SMALL GTPASES 2016, VOL. 7, NO. 3, 164–167 http://dx.doi.org/10.1080/21541248.2016.1175539
COMMENTARY
A modification switch on a molecular switch: Phosphoregulation of Rab7 during endosome maturation Swapnil Rohidas Shindea,b and Subbareddy Maddikaa a Laboratory of Cell Death & Cell Survival, Centre for DNA Fingerprinting and Diagnostics (CDFD), Nampally, Hyderabad, India; bGraduate Studies, Manipal University, Manipal, India
ABSTRACT
ARTICLE HISTORY
Rab GTPases, the highly conserved members of Ras GTPase superfamily are the pivotal regulators of vesicle-mediated trafficking. Rab GTPases, each with a specific subcellular localization, exert tremendous control over various aspects of vesicular transport, identity and dynamics. Several lines of research have established that GDI, GEFs and GAPs are the critical players to orchestrate Rab GTPase activity and function. The importance of post translational modifications in Rab GTPase functional regulation is poorly or not yet been addressed except for prenylation. Our recent study has revealed a novel dephosphorylation dependent regulatory mechanism for Rab7 activity and function. We have shown the importance of PTEN mediated dephosphorylation of Rab7 on highly conserved S72 and Y183 residues, which is essential for its GDI mediated membrane targeting and further activation by GEF. In conclusion, our study highlighted the importance of a phosphorylation/ dephosphorylation switch in controlling timely Rab7 localization and activity on endosomes.
Received 2 April 2016 Revised 2 April 2016 Accepted 3 April 2016 KEYWORDS
endosome; membrane trafficking; phosphorylation; PTEN; Rab7
Introduction The biogenesis and trafficking of intracellular vesicles and their spatio temporal regulation is critical for maintenance of cellular homeostasis. Rab GTPases, a large family of small GTPases are central to the process of endocytosis which ensures the delivery of cargos to their appropriate destinations.1,2 Since the identification of YPT1, the first Ras related GTPase in yeast 3 and the demonstration of possible role of Ras related GTPases in membrane trafficking,4 66 human Rab proteins have been identified so far. Rab GTPases are the molecular switches which cycle between active GTP-bound state or inactive GDP-bound state and oscillate between different cellular localizations. Rab proteins are activated by GEFs (guanine nucleotide exchange factors) that replaces GDP with GTP and are inactivated by GAPs (GTPase activating proteins).5 Rab GTPases associate with the coat protein components, molecular motors and different SNAREs (Soluble N-ethylmaleimide-sensitive factor attachment protein receptor), and thereby serve as multifaceted organizers of almost all membrane trafficking processes.6-9 Rab7, a member of Rab GTPase family is a key regulator of trafficking, maturation, and fusion of endocytic and autophagic vesicles. It is evident from several studies
that Rab7 localizes primarily to late endosomes, lysosomes, and autophagolysosomes.10-13 Essentially, Rab7 activity specifically controls the maturation of cargo containing early endosomes to late endosomes and their subsequent degradation in lysosomes. Rab7 activity is critical to regulate the lysosome-mediated degradation of endocytic cargoes such as activated EGF receptors.12,14 Loss of Rab7 activity leads to loss of lysosomal integrity as well as delayed or slow degradation of internalized cargos such as EGF receptors.10,12 The importance of Rab7 activity has further been demonstrated by its depletion in the cell which leads to trapping of cargos in late endosomes.15 The entrapment of cargo results in the accumulation of enlarged late endosomes/multivesicular bodies with persistent cellular signaling. Rab GTPases have been extensively studied over the years but our understanding of their regulation by post translational modifications is limited. Prenylation is the most characterized posttranslational modification of Rabs. One or 2 prenyl groups are added to successive cysteine residues present at the extreme C-terminus of the protein which is essential for initial targeting of the Rabs to membranes.16,17 Recently, in an interesting study, Lachance et al have unraveled a novel role for
