Rab2 Interacts Directly with Atypical Protein Kinase C (aPKC) ι/λ and Inhibits aPKCι/λ-dependent Glyceraldehyde-3-phosphate Dehydrogenase Phosphorylation*

Atypical protein kinase C ι/λ (PKCι/λ) is essential for protein transport in the early secretory pathway. The small GTPase Rab2 selectively recruits the kinase to vesicular tubular clusters (VTCs) where PKCι/λ phosphorylates glyceraldehyde-3-phosphate dehydrogenase (GAPDH). VTCs are composed of small vesicles and tubules and serve as transport intermediates that shuttle cargo from the endoplasmic reticulum to the Golgi complex. These structures are the first site of segregation of the anterograde and retrograde pathways. When Rab2 binds to a VTC subcompartment, the subsequent recruitment of PKCι/λ and soluble components, including COPI (coatomer and ADP-ribosylation factor), results in the release of retrograde-directed vesicles. Because Rab2 stimulates PKCι/λ membrane association in a dose-dependent manner, we investigated whether the two proteins physically interact. Using a combination of in vivo and in vitro assays, we found that Rab2 interacts directly with PKCι/λ and that this interaction occurs through the Rab2 amino terminus (residues 1–19) and the PKCι/λ regulatory domain. A mutant lacking the PKCι/λ binding domain (Rab2N′Δ19) was functionally characterized. In contrast to Rab2, Rab2N′Δ19 failed to recruit PKCι/λ to normal rat kidney microsomes in a quantitative binding assay. To determine whether Rab2 modulates the ability of PKCι/λ to phosphorylate GAPDH, an in vitro kinase assay was supplemented with Rab2 or Rab2N′Δ19. Rab2 inhibited PKCι/λ-dependent GAPDH phosphorylation, whereas no effect was observed when the assay was performed with the aminoterminal truncation mutant. These results suggest that a downstream effector recruited to the VTC stimulates PKCι/λ-mediated GAPDH phosphorylation by alleviating the inhibition imposed by Rab2-PKCι/λ interaction.

The Rab family of small GTPases regulates membrane traffic in the exocytic and endocytic pathways (1,2). These essential proteins alternate between a GTP-and GDP-bound conformation, which catalyzes a cycle of membrane association and release to the cytosol. Rab proteins bind to specific intracellular compartments and promote recruitment of soluble components that facilitate membrane fission, cytoskeletal interaction, and vesicle tethering/docking (2)(3)(4). Multiple sequence alignments indicate that the amino-and carboxyl-terminal regions of Rab proteins are highly divergent and therefore potential sites for proteinprotein interaction with unique effectors that regulate compartment-specific transport events. In that regard, the amino terminus of Rab5 was found to play a critical role in Rab5-dependent early endosome fusion (5,6), whereas residues 19 -22 in Rab3A are required for interaction with the effector Rabphilin-3A (7). The amino terminus of Rab2 is also required for function: Deletion of residues 1-14 attenuated the inhibitory properties of the Rab2 trans dominant mutant (N119I) (8). Moreover, a peptide corresponding to the deleted amino acids (residues 2-14) was a potent and irreversible inhibitor of ER 1 to Golgi traffic when introduced into an in vitro transport assay (8). Studies were initiated to elucidate the mechanism by which the Rab2 (13-mer) arrests transport and included employing a quantitative binding assay that measures recruitment of transport-related proteins to membranes incubated with the peptide. The Rab2 (13-mer) markedly stimulated recruitment of COPI (coatomer and ADPribosylation factor) to normal rat kidney (NRK) cell microsomes in a protein kinase C (PKC)-dependent manner (9,10). We have since identified the participating isoform as PKC/, a member of the atypical subgroup of PKC (11). This subfamily of kinases plays a critical role in controlling cell growth by interacting with proteins that ultimately link Cdc42-coordinated signaling cascades with cytoskeletal rearrangement and vesicle transport (12)(13)(14)(15). Interestingly, the Rab2 (13-mer) not only stimulates COPI membrane association but also promotes membrane binding of PKC/ and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (10, 11). These combined results suggest that the Rab2 amino terminus is necessary for Rab2 activity and that the Rab2 (13-mer) functions as a bona fide Rab2 domain that interacts with one or more components of the trafficking machinery.
Rab2 is required for membrane transport in the early secretory pathway. This protein immunolocalizes to vesicular tubular clusters (VTCs) that function as transport intermediates between the endoplasmic reticulum and the Golgi complex (16,17). These structures are the first site of segregation of the anterograde and retrograde pathways and thereby sort and recycle resident proteins from itinerate proteins destined for secretion (18,19). In our ongoing studies, we found that Rab2 requires PKC/ kinase activity to promote retrograde vesicle formation from a VTC subcompartment (11,20). The Rab2generated vesicles lacked anterograde-directed cargo but contained PKC/, COPI, GAPDH, and the recycling protein p53/ p58 (10, 21). It is noteworthy that GAPDH is a substrate for PKC/ and that GAPDH interacts directly with the PKC/ regulatory domain (22).
