Rab2 Utilizes Glyceraldehyde-3-phosphate Dehydrogenase and Protein Kinase Cι to Associate with Microtubules and to Recruit Dynein
2008; Elsevier BV; Volume: 284; Issue: 9 Linguagem: Inglês
10.1074/jbc.m807756200
ISSN1083-351X
AutoresEllen J. Tisdale, Fouad Azizi, Cristina R. Artalejo,
Tópico(s)RNA Research and Splicing
ResumoRab2 requires glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and atypical protein kinase Cι (aPKCι) for retrograde vesicle formation from vesicular tubular clusters that sort secretory cargo from recycling proteins returned to the endoplasmic reticulum. However, the precise role of GAPDH and aPKCι in the early secretory pathway is unclear. GAPDH was the first glycolytic enzyme reported to co-purify with microtubules (MTs). Similarly, aPKC associates directly with MTs. To learn whether Rab2 also binds directly to MTs, a MT binding assay was performed. Purified Rab2 was found in a MT-enriched pellet only when both GAPDH and aPKCι were present, and Rab2-MT binding could be prevented by a recombinant fragment made to the Rab2 amino terminus (residues 2-70), which directly interacts with GAPDH and aPKCι. Because GAPDH binds to the carboxyl terminus of α-tubulin, we characterized the distribution of tyrosinated/detyrosinated α-tubulin that is recruited by Rab2 in a quantitative membrane binding assay. Rab2-treated membranes contained predominantly tyrosinated α-tubulin; however, aPKCι was the limiting and essential factor. Tyrosination/detyrosination influences MT motor protein binding; therefore, we determined whether Rab2 stimulated kinesin or dynein membrane binding. Although kinesin was not detected on membranes incubated with Rab2, dynein was recruited in a dose-dependent manner, and binding was aPKCι-dependent. These combined results suggest a mechanism by which Rab2 controls MT and motor recruitment to vesicular tubular clusters. Rab2 requires glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and atypical protein kinase Cι (aPKCι) for retrograde vesicle formation from vesicular tubular clusters that sort secretory cargo from recycling proteins returned to the endoplasmic reticulum. However, the precise role of GAPDH and aPKCι in the early secretory pathway is unclear. GAPDH was the first glycolytic enzyme reported to co-purify with microtubules (MTs). Similarly, aPKC associates directly with MTs. To learn whether Rab2 also binds directly to MTs, a MT binding assay was performed. Purified Rab2 was found in a MT-enriched pellet only when both GAPDH and aPKCι were present, and Rab2-MT binding could be prevented by a recombinant fragment made to the Rab2 amino terminus (residues 2-70), which directly interacts with GAPDH and aPKCι. Because GAPDH binds to the carboxyl terminus of α-tubulin, we characterized the distribution of tyrosinated/detyrosinated α-tubulin that is recruited by Rab2 in a quantitative membrane binding assay. Rab2-treated membranes contained predominantly tyrosinated α-tubulin; however, aPKCι was the limiting and essential factor. Tyrosination/detyrosination influences MT motor protein binding; therefore, we determined whether Rab2 stimulated kinesin or dynein membrane binding. Although kinesin was not detected on membranes incubated with Rab2, dynein was recruited in a dose-dependent manner, and binding was aPKCι-dependent. These combined results suggest a mechanism by which Rab2 controls MT and motor recruitment to vesicular tubular clusters. The small GTPase Rab2 is essential for membrane trafficking in the early secretory pathway and associates with vesicular tubular clusters (VTCs) 2The abbreviations used are: VTC, vesicular