myotubularin phosphatase MTMR4 regulates sorting from early endosomes

“…In addition, six catalytically inactive myotubularins have been described that contain mutations in the CX 5 R motif (Robinson and Dixon, 2006). Heteromeric interactions between active and non-active myotubularins enhances the lipid 3-phosphatase activity and/or subcellular localization of the catalytically active phosphatases Mochizuki and Majerus, 2003;Nandurkar et al, 2003;Robinson and Dixon, 2005;Berger et al, 2006). The gene expressing founding family member myotubularin (MTM1) is the candidate gene mutated in X-linked myotubular myopathy, a syndrome associated with muscle weakness, hypotonia and early death (Laporte et al, 1996).…”