CONTACT Subbareddy Maddika [email protected] CDFD, Bldg. 7, Gruhakalpa, Nampally, Hyderabad, India. Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/ksgt. © 2016 Taylor & Francis
SMALL GTPASES
ubiquitination of Rab11a by a b2AR/ HACE1 complex in regulating Rab11a activity and b2AR trafficking.18 Lately, Lai et al, reported PINK1 mediated phosphorylation of Rab8A at Ser111 and its implication on Rab8A activation.19 These studies underline the fact that posttranslational modifications such as phosphorylation indeed plays critical role in regulation of Rab GTPase activity. Interestingly, our recent studies have unraveled dephosphorylation of Rab7 by a tumor suppressor phosphatase PTEN.20
Phosphoregulation of Rab7 While trying to identify novel cellular functions of phosphatase PTEN based on the interacting proteins using proteomic analysis, we identified Rab7 as one of the critical PTEN associated proteins. Although PTEN is known to be a dual-specific phosphatase that acts on both lipid and protein substrates, majority of its functions are attributed to its lipid phosphatase activity. PTEN converts phosphatidylinositol-3,4,5-trisphosphate (PIP3) to phosphatidylinositol-4,5-bisphosphate (PIP2) at the cellular membrane and thereby negatively regulates PI3KAKT signaling. In our study, we elegantly showed that Rab7 as a functional protein substrate for PTEN phosphatase activity.20 Subsequently, we identified 2 conserved residues S72 and Y183 on Rab7 as potential dephosphorylation sites by PTEN. As timely and accurate localization of Rab7 on to the endosomal membranes is known to be critical for its activation, we focused on testing if the dephosphorylation event by PTEN is important in controlling Rab7 localization. Proper localization of a Rab GTPase onto the specific membrane is a complex process coordinated by several Rab associated proteins. The accepted model for Rab recruitment to the endosomal membrane suggests that cytoplasmic GDP-associated inactive Rab is presented by GDI (GDP dissociation inhibitor) to the membrane.21-25 At the membrane GDI displacement factor (GDF) displaces GDI and presents it to GEF (GDP/GTP exchange factor) for activation.26-28 Further, the GTP bound Rab associates with its effector protein which stabilizes it on the endosomal membrane. We tested our hypothesis of phosphorylation/dephosphorylation switch in controlling Rab7 membrane targeting by first analyzing the interaction of Rab7 with GDI, its membrane delivery component. While Rab7 wild type and a non-phosphorylatable mutant (S72A/ Y183F) interacts with GDI, a consitutive phosphomimetic mutant (S72E/Y183D) fails to do so. Consequently, the constitutive phosphorylation mutant fails to localize to endosomal membranes, thus suggesting that dephosphorylation of these sites is prerequisite for GDI
165
dependent delivery of Rab on to the membrane. Further by using various biochemical and mutational approaches, we clearly demonstrated that while dephosphorylation of S72 is important for Rab7 delivery to the membrane, Y183 dephosphorylation is essential for its stable association at the endosomes via interaction with its downstream effector RILP. Importantly, we observed that a constitutive phosphorylation mutant of Rab7 occurs predominantly in GDP-bound inactive state and a non-phosphorylatable mutant in active GTP bound state. This data clearly suggests the presence of a phospho/dephosphorylation switch in controlling the inactive/active cycles of Rab7 in cytosol and the endosomal membranes. Further the phenotypic implication of Rab7 phosphoregulation was addressed by using epidermal growth factor (EGF) receptor as a model cargo. Expression of Rab7 phosphomimetic mutant results in accumulation of ligand bound EGFR in early endosomes due to defective transition into late endosomes and lysosomes. Consequently, expression of Rab7 phosphomimetic mutants enhances proliferative signaling by constitutive Akt phosphorylation mediated by accumulated EGFR. Thus, by guiding Rab7 localization on to endosomes, PTEN promotes the degradation of growth factor receptors through endosome maturation and blocks the proliferative signaling, possibly providing alternate mechanisms of how PTEN may act as a tumor suppressor.