tubular cluster; ER, endoplasmic reticulum; aPKCι, atypical protein kinase C iota; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MAPs, microtubule associated proteins; MTs, microtubules; Tyr-tubulin, tyrosinated α-tubulin; Glu-tubulin, detyrosinated tubulin; GTPγS, guanosine 5′-3-O-(thio)triphosphate; PBS, phosphate-buffered saline; Pipes, 1,4-piperazinediethanesulfonic acid.2The abbreviations used are: VTC, vesicular tubular cluster; ER, endoplasmic reticulum; aPKCι, atypical protein kinase C iota; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MAPs, microtubule associated proteins; MTs, microtubules; Tyr-tubulin, tyrosinated α-tubulin; Glu-tubulin, detyrosinated tubulin; GTPγS, guanosine 5′-3-O-(thio)triphosphate; PBS, phosphate-buffered saline; Pipes, 1,4-piperazinediethanesulfonic acid. located between the endoplasmic reticulum (ER) and the cis-Golgi compartment (1Tisdale E.J. Bourne J.R. Khosravi-Far R. Der C.J. Balch W.E. J. Cell Biol. 1992; 119: 749-761Crossref PubMed Scopus (417) Google Scholar, 2Tisdale E.J. Balch W.E. J. Biol. Chem. 1996; 271: 29372-29379Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). VTCs are pleomorphic structures that sort anterograde-directed cargo from recycling proteins and trafficking machinery retrieved to the ER (3Balch W.E. McCaffery J.M. Plutner H. Farquhar M.G. Cell. 1994; 76: 841-852Abstract Full Text PDF PubMed Scopus (331) Google Scholar, 4Altan-Bonnet N. Sougrat R. Lippincott-Schwartz J. Curr. Opin. Cell Biol. 2004; 16: 364-372Crossref PubMed Scopus (145) Google Scholar, 5Lee M.C.S. Miller E.A. Goldberg J. Orci L. Schekman R. Annu. Rev. Cell Dev. Biol. 2004; 20: 87-123Crossref PubMed Scopus (687) Google Scholar, 6Appenzeller-Herzog C. Hauri H.P. J. Cell Sci. 2006; 119: 2173-2183Crossref PubMed Scopus (306) Google Scholar). Rab2 bound to a VTC microdomain stimulates recruitment of soluble factors that results in the release of vesicles containing the recycling protein p53/p58 (7Tisdale E.J. Mol. Biol. Cell. 1999; 10: 1837-1849Crossref PubMed Scopus (64) Google Scholar). In that regard, we have previously reported that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and atypical PKC ι (aPKCι) are Rab2 effectors that interact directly with the Rab2 amino terminus and with each other (8Tisdale E.J. J. Biol. Chem. 2003; 278: 52524-52530Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 9Tisdale E.J. Kelly C. Artalejo C.R. J. Biol. Chem. 2004; 279: 54046-54052Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Their interaction requires Src-dependent tyrosine phosphorylation of GAPDH and aPKCι (10Tisdale E.J. Artalejo C.R. Traffic. 2007; 8: 733-741Crossref PubMed Scopus (47) Google Scholar). Moreover, GAPDH is a substrate for aPKCι (11Tisdale E.J. J. Biol. Chem. 2002; 277: 3334-3341Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). GAPDH catalytic activity is not required for ER to Golgi transport indicating that GAPDH provides a specific function essential for membrane trafficking from VTCs independent of glycolytic function (9Tisdale E.J. Kelly C. Artalejo C.R. J. Biol. Chem. 2004; 279: 54046-54052Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Indeed, phospho-GAPDH influences MT dynamics in the early secretory pathway (11Tisdale E.J. J. Biol. Chem. 2002; 277: 3334-3341Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). GAPDH was the first glycolytic enzyme reported to co-purify with microtubules (MTs) (12Kumagai H. Sakai H. J. Biochem. (Tokyo). 1983; 93: 1259-1269Crossref PubMed Scopus (5) Google Scholar) and subsequently was shown to interact with the carboxyl terminus of α-tubulin (13Volker K.W. Knull H.R. Arch. Biochem. Biophys. 