The VPS-34 PI3 kinase negatively regulates RAB-5 during endosome maturation

The Rab5 GTPase and phosphatidylinositol-3 phosphate (PI(3)P) coordinately regulate endosome trafficking. Rab5 recruits Vps34, the class III PI3-kinase (PI3K), to generate PI(3)P and recruit PI(3)P binding proteins. Loss of Rab5 and loss of Vps34 have opposite effects on endosome size suggesting that our understanding of how Rab5 and PI(3)P cooperate is incomplete. Here we report a novel regulatory loop, whereby C. elegans VPS34 inactivates RAB-5 via recruitment of the TBC-2 Rab GTPase Activating Protein. We found that loss of VPS-34 caused a large late endosome phenotype like that of loss of TBC-2, and that Rab5 activity is increased in Vps34 knockout mouse embryonic fibroblasts. We found that VPS-34 is required for TBC-2 endosome localization and that the Pleckstrin Homology (PH) domain of TBC-2 bound PI(3)P. Deletion of the PH domain enhanced TBC2 localization to endosomes in a VPS-34 dependent manner. Thus, PI(3)P binding the PH domain maybe permissive for another PI(3)P regulated interaction that recruits TBC-2 to endosomes. Therefore, VPS-34 recruits TBC-2 to endosomes to inactivate RAB-5 to ensure the directionality of endosome maturation. Jo ur na l o f C el l S ci en ce • A dv an ce a rt ic le INTRODUCTION The Vps34 class III phosphatidylinositol 3-kinase (PI3K) regulates endosome and phagosome trafficking, and the initiation of autophagy (Vieira et al., 2001, Kihara et al., 2001, Peterson et al., 1999, Futter et al., 2001, Schu et al., 1993). Vps34 forms a complex with Vps15, a serine/threonine kinase (Stack et al., 1993, Volinia et al., 1995), and Beclin1 (yeast Atg6/Vps30), a key regulator of autophagy (Kihara et al., 2001). Vps34 specifically phosphorylates phosphatidylinositol (PI) to generate phosphatidylinositol 3-phosphate (PI(3)P) (Schu et al., 1993, Volinia et al., 1995), which serves as a substrate for proteins with PI(3)P binding domains such as FYVE and Phox (PX) domains (Burd and Emr, 1998, Gaullier et al., 1998, Patki et al., 1998, Ellson et al., 2001, Cheever et al., 2001, Kanai et al., 2001, Song et al., 2001). Myotubularins are PI3-phosphatases that counteract the effects of Vps34 by dephosphorylating PI(3)P and PI(3,5)P2 to generate PI and PI(5)P, respectively (Robinson and Dixon, 2006). Thus, Vps34 and myotubularins regulate the lipid and protein composition of endosomes. The Rab5 and Rab7 GTPases respectively localize to early and late endosomes where they drive multiple aspects of endosome trafficking to the lysosome (Huotari and Helenius, 2011, Stenmark, 2009). In their GTP-bound “active” state, they bind and recruit effector proteins to endosomes. Both Rab5 and Rab7 GTPases can interact with Vps15 to recruit and/or activate Vps34 on early and late endosomes, respectively (Christoforidis et al., 1999, Murray et al., 2002, Stein et al., 2003). Many Rab5 and Rab7 effectors also bind PI(3)P, indicating a cooperative function for the Rab GTPases and Vps34 on endosomes (Pankiv et al., 2010, Simonsen et al., 1998, Patki et al., 1997, Nielsen et al., 2000). For example, EEA1 promotes homotypic fusion of early endosomes, and it is recruited to endosomes via interactions with PI(3)P and GTP-bound Rab5 (Mills et al., 1998, Simonsen et al., 1998, Patki et al., 1997). Interestingly, inhibition of Vps34 via Wortmannin caused a large late endosome phenotype (Futter et al., 2001, Brown et al., 1995, Reaves et al., 1996) that was reversed by expression of dominant negative Rab5(S34N) (Fernandez-Borja et al., 1999). Constitutively active Rab5(Q79L) also induced large early and late endosome phenotypes (Wegner et al., 2010, Rink et al., 2005, Stenmark et al., 1994, Lawe et al., 2002) and was able to recruit EEA1 to membranes in the presence of Wortmannin (Simonsen et al., 1998). The above data