Future perspectives Our study has provided an interesting observation that phosphorylated Rab7 is inactive and functionally incompetent in the cytosol as it is unable to drive the ligand bound receptor trafficking through endosomes. Further, the investigation of potential kinase that phosphorylates Rab7 on those conserved residues will certainly contribute in better understanding of Rab7 activity cycle as well as its function. Given that S72 site is conserved among many other Rab proteins, it would be interesting to further test the presence of phosphorylation/dephosphorylation switch in global regulation of membrane localization and activity of other Rab proteins as well. In fact, a recent study showing phosphorylation mediated regulation of Rab8 activity19 strengthens the argument that phosphorylation might be an important regulatory mechanism of different Rab proteins. The available literature including our study points that phosphorylation may negatively regulate Rab GTPases activity and function. Further, it can be derived that probably phosphorylation prohibits the access of GDI or GEF to the Rab GTPases which keeps them cytosolic and in inactive state. Based on the mechanistic
166
S. R. SHINDE AND S. MADDIKA
Figure 1. A schematic to present probable regulatory role of phosphorylation in Rab GTPase activity cycle.
evidences provided by our study we propose that dephosphorylation by phosphatases is indeed essential for Rab GTPase activation. But, the enigma is to understand the phosphorylation event in details. Firstly, it is important to identify the kinases which phosphorylate Rab7 and secondly it is critical to point the step at which it happens during Rab7 activity cycle. It is possible that phosphorylation of Rab7 happens on the endosomal membrane which is followed by GTP hydrolysis by GAPs leading to inactivation of Rabs. However, our results showing defective binding of GDI with phosphorylated Rab7 version and given that GDI is important for not only the delivery of GDP bound Rab7 to the membrane but also the retrieval of Rab7 from the membrane after GTP hydolysis by GAP, phosphorylation at the membrane might be an inviable possiblity. On the other hand, it is tempting to speculate that Rab phosphorylation might happen immediately after its retrieval from the membrane by GDI and then Rab is kept in a fully inactive GDP bound state in the cytosol. Upon receipt of appropriate activation signal, phosphatase dephosphorylates and present the Rab to GDI for further activation cycle (Fig. 1). Further studies are required to test this model of Rab activation cycle. Also, future studies need to explore the machinery performing phospho-dephosphorylation of other members of Rab GTPase family that may provide new insights related to their functional regulation. The failure of Rabs to perform their function leads to various disease conditions such as cancer. Thus unravelling of other regulators will provide us with better therapeutic targets to combat various human malignancies.
Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.
Funding This work was fully supported by Wellcome Trust/DBT India Alliance grant (Intermediate fellowship to S.M; 500230/Z/11/ Z). S.M is a recipient of Department of Biotechnology’s IYBA award (BT/01/IYBA/2009). SRS acknowledges the support of Senior Research Fellowship from University Grants Commission (UGC), India.