1997; 338: 237-243Crossref PubMed Scopus (68) Google Scholar). The binding of GAPDH to MTs promotes formation of cross-linked parallel MT arrays or bundles (14Somers M. Engelborghs Y. Baert J. Eur. J. Biochem. 1990; 193: 437-444Crossref PubMed Scopus (59) Google Scholar, 15MacRae T.H. Biochim. Biophys. Acta. 1992; 1160: 145-155Crossref PubMed Scopus (40) Google Scholar). GAPDH has also been reported to possess membrane fusogenic activity, which is inhibited by tubulin (16Glaser P. Han X. Gross R.W. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14104-14109Crossref PubMed Scopus (59) Google Scholar). Similarly, aPKC associates directly with tubulin and promotes MT stability and MT remodeling at specific intracellular sites (17Garcia-Rocha M. Avila J. Lozano J. Exp. Cell Res. 1997; 230: 1-8Crossref PubMed Scopus (29) Google Scholar, 18Seibenhener M.L. Roehm J. White W. Neidigh K. Vandenplas M. Wooten M.W. Mol. Cell Biol. Res. Commun. 1999; 2: 28-31Crossref PubMed Scopus (22) Google Scholar, 19Ruiz-Canada C. Ashley J. Moeckel-Cole S. Drier E. Yin J. Budnik V. Neuron. 2004; 42: 567-580Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 20Harris T. Peifer M. Dev. Cell. 2007; 12: 727-738Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 21Etienne-Manneville S. Hall A. Curr. Opin. Cell Biol. 2003; 15: 67-72Crossref PubMed Scopus (250) Google Scholar). It may not be coincidental that these two Rab2 effectors influence MT dynamics because recent studies indicate that the cytoskeleton plays a central role in the organization and operation of the secretory pathway (22Caviston J. Holzbaur E. Trends Cell Biol. 2006; 16: 530-537Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). MTs are dynamic structures that grow or shrink by the addition or loss of α- and β-tubulin heterodimers from the ends of protofilaments (23Wade R.H. Methods Mol. Med. 2007; 137: 1-16Crossref PubMed Scopus (45) Google Scholar). Their assembly and stability is regulated by a variety of proteins traditionally referred to as microtubule-associated proteins (MAPs). In addition to the multiple α/β isoforms that are present in eukaryotes, MTs undergo an assortment of post-translational modifications, including acetylation, glycylation, glutamylation, phosphorylation, palmitoylation, and detyrosination, which further contribute to their biochemical heterogeneity (24Verhey K. Gaertig J. Cell Cycle. 2007; 6: 2152-2160Crossref PubMed Scopus (384) Google Scholar, 25Hammond J. Cai D. Verhey K. Curr. Opin. Cell Biol. 2007; 19: 1-6Crossref Scopus (39) Google Scholar). It has been proposed that these tubulin modifications regulate intracellular events by facilitating interaction with MAPs and with other specific effector proteins (24Verhey K. Gaertig J. Cell Cycle. 2007; 6: 2152-2160Crossref PubMed Scopus (384) Google Scholar). For example, the reversible addition of tyrosine to the carboxyl terminus of α-tubulin regulates MT interaction with plus-end tracking proteins (+TIPs) containing the cytoskeleton-associated protein glycine-rich (CAP-Gly) motif and with dynein-dynactin (27Honnappa S. Okhrimenko O. Jaussi R. Jawhari H. Jeselarov I. Winkler F. Steinmetz M. Mol. Cell. 2006; 23: 663-671Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 28Peris L. Thery M. Faure J. Saoudi Y. Lafanechere L. Chilton J. Gordon-Weeks P. Galijart N. Bornens M. Wordeman L. Wehland J. Andrieux A. Job D. J. Cell Biol. 2007; 174: 839-849Crossref Scopus (232) Google Scholar, 29Weisbrich A. Honnappa S. Jaussi R. Okhrimenko O. Frey D. Jelesarov I. Akhmanova A. Steinmetz M. Nat. Struct. Mol. Biol. 