Sequential Actions of Myotubularin Lipid Phosphatases Regulate Endosomal PI(3)P and Growth Factor Receptor Trafficking

Abstract

Two different human diseases, X-linked myotubular myopathy and Charcot-Marie-Tooth disease, result from mutant MTM1 or MTMR2 lipid phosphatases. Although events involved in endosomal PI(3)P and PI(3,5)P 2 synthesis are well established and pivotal in receptor signaling and degradation, enzymes involved in phosphoinositide degradation and their roles in trafficking are incompletely characterized. Here, we dissect the functions of the MTM1 and MTMR2 myotubularins and establish how they contribute to endosomal PI(3)P homeostasis. By mimicking loss of function in disease through siRNA-mediated depletion of the myotubularins, excess PI(3)P accumulates on early (MTM1) and late (MTMR2) endosomes. Surprisingly, the increased PI(3)P blocks the egress of epidermal growth factor receptors from early or late endosomes, suggesting that the accumulation of signaling receptors in distinct endosomes may contribute to the unique disease etiologies when MTM1 or MTMR2 are mutant. We further demonstrate that direct myotubularin binding to the type III PI 3-kinase complex hVps34/hVps15 leads to phosphatase inactivation. The lipid kinase-phosphatase interaction also precludes interaction of the PI 3-kinase with Rab GTPase activators. Thus, unique molecular complexes control kinase and phosphatase activation and locally regulate PI(3)P on discrete endosome populations, thereby providing a molecular rationale for related human myo-and neuropathies. INTRODUCTIONHow phophoinositide synthesis and degradation are coordinately regulated remains a key question in endocytic membrane trafficking. The importance of phosphoinositides in endocytic trafficking has been largely studied by inhibiting lipid kinases or overexpressing lipid phosphatases to reduce lipid levels (Futter et al., 2001;Chaussade et al., 2003;Lu et al., 2003;Petiot et al., 2003;Tsujita et al., 2004;Johnson et al., 2006). The fact that inactivation of lipid phosphatases results in disease points to the importance of optimal phosphoinositide levels in homeostasis (Laporte et al., 1996;Bolino et al., 2000;Bitoun et al., 2005;Niemann et al., 2006;Spinosa et al., 2008).Endocytic membrane trafficking is critically dependent on the local synthesis of phosphatidylinositol 3-phosphate (PI(3)P) and phosphatidylinositol 3,5-phosphate (PI(3,5)P 2 ) (Futter et al., 2001;Chaussade et al., 2003;Ikonomov et al., 2003;Lu et al., 2003;Petiot et al., 2003;Stein et al., 2003;Tsujita et al., 2004;Johnson et al., 2006;Shisheva, 2008). PI(3)P is generated on early endosomes as well as on late endosomes by the type III PI 3-kinase complex hVps34/hVps15 (Gillooly et al., 2000;Simonsen et al., 2001;Stein et al., 2003). PI(3)P is responsible for the temporal recruitment of proteins required for endosome fusion and membrane invagination. Endosomal synthesis of PI(3)P is initiated with the activation of the Rab5 and Rab7 GTPases on early and late endosomes, respectively (Christoforidis et al., 1999;Feng et al., 2001;Murray et al., 2002;Stein et al., 2003Stein et al., , 2005. The activated GTPases bind an...

A phosphoinositide conversion mechanism for exit from endosomes

“…PI(4)P, PI(3,4)P 2 and PI(4,5)P 2 , remained unaffected (Figure 21a). These data are consistent with previously published biochemical measurements of PI level in MTM1-depleted cells using [H 3 ]-inositol labeling (Cao, Backer et al 2008). Furthermore, sub-plasma membrane TfR-endosomes were specifically enriched in PI(3)P upon loss of MTM1 ( Figure 21a), matching the prominent increase in PI(3)P-effector proteins (see Figure 17, i.e.…”

“…Moreover, PIKfyve depletion mimicked the peripheral accumulation of TfR-endosomes, although less prominently than the loss of MTM1, while exocytosis was not affected (Figure 26a-c). Thus, we conclude that exit from endosomes en route to late endosomes/ lysosomes functions as a save guard in MTM1-depleted cells to avoid accumulation of early endosomal PI(3)P. Despite the fact that PI(3,5)P 2 could be an additional substrate for MTM1 3-phosphatase activity in vitro (Cao, Backer et al 2008;Hnia, Tronchere et al 2011), lowering endosomal PI(3,5)P 2 levels by co-depletion of PIKfyve did not affect phenotypic changes observed upon loss of MTM1. Thus, endosomal defects caused by loss of MTM1 are primarily due to elevated PI(3)P, but not PI(3,5)P 2 levels.…”

“…A similar, although less severe phenotype was seen upon depletion of the closely related PI 3-phosphatase MTMR1, an enzyme of so far unknown function, while loss of MTMR2 (a PI(3)P and PI(3,5)P 2 3-phosphatase, localized to early and late endosomes and implicated in degradative sorting (Cao, Backer et al 2008)), MTMR4 (a PI(3)P 3-phosphatase, localized to early and recycling endosomes and implicated in endosomal sorting (Naughtin, Sheffield et al 2010)) or MTMR7 (a PI(3)P and I(1,3)P 2 3-phospahatse, associated with Golgi-like structures (Mochizuki and Majerus 2003)) had no effect on TfR distribution or exocytosis (Figure 15). One might speculate that differences in tissue-specific and developmentally regulated expression patterns account for the need of MTM1 and MTMR1 in vivo.…”

“…MTMR2 preferentially binds to PI(3,5)P 2 via its PH-domain (Begley, Taylor et al 2003), predominantly accepts PI(3,5)P 2 as a substrate and localizes to early and late endosomes (Figure 4) (Franklin, Taylor et al 2011). Through association with Vps34 MTMR2 is thought to balance endosomal PI(3)P and PI(3,5)P 2 levels and regulates early to late endosomal trafficking (Cao, Backer et al 2008). In addition, MTMR2 recruits the PI(3)P-and PI(3,5)P 2 -effector RME-8 (receptor mediated endocytosis 8) and, thus, links PI-conversion to retromer-dependent endosomal tubulation (Cao, Backer et al 2008;Xhabija, Taylor et al 2011).…”

“…Through association with Vps34 MTMR2 is thought to balance endosomal PI(3)P and PI(3,5)P 2 levels and regulates early to late endosomal trafficking (Cao, Backer et al 2008). In addition, MTMR2 recruits the PI(3)P-and PI(3,5)P 2 -effector RME-8 (receptor mediated endocytosis 8) and, thus, links PI-conversion to retromer-dependent endosomal tubulation (Cao, Backer et al 2008;Xhabija, Taylor et al 2011). In Schwann cells, MTMR2…”