References [1] Schwartz SL, Cao C, Pylypenko O, Rak A, WandingerNess A. Rab GTPases at a glance. J Cell Sci 2007; 120:3905-10; PMID:17989088; http://dx.doi.org/10.1242/ jcs.015909 [2] Numrich J, Ungermann C. Endocytic Rabs in membrane trafficking and signaling. Biol Chem 2014; 395:327-33; PMID:24158421; http://dx.doi.org/10.1515/hsz-2013-0258 [3] Schmitt HD, Wagner P, Pfaff E, Gallwitz D. The rasrelated YPT1 gene product in yeast: a GTP-binding protein that might be involved in microtubule organization. Cell 1986; 47:401-12; PMID:3094963; http://dx.doi.org/ 10.1016/0092-8674(86)90597-0 [4] Salminen A, Novick PJ. A ras-like protein is required for a post-Golgi event in yeast secretion. Cell 1987; 49:52738; PMID:3552249; http://dx.doi.org/10.1016/0092-8674 (87)90455-7 [5] Barr F, Lambright DG. Rab GEFs and GAPs. Curr Opin Cell Biol 2010; 22:461-70; PMID:20466531; http://dx.doi. org/10.1016/j.ceb.2010.04.007 [6] Cai Y, Chin HF, Lazarova D, Menon S, Fu C, Cai H, Sclafani A, Rodgers DW, De La Cruz EM, Ferro-Novick S,
SMALL GTPASES
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
et al. The structural basis for activation of the Rab Ypt1p by the TRAPP membrane-tethering complexes. Cell 2008; 133:1202-13; PMID:18585354; http://dx.doi.org/ 10.1016/j.cell.2008.04.049 Callaghan J, Simonsen A, Gaullier JM, Toh BH, Stenmark H. The endosome fusion regulator early-endosomal autoantigen 1 (EEA1) is a dimer. Biochem J 1999; 338 (Pt 2):539-43; PMID:10024533; http://dx.doi.org/10.1042/ bj3380539 Roland JT, Kenworthy AK, Peranen J, Caplan S, Goldenring JR. Myosin Vb interacts with Rab8a on a tubular network containing EHD1 and EHD3. Mol Biol Cell 2007; 18:2828-37; PMID:17507647; http://dx.doi.org/ 10.1091/mbc.E07-02-0169 Nielsen E, Christoforidis S, Uttenweiler-Joseph S, Miaczynska M, Dewitte F, Wilm M, et al. Rabenosyn-5, a novel Rab5 effector, is complexed with hVPS45 and recruited to endosomes through a FYVE finger domain. J Cell Biol 2000; 151:601-12; PMID:11062261; http://dx. doi.org/10.1083/jcb.151.3.601 Feng Y, Press B, Wandinger-Ness A. Rab 7: an important regulator of late endocytic membrane traffic. J Cell Biol 1995; 131:1435-52; PMID:8522602; http://dx.doi.org/ 10.1083/jcb.131.6.1435 Vitelli R, Santillo M, Lattero D, Chiariello M, Bifulco M, Bruni CB, Bucci C. Role of the small GTPase Rab7 in the late endocytic pathway. J Biol Chem 1997; 272:4391-7; PMID:9020161; http://dx.doi.org/ 10.1074/jbc.272.7.4391 Bucci C, Thomsen P, Nicoziani P, McCarthy J, van Deurs B. Rab7: a key to lysosome biogenesis. Mol Biol Cell 2000; 11:467-80; PMID:10679007; http://dx.doi.org/ 10.1091/mbc.11.2.467 Harrison RE, Bucci C, Vieira OV, Schroer TA, Grinstein S. Phagosomes fuse with late endosomes and/or lysosomes by extension of membrane protrusions along microtubules: role of Rab7 and RILP. Mol Cell Biol 2003; 23:6494-506; PMID:12944476; http://dx.doi.org/10.1128/ MCB.23.18.6494-6506.2003 Ceresa BP, Bahr SJ. rab7 activity affects epidermal growth factor:epidermal growth factor receptor degradation by regulating endocytic trafficking from the late endosome. J Biol Chem 2006; 281:1099-106; PMID:16282324; http:// dx.doi.org/10.1074/jbc.M504175200 Vanlandingham PA, Ceresa BP. Rab7 regulates late endocytic trafficking downstream of multivesicular body biogenesis and cargo sequestration. J Biol Chem 2009; 284:12110-24; PMID:19265192; http://dx.doi.org/ 10.1074/jbc.M809277200 Rak A, Pylypenko O, Niculae A, Pyatkov K, Goody RS, Alexandrov K. Structure of the Rab7:REP-1 complex: insights into the mechanism of Rab prenylation and choroideremia disease. Cell 2004; 117:749-60; PMID:15186776; http://dx. doi.org/10.1016/j.cell.2004.05.017 Rak A, Pylypenko O, Durek T, Watzke A, Kushnir S, Brunsveld L, Waldmann H, Goody RS, Alexandrov K.