2007; 14: 959-967Crossref PubMed Scopus (155) Google Scholar). Additionally, MT motility and cargo transport rely on the cooperation of the motor proteins kinesin and dynein (30Ross J. Ali M.Y. Warshaw D. Curr. Opin. Cell Biol. 2008; 20: 41-47Crossref PubMed Scopus (237) Google Scholar). Kinesin is a plus-end directed MT motor, whereas cytoplasmic dynein is a minus-end MT-based motor, and therefore the motors transport vesicular cargo toward the opposite end of a MT track (31Gross S. Vershinin M. Shubeita G. Curr. Biol. 2007; 17: R478-R486Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Although MT assembly does not appear to be directly regulated by small GTPases, Rab proteins provide a molecular link for vesicle movement along MTs to the appropriate target (22Caviston J. Holzbaur E. Trends Cell Biol. 2006; 16: 530-537Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 32Echard A. Jollivet F. Martinez O. Lacapere J.J. Rousselet A. Janoueix-Lerosey I. Goud B. Science. 1998; 279: 580-585Crossref PubMed Scopus (408) Google Scholar, 33Short B. Preisinger C. Schaletzky J. Kopajtich R. Barr F. Curr. Biol. 2002; 12: 1792-1795Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 34Hoepfner S. Severin F. Cabezas A. Habermann B. Runge A. Gillooly D. Stenmark H. Zerial M. Cell. 2005; 121: 437-450Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). In this study, the potential interaction of Rab2 with MTs and motor proteins was characterized. We found that Rab2 does not bind directly to preassembled MTs but does associate when both GAPDH and aPKCι are present and bound to MTs. Moreover, the MTs predominantly contained tyrosinated α-tubulin (Tyr-tubulin) suggesting that a dynamic pool of MTs that differentially binds MAPs/effector proteins/motors associates with VTCs in response to Rab2. To that end, we determined that Rab2-promoted dynein/dynactin binding to membranes and that the recruitment required aPKCι. Quantitative Membrane Binding Assay—HeLa membranes were prepared as described previously (10Tisdale E.J. Artalejo C.R. Traffic. 2007; 8: 733-741Crossref PubMed Scopus (47) Google Scholar). Membranes (∼30 μg of total protein) were added to a reaction mixture that contained 27.5 mm Hepes (pH 7.4), 2.75 mm MgOAc, 65 mm KOAc, 5 mm EGTA, 1.8 mm CaCl2, 1 mm ATP, 5 mm creatine phosphate, and 0.2 IU rabbit muscle creatine phosphokinase (35Tisdale E.J. Jackson M.R. J. Biol. Chem. 1998; 273: 17269-17277Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Purified recombinant Rab2, aPKCι, and aPKCι (K274W) (7Tisdale E.J. Mol. Biol. Cell. 1999; 10: 1837-1849Crossref PubMed Scopus (64) Google Scholar, 36Tisdale E.J. Traffic. 2000; 1: 702-712Crossref PubMed Scopus (41) Google Scholar) or purified recombinant Rab2 amino-terminal fragment, prepared as described below, was added at the concentrations indicated under "Results," and the reaction mix was incubated on ice for 10 min. Rat liver cytosol (∼25 μg of total protein) and 2.0 μm GTPγS (Sigma) was then added, and the reactions were shifted to 32 °C and incubated for 12 min. The binding reaction was layered onto a 20% sucrose cushion and centrifuged at 35,000 × g for 20 min at 25 °C. The pellet was separated by SDS-PAGE and transferred to nitrocellulose in 25 mm Tris (pH 8.3), 192 mm glycine, and 20% methanol. The membrane was blocked in Tris-buffered saline that contained 5% nonfat dry milk or 5% bovine serum albumin and 0.5% Tween 20; then incubated with anti-dynein intermediate chain (clone 70.1) (Sigma), or anti-kinesin (clone IBII) (Sigma), or anti-detyrosinated α-tubulin (Glu-tubulin) (Millipore Corp, Billerica, MA), or