suggests that the enlarged endosomes seen with loss of Vps34/PI(3)P could result from increased Rab5 activity. The Caenorhabditis elegans intestine consists of a single layer of polarized epithelial cells that provides an in vivo model to study endosome trafficking (Sato et al., 2014). As in Jo ur na l o f C el l S ci en ce • A dv an ce a rt ic le mammalian cells, expression of constitutively active RAB-5(Q78L-analogous to mammalian Rab5 Q79L) in the C. elegans intestine results in the formation of enlarged late endosomes (Chotard et al., 2010a). Similarly, loss of C. elegans TBC-2, a RAB-5 GTPase Activating Protein (GAP), also results in the formation of enlarged late endosomes in the intestine (Chotard et al., 2010a). TBC-2 localizes to late endosomes in a RAB-7-dependent manner, where it may facilitate RAB-5 to RAB-7 conversion by inactivating RAB-5. In addition to facilitating early to late endosome maturation, TBC-2 also regulates phagosome maturation during apoptotic cell clearance (Li et al., 2009), endosome recycling (Sun et al., 2012, Liu and Grant, 2015), yolk protein trafficking/storage (Chotard et al., 2010b) and neuropeptide release via dense core vesicle trafficking (Sasidharan et al., 2012). TBC-2 is homologous to human Armus/TBC1D2A and TBC1D2B proteins and comprises a unique family of Rab GAPs with an N-terminal Pleckstrin Homology (PH) domain, a central Coiled-Coil (CC) domain and a C-terminal Tre2/Bub2/Cdc16 (TBC) catalytic domain (Chotard et al., 2010a). Armus/TBC1D2A has Rab7 GAP activity and interacts with Rac1 to regulate degradation of E-cadherin and interacts with LC3 and LRRK1 to regulate autophagy (Carroll et al., 2013, Frasa et al., 2010, Toyofuku et al., 2015). TBC1D2B interacts with Rab22 in a nucleotideindependent manner, but its cellular function is not known (Kanno et al., 2010). C. elegans VPS-34/LET-512 is essential for development. It has been shown to regulate trafficking of the LRP-1 LDL receptor, nuclear membrane morphology, and phagosome maturation during engulfment of apoptotic cells (Roggo et al., 2002, Kinchen et al., 2008). Loss of vps-34 was reported to result in accumulation of large vesicles, the nature of which has not been described (Roggo et al., 2002). Here we determine the requirements for VPS-34 PI3K on endosome size in the intestine and its relationship with RAB-5 and TBC-2. We show that knockdown of the C. elegans class III PI3K components vps-34, vps-15 or bec1 resulted in the formation of enlarged late endosomes similar in nature to that seen by loss of tbc-2 or expression of constitutively active RAB-5(Q78L). This large late endosome phenotype required the late endosomal RAB-7 GTPase. Overexpression of the MTM-1 myotubularin PI3-phosphatase induced the same phenotype as knockdown of the PI3K components indicating that the large late endosome phenotype was due to loss of PI(3)P. We show that the PH domain of TBC-2 bound PI(3)P in vitro and that VPS-34 promoted TBC-2 localization to endosomes. Truncation of the PH domain enhanced TBC-2 endosome localization in a VPS-34 dependent manner, and further truncation of the PH and CC domains abrogated TBC-2 endosome localization. We demonstrate that PI(3)P has dual roles in regulating TBC-2 endosome localization. A direct interaction between PI(3)P and the PH Jo ur na l o f C el l S ci en ce • A dv an ce a rt ic le domain of TBC-2 may result in a conformational change allowing the CC domain to mediate a protein-protein interaction with an endosomal protein whose localization or activity is regulated by PI(3)P. Consistent with Vps34 antagonizing the activity of Rab5, we found that Rab5 activity is increased in Vps34 KO MEFs. Therefore, the VPS-34/VPS-15/BEC-1 PI3K complex may negatively regulate RAB-5 via recruitment of TBC-2 to endosomes. Jo ur na l o f C el l S ci en ce • A dv an ce a rt ic le