In this study, we learned that the PKC/ regulatory domain not only binds to GAPDH, but also directly interacts with Rab2 via the Rab2 amino terminus. A Rab2 truncation mutant missing the first 19 residues was unable to bind the kinase. Unlike intact Rab2, Rab2NЈ⌬19 failed to recruit PKC/ and GAPDH to NRK microsomes in a quantitative binding assay. Because PKC-binding proteins are potential regulators of kinase activity, we determined whether Rab2 modulates PKC/ enzymatic activity, in vitro. Rab2 caused a dramatic reduction of PKC/mediated GAPDH phosphorylation. A similar reduction in phosphorylation activity occurred in the presence of the Rab2 (13-mer). However, PKC/-dependent phosphorylation of GAPDH was restored when the in vitro kinase assay was performed in the presence of Rab2NЈ⌬19. We propose that Rab2 directly interacts with PKC/ and inhibits PKC/ kinase activity at the VTC. The subsequent recruitment of one or more Rab2 downstream effectors relieves the negative regulation imposed by Rab2 and stimulates PKC/ to phosphorylate VTCassociated GAPDH and promote microtubule nucleation.

EXPERIMENTAL PROCEDURES
Materials and Methods-Rab2 amino-terminal peptides were synthesized at the University of Michigan Protein and Carbohydrate Structure Facility (Ann Arbor, MI). The cDNA to PKC was a gift from Dr. Trevor Biden (Garvan Institute of Medical Research, Sydney, Australia). The mammalian Matchmaker two-hybrid assay kit was purchased from Clontech Laboratories, Inc. (Palo Alto, CA). Horseradish peroxidase-and alkaline phosphatase-conjugated secondary antibodies were purchased from Pierce (Rockford, IL).
Immunoprecipitation-HeLa cells (3 ϫ 10 6 ) were lysed in 50 mM Tris-buffered saline (TBS) (pH 8.0) and 1% Triton X-100 for 10 min on ice and then clarified by centrifugation at 20,000 ϫ g for 10 min at 4°C. The soluble fraction was precleared with Protein G plus/Protein Aagarose (Novagen, Madison, WI) and subjected to immunoprecipitation for 4 h at RT with an affinity-purified anti-Rab2 polyclonal antibody or with preimmune serum and Protein G plus/Protein A-agarose. The immune complexes were collected by centrifugation at 5000 rpm for 5 min, washed 3ϫ with TBS, 1% Triton X-100, and 100 mM NaCl, and then boiled in sample buffer. The immunoprecipitates were separated by SDS-PAGE and transferred to nitrocellulose. The blot was blocked in TBS that contained 5% BSA and 0.5% Tween 20, incubated with an anti-Rab2 polyclonal antibody and a monoclonal antibody to PKC/ (BD Biosciences, San Diego, CA), washed, further incubated with a horseradish peroxidase (HRP)-conjugated anti-rabbit or an anti-mouse antibody and then developed with enhanced chemiluminescence (ECL) (Amersham Biosciences, Piscataway, NJ).
Mammalian Two-hybrid Assay-Human Rab2 cDNA was cloned inframe to the EcoRI site of the GAL4 DNA-binding domain in the pM vector and PKC/ cDNA cloned in-frame to the EcoRI site of the activation domain in pVP16 (Clontech Laboratories, Inc.). The two constructs (5 g each) were co-transfected with the reporter vector pG5CAT (5 g) that contains the chloramphenicol acetyltransferase (CAT) gene into HeLa cells (10 6 ) using a calcium phosphate transfection protocol (23). Control cells were transfected with a combination of vectors as described under "Results." The cells were collected 72 h post-transfection, lysed in sample buffer, separated by SDS-PAGE, and then transferred to nitrocellulose. The blot was blocked in TBS that contained 5% BSA and 0.5% Tween 20, incubated with an anti-CAT polyclonal antibody (Invitrogen), washed, further incubated with an HRP-conjugated secondary antibody, and then developed with ECL (Amersham Biosciences).