anti-tyrosinated α-tubulin (Tyr-tubulin) (Millipore), or anti-GAPDH (Chemicon International, Temecula, CA), or anti-aPKCι (BD Biosciences), or anti-p50/dynamitin (Millipore); washed; further incubated with the appropriate horseradish peroxidase-conjugated secondary antibody; developed with enhanced chemiluminescence (ECL) (Pierce); and then quantified by densitometry using the ImageQuant program (GE Healthcare). Indirect Immunofluorescence—HeLa cells were plated on coverslips (5 × 105 cells/75-mm dish) and then transiently mock-transfected or transfected with pCR3.1-Rab2 or with pcDNA4/HisMax-PKCι using Lipofectamine (Invitrogen) for 36 h at 37 °C in a 5% CO2 incubator. The coverslips were transferred to 0.1% Triton X-100, 80 mm Pipes (pH 6.9), 5 mm EGTA, 1 mm MgCl2, for 3 min to permeabilize the cells, fixed for 20 min with 4% formaldehyde, washed three times with PBS, and then blocked for 1 h in PBS, 5% normal goat serum. The cells were then incubated for 30 min with either anti-Tyr-tubulin (Millipore), or an affinity-purified Rab2 polyclonal antibody (35Tisdale E.J. Jackson M.R. J. Biol. Chem. 1998; 273: 17269-17277Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), or an affinity-purified polyclonal to p53/p58 (37Tisdale E.J. Plutner H. Matteson J. Balch W.E. J. Cell Biol. 1997; 137: 581-593Crossref PubMed Scopus (76) Google Scholar), or anti-GAPDH (Cell Signaling, Danvers, MA), anti-aPKCι (GenScript Corp., Piscataway, NJ), or fluorescein isothiocyanate-conjugated anti-Xpress (Invitrogen); washed extensively with PBS; incubated with Alexa Fluor 488 chicken anti-mouse antibody and Alex Fluor 594 chicken anti-rabbit; washed extensively with PBS; mounted in Mowiol containing 1,4-diazabicyclo[2.2.2]octane (Sigma); and then viewed with a Zeiss AxioImager epifluorescence microscope (Carl Zeiss, Gottingen, Germany) and photographed with an AxioCamMRm camera (Zeiss Microimaging, Thornwood, NY) using AxioVision Z-stack software to capture 20 images of ∼3.6 μm (Zeiss Microimaging). The image threshold was obtained for both channels, and the extent of true co-localization in the boxed areas (yellow pixels in merged image) was analyzed using the Manders overlap co-localization coefficient (co-localization module, Zeiss Microimaging). A value of 1 is high co-localization, and a value of 0 is low co-localization. MT Binding Assay—MTs were assembled from 1 μg of pure bovine brain tubulin (Cytoskeleton, Inc., Denver, CO) in 20 μl of Buffer B (100 mm Pipes (pH 6.8), 2 mm MgCl2, and 0.5 mm EGTA) containing 20 μm paclitaxel (Sigma) for 20 min at 37 °C. The MTs were then incubated with purified recombinant Rab2, His6-aPKCι, or His6-GAPDH (200 ng) in a total volume of 50 μl for 30 min at 37 °C. The reaction mixtures were layered onto 250 μl of a 20% sucrose cushion in Buffer B containing 20 μm paclitaxel, and then centrifuged for 30 min at 100,000 × g in a Sorvall M120 Discovery ultra-microcentrifuge using a S120-AT2 rotor. The resultant supernatant and pellet were separated by SDS-PAGE and transferred to nitrocellulose. The blot was probed with the appropriate antibody as indicated under "Results," washed, further incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibody, developed with ECL, and then quantified by densitometry, as above. Construction of the Rab2 Amino-terminal Fragment/Deletion Mutant—The Rab2 amino-terminal domain was generated by PCR using the 5′ oligonucleotide primer, 5′-CACCATGGCGTACGCCTATCTC-3′, in tandem with the 3′ antisense oligonucleotide, 5′-AATAGTTATCATTCGAGCACCGAA-3′. The amplified product was subcloned into pET102/D-TOPO (C-term) (Invitrogen), and the