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[28]
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Structure of Rab GDP-dissociation inhibitor in complex with prenylated YPT1 GTPase. Science 2003; 302:646-50; PMID:14576435; http://dx.doi.org/10.1126/science.1087761 Lachance V, Degrandmaison J, Marois S, Robitaille M, Genier S, Nadeau S, Angers S, Parent JL. Ubiquitylation and activation of a Rab GTPase is promoted by a beta(2) AR-HACE1 complex. J Cell Sci 2014; 127:111-23; PMID:24190883; http://dx.doi.org/10.1242/jcs.132944 Lai YC, Kondapalli C, Lehneck R, Procter JB, Dill BD, Woodroof HI, Gourlay R, Peggie M, Macartney TJ, Corti O, et al. Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1. EMBO J 2015; 34:2840-61; PMID:26471730; http://dx.doi.org/ 10.15252/embj.201591593 Shinde SR, Maddika S. PTEN modulates EGFR late endocytic trafficking and degradation by dephosphorylating Rab7. Nat Commun 2016; 7:10689; PMID:26869029; http://dx.doi.org/10.1038/ncomms10689 Sasaki T, Kikuchi A, Araki S, Hata Y, Isomura M, Kuroda S, Takai Y. Purification and characterization from bovine brain cytosol of a protein that inhibits the dissociation of GDP from and the subsequent binding of GTP to smg p25A, a ras p21-like GTP-binding protein. J Biol Chem 1990; 265:2333-7; PMID:2105320 Ullrich O, Horiuchi H, Bucci C, Zerial M. Membrane association of Rab5 mediated by GDP-dissociation inhibitor and accompanied by GDP/GTP exchange. Nature 1994; 368:157-60; PMID:8139660; http://dx.doi.org/ 10.1038/368157a0 Ullrich O, Stenmark H, Alexandrov K, Huber LA, Kaibuchi K, Sasaki T, Takai Y, Zerial M. Rab GDP dissociation inhibitor as a general regulator for the membrane association of rab proteins. J Biol Chem 1993; 268:18143-50; PMID:8349690 Pfeffer S. A model for Rab GTPase localization. Biochem Soc Trans 2005; 33:627-30; PMID:16042559; http://dx. doi.org/10.1042/BST0330627 Matsui Y, Kikuchi A, Araki S, Hata Y, Kondo J, Teranishi Y, Takai Y. Molecular cloning and characterization of a novel type of regulatory protein (GDI) for smg p25A, a ras p21-like GTP-binding protein. Mol Cell Biol 1990; 10:4116-22; PMID:2115118; http://dx.doi.org/10.1128/ MCB.10.8.4116 Soldati T, Shapiro AD, Svejstrup AB, Pfeffer SR. Membrane targeting of the small GTPase Rab9 is accompanied by nucleotide exchange. Nature 1994; 369:76-8; PMID:8164745; http://dx.doi.org/10.1038/369076a0 Itzen A, Pylypenko O, Goody RS, Alexandrov K, Rak A. Nucleotide exchange via local protein unfolding–structure of Rab8 in complex with MSS4. EMBO J 2006; 25:1445-55; PMID:16541104; http://dx.doi.org/10.1038/ sj.emboj.7601044 Delprato A, Lambright DG. Structural basis for Rab GTPase activation by VPS9 domain exchange factors. Nat Struct Mol Biol 2007; 14:406-12; PMID:17450153; http://dx.doi.org/10.1038/nsmb1232
COMMENTARY
A modification switch on a molecular switch: Phosphoregulation of Rab7 during endosome maturation Swapnil Rohidas Shindea,b and Subbareddy Maddikaa a Laboratory of Cell Death & Cell Survival, Centre for DNA Fingerprinting and Diagnostics (CDFD), Nampally, Hyderabad, India; bGraduate Studies, Manipal University, Manipal, India
ABSTRACT
ARTICLE HISTORY
Rab GTPases, the highly conserved members of Ras GTPase superfamily are the pivotal regulators of vesicle-mediated trafficking. Rab GTPases, each with a specific subcellular localization, exert tremendous control over various aspects of vesicular transport, identity and dynamics. Several lines of research have established that GDI, GEFs and GAPs are the critical players to orchestrate Rab GTPase activity and function. The importance of post translational modifications in Rab GTPase functional regulation is poorly or not yet been addressed except for prenylation. Our recent study has revealed a novel dephosphorylation dependent regulatory mechanism for Rab7 activity and function. We have shown the importance of PTEN mediated dephosphorylation of Rab7 on highly conserved S72 and Y183 residues, which is essential for its GDI mediated membrane targeting and further activation by GEF. In conclusion, our study highlighted the importance of a phosphorylation/ dephosphorylation switch in controlling timely Rab7 localization and activity on endosomes.