sequence was verified by DNA sequence analysis. BL21 (DE3) pLysS cells (EMD Biosciences, La Jolla, CA) transformed with the clone were grown at 37 °C to 0.6 A600 and then induced with 0.4 mm isopropyl β-d-thiogalactopyranoside for 3 h at 37 °C. The liquid culture was centrifuged at 6,000 rpm for 30 min, and the pellet was resuspended in 50 mm Tris (pH 7.4), 1 mm dithiothreitol, 1 mm EDTA, 1% Triton X-100, and 1 mm phenylmethylsulfonyl fluoride; sonicated; centrifuged at 22,000 × g for 30 min; and then the supernatant was applied to a 1-ml column of Ni2+-nitrilotriacetic acid-agarose (Qiagen, Valencia, CA) equilibrated in Buffer A (10 mm Hepes (pH 7.9), 5 mm MgCl2, 0.1 mm EDTA, 50 mm NaCl, and 0.8 mm imidazole). The column was washed with 10 volumes of Buffer A containing 25 mm imidazole. The tagged protein was eluted with Buffer A supplemented with 200 mm imidazole. An aliquot of the collected fractions (100 μl) was analyzed by SDS-PAGE and immunoblotted with an anti-His6 monoclonal antibody (Cell Signaling). His6-Rab2 fragment enriched fractions were pooled and concentrated, and the protein concentration was determined by Micro BCA protein assay reagent (Pierce). The Rab2 amino-terminal deletion mutant was generated, as outlined previously (9Tisdale E.J. Kelly C. Artalejo C.R. J. Biol. Chem. 2004; 279: 54046-54052Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Rab2 Requires GAPDH and aPKCι to Associate with MTs—To learn if Rab2 binds directly to MTs, an in vitro MT binding assay was performed in which purified recombinant Rab2 was incubated with paclitaxel assembled MTs for 30 min at 37 °C. The binding reaction was centrifuged through a 20% sucrose cushion, and then the pellet containing MTs and associated protein and the supernatant containing unbound protein were analyzed by SDS-PAGE and Western blot. As shown in Fig. 1A, Rab2 was found almost exclusively in the high speed supernatant, which contained a negligible amount of tubulin. In contrast, when the MT binding assay was performed with purified aPKCι or with purified GAPDH, both proteins co-sedimented with MTs in the high speed pellet consistent with previous reports that showed GAPDH and aPKC interact directly with MTs (12Kumagai H. Sakai H. J. Biochem. (Tokyo). 1983; 93: 1259-1269Crossref PubMed Scopus (5) Google Scholar, 13Volker K.W. Knull H.R. Arch. Biochem. Biophys. 1997; 338: 237-243Crossref PubMed Scopus (68) Google Scholar, 17Garcia-Rocha M. Avila J. Lozano J. Exp. Cell Res. 1997; 230: 1-8Crossref PubMed Scopus (29) Google Scholar, 38Volker K.W. Reinitz C.A. Knull H.R. Comp. Biochem. Physiol. B. 1995; 112: 503-514Crossref PubMed Scopus (59) Google Scholar). Because GAPDH and aPKCι bind directly to the Rab2 amino terminus, we determined whether either protein could promote Rab2-MT association by supplementing the assay with either purified GAPDH or purified aPKCι (Fig. 1B). As observed previously, GAPDH and aPKCι independently bound to MTs, whereas Rab2 displayed no binding affinity for MTs and fractionated with the supernatant. However, Rab2 was found in the MT-enriched pellet when the MT binding assay was conducted in the presence of both GAPDH and aPKCι (Fig. 1B). Rab2 indirect association with MTs was prevented when the binding assay was supplemented with a recombinant protein fragment corresponding to the Rab2 amino terminus (residues 2-70) that directly interacts with GAPDH and aPKCι (Fig. 1C) (8Tisdale E.J. J. Biol. Chem. 2003; 278: 52524-52530Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 9Tisdale E.J. Kelly C. Artalejo C.R. J. Biol. Chem. 2004; 279: 54046-54052Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). The amount of Rab2 bound to MTs was reduced ∼50% in the presence of Rab2 (residues 2-70) (125 ng), and ∼70% of Rab2 was found in the high speed supernatant when co-incubated with 250 ng of the Rab2 fragment (Fig. 1C). Moreover, Rab2 (residues 2-70) was found in the GAPDH-aPKCι-MT pellet indicating that the recombinant fragment interfered with Rab2-MT binding by competition for GAPDH and aPKCι interaction. These combined results suggest that Rab2 requires and utilizes both GAPDH and aPKCι as adaptor/scaffolding proteins to indirectly associate with MTs. Rab2 via aPKCι Preferentially Recruits Tyrosinated α-Tubulin and Dynein to Membranes—Tubulin can undergo a variety of post-translational modifications, including removal of the tyrosine residue from the carboxyl terminus of α-tubulin and subsequent tyrosine re-addition (detyrosination/tyrosination cycle) that influences interaction with specific MAPs and motor proteins (25Hammond J. Cai D. Verhey K. Curr. Opin. Cell Biol. 2007; 19: 1-6Crossref Scopus (39) Google Scholar). It is notable that GAPDH has been reported to bind specifically to the carboxyl terminus of α-tubulin (13Volker K.W. Knull H.R. Arch. Biochem. Biophys. 1997; 338: 237-243Crossref PubMed Scopus (68) Google Scholar). Therefore, we analyzed the distribution of Rab2 recruited tyrosinated α-tubulin (Tyr-tubulin) versus detyrosinated α-tubulin (Glu-tubulin) to VTCs by performing a quantitative membrane binding assay (10Tisdale E.J. Artalejo C.R. Traffic. 2007; 8: 733-741Crossref PubMed Scopus (47) Google Scholar, 39Tisdale E.J. Artalejo C.R. J. Biol. Chem. 2006; 281: 8436-8442Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Salt-washed HeLa cell membranes were incubated with rat liver cytosol and GTPγS in the absence or presence of increasing Rab2 concentrations. The reaction was then centrifuged through a 20% sucrose cushion, and the pellet containing membranes and associated proteins, including MTs, was subjected to SDS-PAGE and Western blot analysis. The relative level of Tyr- and Glu-tubulin recruited to membranes is reported as percent of total pool because of the use of two monoclonal antibodies with different binding affinities. As shown in Fig. 2A, Rab2 promoted a dose-dependent increase in membrane-bound Tyr-tubulin. From the total pool of cytosolic Tyr-tubulin added to the binding assay and therefore available for Rab2-dependent recruitment, ∼9-11% of Tyr-tubulin became membrane-associated after co-incubation with Rab2 (75 ng), and addition of a higher Rab2 concentration (150 ng) resulted in ∼17-20% increase in membrane-bound Tyr-tubulin. In contrast, ∼3-6% of Glu-Tyr was bound to Rab2-incubated membranes regardless of the Rab2 concentration. Because Rab2-treated membranes contained a significant amount of Tyr-tubulin, HeLa cells were transfected with Rab2 cDNA, and the distribution of Rab2 and Tyr-tubulin in vivo was analyzed by indirect immunofluorescence. Cells ectopically expressing Rab2 displayed prominent Rab2- and Tyr-tubulin-labeled pleomorphic elements (boxed area; Manders coefficient of 0.942) that were primarily juxtaposed to the nucleus as well as smaller Tyr-tubulin-labeled punctate structures dispersed throughout the cell periphery (Fig. 2B). These Tyr-tubulin/Rab2-containing entities did not stain with anti-Glu-tubulin antibody (data not shown) but did co-distribute with GAPDH (boxed area; Manders coefficient of 0.921) and aPKCι (boxed area; Manders coefficient of 0.925) (Fig. 2C). Importantly, the Tyr-tubulin/Rab2 structures co-labeled with anti-p53/p58 (boxed area; Manders coefficient of 0.928) demonstrating