Received 2 April 2016 Revised 2 April 2016 Accepted 3 April 2016 KEYWORDS
endosome; membrane trafficking; phosphorylation; PTEN; Rab7
Introduction The biogenesis and trafficking of intracellular vesicles and their spatio temporal regulation is critical for maintenance of cellular homeostasis. Rab GTPases, a large family of small GTPases are central to the process of endocytosis which ensures the delivery of cargos to their appropriate destinations.1,2 Since the identification of YPT1, the first Ras related GTPase in yeast 3 and the demonstration of possible role of Ras related GTPases in membrane trafficking,4 66 human Rab proteins have been identified so far. Rab GTPases are the molecular switches which cycle between active GTP-bound state or inactive GDP-bound state and oscillate between different cellular localizations. Rab proteins are activated by GEFs (guanine nucleotide exchange factors) that replaces GDP with GTP and are inactivated by GAPs (GTPase activating proteins).5 Rab GTPases associate with the coat protein components, molecular motors and different SNAREs (Soluble N-ethylmaleimide-sensitive factor attachment protein receptor), and thereby serve as multifaceted organizers of almost all membrane trafficking processes.6-9 Rab7, a member of Rab GTPase family is a key regulator of trafficking, maturation, and fusion of endocytic and autophagic vesicles. It is evident from several studies
that Rab7 localizes primarily to late endosomes, lysosomes, and autophagolysosomes.10-13 Essentially, Rab7 activity specifically controls the maturation of cargo containing early endosomes to late endosomes and their subsequent degradation in lysosomes. Rab7 activity is critical to regulate the lysosome-mediated degradation of endocytic cargoes such as activated EGF receptors.12,14 Loss of Rab7 activity leads to loss of lysosomal integrity as well as delayed or slow degradation of internalized cargos such as EGF receptors.10,12 The importance of Rab7 activity has further been demonstrated by its depletion in the cell which leads to trapping of cargos in late endosomes.15 The entrapment of cargo results in the accumulation of enlarged late endosomes/multivesicular bodies with persistent cellular signaling. Rab GTPases have been extensively studied over the years but our understanding of their regulation by post translational modifications is limited. Prenylation is the most characterized posttranslational modification of Rabs. One or 2 prenyl groups are added to successive cysteine residues present at the extreme C-terminus of the protein which is essential for initial targeting of the Rabs to membranes.16,17 Recently, in an interesting study, Lachance et al have unraveled a novel role for
CONTACT Subbareddy Maddika [email protected] CDFD, Bldg. 7, Gruhakalpa, Nampally, Hyderabad, India. Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/ksgt. © 2016 Taylor & Francis
SMALL GTPASES
ubiquitination of Rab11a by a b2AR/ HACE1 complex in regulating Rab11a activity and b2AR trafficking.18 Lately, Lai et al, reported PINK1 mediated phosphorylation of Rab8A at Ser111 and its implication on Rab8A activation.19 These studies underline the fact that posttranslational modifications such as phosphorylation indeed plays critical role in regulation of Rab GTPase activity. Interestingly, our recent studies have unraveled dephosphorylation of Rab7 by a tumor suppressor phosphatase PTEN.20