that the pleomorphic elements are VTCs and not tubulin aggregates (Fig. 2D). In that regard, we observed in Rab2-overexpressing cells VTCs associated with and aligned with long MT tracks (Fig. 2C). Rab2 recruits GAPDH upstream of aPKCι, and aPKCι membrane association requires GAPDH. Interestingly, increasing amounts of GAPDH added to the binding assay did not significantly increase the level of membrane-associated Tyr-tubulin, indicating that GAPDH is not a limiting factor (data not shown). However, the ability of Rab2 to promote Tyr-tubulin membrane binding was eliminated when purified recombinant Rab2 N′Δ19, a mutant protein that fails to recruit aPKCι to membranes, was added to the assay (8Tisdale E.J. J. Biol. Chem. 2003; 278: 52524-52530Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) (Fig. 3A). This truncation in Rab2 reduces association with GAPDH but does not abolish Rab2-GAPDH interaction (9Tisdale E.J. Kelly C. Artalejo C.R. J. Biol. Chem. 2004; 279: 54046-54052Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). To learn whether aPKCι played a specific role in Tyr-tubulin recruitment, the membrane binding assay was supplemented with purified recombinant aPKCι. In this case and unlike GAPDH, increasing amounts of membrane-associated aPKCι caused ∼5-8-fold increase in membrane-bound Tyr-tubulin and a minimal increase (∼1-1.5-fold) in the amount of membrane-bound Glu-tubulin (Fig. 3B). The putative role of aPKCι in Tyr-tubulin binding was further assessed in the binding reaction by adding anti-aPKCι. Fig. 3C shows that increasing amounts of anti-aPKCι reduced membrane association of aPKCι and Tyr-tubulin; however, anti-PKCι had no effect on Glu-tubulin recruitment. These combined biochemical results are highly suggestive that membrane-bound aPKCι influences dynamic MTs enriched in Tyr-tubulin to associate with VTCs. Rab proteins regulate MT motor protein recruitment, and MT motor proteins preferentially bind to either Glu or Tyr-MTs (22Caviston J. Holzbaur E. 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Marotta A. Conrad P. Bloom G.S. J. Cell Biol. 1995; 128: 293-306Crossref PubMed Scopus (213) Google Scholar, 43Roghi C. Allan V.J. J. Cell Sci. 1999; 112: 4673-4685Crossref PubMed Google Scholar, 44Stauber T. Simpson J. Pepperkok R. Vernos I. Curr. Biol. 2006; 16: 2245-2251Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), we did not detect any kinesin or any Rab2-recruited kinesin on the membranes (data not shown). In contrast, Rab2 promoted a dose-dependent increase in membrane-associated dynein as determined by probing the blot with anti-dynein intermediate chain (Fig. 4A). Similarly, Rab2 stimulated membrane association of p50/dynamitin, a component of the dynactin complex (Fig. 4A). However, addition of Rab2 N′Δ19 to the assay failed to increase dynein or p50/dynamitin binding to membranes suggesting that aPKCι was necessary for their downstream recruitment (Fig. 4A). To investigate the potential role of aPKCι in dynein/dynactin recruitment, anti-GAPDH or anti-aPKCι was added to the membrane binding assay. The anti-GAPDH reagent blocked membrane association of GAPDH, and consequently aPKCι and dynein/dynamitin, whereas anti-PKCι inhibited aPKCι and dynein/dynamitin membrane binding but had no effect on GAPDH recruitment (Fig. 4B). Because aPKCι-dependent GAPDH phosphorylation plays a role in MT binding to membranes, we determined whether aPKCι kinase activity was required for dynein/dynamitin recruitment by incubating membranes with kinase-dead PKCι (K274W) (36Tisdale E.J. Traffic. 2000;
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