Phosphoregulation of Rab7 While trying to identify novel cellular functions of phosphatase PTEN based on the interacting proteins using proteomic analysis, we identified Rab7 as one of the critical PTEN associated proteins. Although PTEN is known to be a dual-specific phosphatase that acts on both lipid and protein substrates, majority of its functions are attributed to its lipid phosphatase activity. PTEN converts phosphatidylinositol-3,4,5-trisphosphate (PIP3) to phosphatidylinositol-4,5-bisphosphate (PIP2) at the cellular membrane and thereby negatively regulates PI3KAKT signaling. In our study, we elegantly showed that Rab7 as a functional protein substrate for PTEN phosphatase activity.20 Subsequently, we identified 2 conserved residues S72 and Y183 on Rab7 as potential dephosphorylation sites by PTEN. As timely and accurate localization of Rab7 on to the endosomal membranes is known to be critical for its activation, we focused on testing if the dephosphorylation event by PTEN is important in controlling Rab7 localization. Proper localization of a Rab GTPase onto the specific membrane is a complex process coordinated by several Rab associated proteins. The accepted model for Rab recruitment to the endosomal membrane suggests that cytoplasmic GDP-associated inactive Rab is presented by GDI (GDP dissociation inhibitor) to the membrane.21-25 At the membrane GDI displacement factor (GDF) displaces GDI and presents it to GEF (GDP/GTP exchange factor) for activation.26-28 Further, the GTP bound Rab associates with its effector protein which stabilizes it on the endosomal membrane. We tested our hypothesis of phosphorylation/dephosphorylation switch in controlling Rab7 membrane targeting by first analyzing the interaction of Rab7 with GDI, its membrane delivery component. While Rab7 wild type and a non-phosphorylatable mutant (S72A/ Y183F) interacts with GDI, a consitutive phosphomimetic mutant (S72E/Y183D) fails to do so. Consequently, the constitutive phosphorylation mutant fails to localize to endosomal membranes, thus suggesting that dephosphorylation of these sites is prerequisite for GDI
165
dependent delivery of Rab on to the membrane. Further by using various biochemical and mutational approaches, we clearly demonstrated that while dephosphorylation of S72 is important for Rab7 delivery to the membrane, Y183 dephosphorylation is essential for its stable association at the endosomes via interaction with its downstream effector RILP. Importantly, we observed that a constitutive phosphorylation mutant of Rab7 occurs predominantly in GDP-bound inactive state and a non-phosphorylatable mutant in active GTP bound state. This data clearly suggests the presence of a phospho/dephosphorylation switch in controlling the inactive/active cycles of Rab7 in cytosol and the endosomal membranes. Further the phenotypic implication of Rab7 phosphoregulation was addressed by using epidermal growth factor (EGF) receptor as a model cargo. Expression of Rab7 phosphomimetic mutant results in accumulation of ligand bound EGFR in early endosomes due to defective transition into late endosomes and lysosomes. Consequently, expression of Rab7 phosphomimetic mutants enhances proliferative signaling by constitutive Akt phosphorylation mediated by accumulated EGFR. Thus, by guiding Rab7 localization on to endosomes, PTEN promotes the degradation of growth factor receptors through endosome maturation and blocks the proliferative signaling, possibly providing alternate mechanisms of how PTEN may act as a tumor suppressor.
Future perspectives Our study has provided an interesting observation that phosphorylated Rab7 is inactive and functionally incompetent in the cytosol as it is unable to drive the ligand bound receptor trafficking through endosomes. Further, the investigation of potential kinase that phosphorylates Rab7 on those conserved residues will certainly contribute in better understanding of Rab7 activity cycle as well as its function. Given that S72 site is conserved among many other Rab proteins, it would be interesting to further test the presence of phosphorylation/dephosphorylation switch in global regulation of membrane localization and activity of other Rab proteins as well. In fact, a recent study showing phosphorylation mediated regulation of Rab8 activity19 strengthens the argument that phosphorylation might be an important regulatory mechanism of different Rab proteins. The available literature including our study points that phosphorylation may negatively regulate Rab GTPases activity and function. Further, it can be derived that probably phosphorylation prohibits the access of GDI or GEF to the Rab GTPases which keeps them cytosolic and in inactive state. Based on the mechanistic
166
S. R. SHINDE AND S. MADDIKA
Figure 1. A schematic to present probable regulatory role of phosphorylation in Rab GTPase activity cycle.
evidences provided by our study we propose that dephosphorylation by phosphatases is indeed essential for Rab GTPase activation. But, the enigma is to understand the phosphorylation event in details. Firstly, it is important to identify the kinases which phosphorylate Rab7 and secondly it is critical to point the step at which it happens during Rab7 activity cycle. It is possible that phosphorylation of Rab7 happens on the endosomal membrane which is followed by GTP hydrolysis by GAPs leading to inactivation of Rabs. However, our results showing defective binding of GDI with phosphorylated Rab7 version and given that GDI is important for not only the delivery of GDP bound Rab7 to the membrane but also the retrieval of Rab7 from the membrane after GTP hydolysis by GAP, phosphorylation at the membrane might be an inviable possiblity. On the other hand, it is tempting to speculate that Rab phosphorylation might happen immediately after its retrieval from the membrane by GDI and then Rab is kept in a fully inactive GDP bound state in the cytosol. Upon receipt of appropriate activation signal, phosphatase dephosphorylates and present the Rab to GDI for further activation cycle (Fig. 1). Further studies are required to test this model of Rab activation cycle. Also, future studies need to explore the machinery performing phospho-dephosphorylation of other members of Rab GTPase family that may provide new insights related to their functional regulation. The failure of Rabs to perform their function leads to various disease conditions such as cancer. Thus unravelling of other regulators will provide us with better therapeutic targets to combat various human malignancies.
Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.
Funding This work was fully supported by Wellcome Trust/DBT India Alliance grant (Intermediate fellowship to S.M; 500230/Z/11/ Z). S.M is a recipient of Department of Biotechnology’s IYBA award (BT/01/IYBA/2009). SRS acknowledges the support of Senior Research Fellowship from University Grants Commission (UGC), India.
References [1] Schwartz SL, Cao C, Pylypenko O, Rak A, WandingerNess A. Rab GTPases at a glance. J Cell Sci 2007; 120:3905-10; PMID:17989088; http://dx.doi.org/10.1242/ jcs.015909 [2] Numrich J, Ungermann C. Endocytic Rabs in membrane trafficking and signaling. Biol Chem 2014; 395:327-33; PMID:24158421; http://dx.doi.org/10.1515/hsz-2013-0258 [3] Schmitt HD, Wagner P, Pfaff E, Gallwitz D. The rasrelated YPT1 gene product in yeast: a GTP-binding protein that might be involved in microtubule organization. Cell 1986; 47:401-12; PMID:3094963; http://dx.doi.org/ 10.1016/0092-8674(86)90597-0 [4] Salminen A, Novick PJ. A ras-like protein is required for a post-Golgi event in yeast secretion. Cell 1987; 49:52738; PMID:3552249; http://dx.doi.org/10.1016/0092-8674 (87)90455-7 [5] Barr F, Lambright DG. Rab GEFs and GAPs. Curr Opin Cell Biol 2010; 22:461-70; PMID:20466531; http://dx.doi. org/10.1016/j.ceb.2010.04.007 [6] Cai Y, Chin HF, Lazarova D, Menon S, Fu C, Cai H, Sclafani A, Rodgers DW, De La Cruz EM, Ferro-Novick S,
SMALL GTPASES
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
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