GST-PKC/ Pull-down-The cDNA encoding PKC was amplified by PCR using a 5Ј primer that included a BamHI site and sequence complementary to PKC (5Ј-GGATCCATGTCCCACACGGTCGCAGGC) in tandem with a 3Ј-antisense primer that included an EcoRI site (5Ј-CCGAATTCGGATCAGACACATTCTTCTGC), and then cloned inframe to the BamH1 and EcoR1 sites of pGEX-2T (Amersham Biosciences). The PKC/ regulatory domain was generated by PCR using a 5Ј-oligonucleotide primer that contained a BamHI site, 5Ј-GGATCC-ATGTCCCACACGGTCGCAGGC, in tandem with the 3Ј-antisense oligonucleotide, 5Ј-GCGAATTCTCACCAATCAAAATCCTGAAGACC-TAG, and then cloned in-frame to the BamHI and EcoRI sites of pGEX-2T. The PKC/ catalytic domain was generated by PCR using a 5Ј primer that included a BamHI site, 5Ј-CGGGATCCTTGCTCCGGGTA-ATAGGAAAGGAAGT, and the 3Ј-antisense primer that included an EcoRI site, 5Ј-CCGAATTCGGATCAGACACATTCTTCTGC, and then cloned in-frame to the BamHI and EcoRI sites in pGEX-2T. BL21(DE3)pLysS (Novagen) cells that contained the recombinant plasmids were grown at 37°C to 0.6 A 600 and then induced with 0.4 mM isopropyl-␤-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 cold TBS, sonicated, and centrifuged at 12,000 RPM for 20 min, and then the supernatant was applied to a glutathione-Sepharose 4B column (Amersham Biosciences). The column was washed with 10 bed volumes of TBS, and the fusion protein eluted with 5 mM reduced glutathione. GST-PKC/, GST-PKC/ (regulatory (reg)), GST-PKC/ (catalytic (cat)), or GST (5 g) were preincubated with 20 l of glutathione-Sepharose 4B for 1 h at room temperature, collected by centrifugation at 4,000 RPM for min, and then washed 3ϫ with TBS and 1% Triton X-100 to remove any unbound protein. The beads were resuspended in 50 mM Tris (pH 7.5), 5 mM MgCl 2 , 100 mM NaCl, 10 M GTP␥S, or 10 M GDP (ICN Biochemicals Inc., Irvine, CA), then 5 g of purified recombinant Rab2 was added, and the mixture was incubated for an additional 2 h at RT. The beads were washed 4ϫ with 1 ml of 50 mM Tris (pH 7.5), 10 mM MgCl 2 , and 100 mM NaCl, and the bound proteins were boiled in sample buffer, separated by SDS-PAGE, and then transferred to nitrocellulose. The blot was blocked in TBS that contained 5% BSA and 0.5% Tween 20, and the membrane was probed with an affinity-purified Rab2 polyclonal antibody, washed, incubated with an HRP-conjugated secondary antibody, and then developed with ECL.
Construction of Rab2 Amino-terminal Deletion Mutants-Amino-terminal deletions of human Rab2 were generated by PCR using 5Јoligonucleotides primers that included an NdeI site and sequence complementary to Rab2 that introduced a start codon at the respective deletion site, ⌬8, 5Ј-GGCATATGTACATCATAATCGGCGACACA; ⌬14, 5Ј-GGCATATGACAGGTGTTGGTAAATCATGC; and ⌬19, 5Ј-GGCAT-ATGTCATGCTTATTGCTACAGTTTACAGAC, in tandem with the antisense primer that encoded for a BamH1 site: 5Ј-CCGCGGATCCTCA-ACAGCAGCCCCCAGC. The amplified products were subcloned into the NdeI and BamHI sites in pET3a (Novagen), and the sequence was verified by DNA analysis.
Purification of Recombinant Rab2 Protein and in Vitro Prenylation-Purification of recombinant Rab2 was performed as previously described (21). Briefly, pET3A-Rab2 or pET3A-Rab2NЈ⌬19 was introduced into BL21(DE3)pLysS. A 1-liter culture was grown to 0.6 A 600 and induced with 0.4 mM isopropyl-1-thio-␤-D-galactopyranoside for 3 h at 37°C. The cells were centrifuged at 5,000 ϫ g for 20 min at 4°C, and the cell pellet was resuspended in 50 mM Tris, pH 7.4, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 0.1 mM benzamidine, 1 mM EDTA, and 1% Triton X-100, and then homogenized by 20 passes with a Dounce tissue grinder. Lysozyme (400 g/ml), DNase I (40 g/ml), and 25 mM MgCl 2 were added to the homogenate, and the mixture was allowed to digest for 30 min at 4°C and then centrifuged at 22,000 ϫ g for 30 min at 4°C. The supernatant was applied to a 70-ml column containing Q-Sepharose Fast Flow (Amersham Biosciences) equilibrated with Buffer A (50 mM Tris, pH 7.4, 10 mM MgCl 2 , and 1.0 mM EDTA), washed with 2 bed volumes of Buffer A, then eluted with a linear NaCl gradient (0 -400 mM) in Buffer A. 3-ml fractions were collected, and an aliquot of each fraction was separated by SDS-PAGE and immunoblotted with a Rab2 polyclonal antibody. Rab2-enriched fractions were pooled, concentrated, and applied to a 200-ml column containing Sephacryl S-100 (Amersham Biosciences) and eluted with Buffer A. Fractions containing Rab2 or Rab2NЈ⌬19 were identified by SDS-PAGE and immunoblotting, then pooled, and concentrated. Typically, the protein prepared from this procedure was ϳ90% pure. For use in the assays, the purified protein was first prenylated in an in vitro reaction. The isoprenylation reaction was performed in a total volume of 500 l that contained 5.0 g of recombinant Rab2, 100 g of geranylgeranyl pyrophosphate (Sigma), or 30 pmol of [ 3 H]geranylgeranyl pyrophosphate (10 Ci/mmol, American Radiolabeled Chemicals, Inc., St. Louis, MO), 1 mM dithiothreitol, 250 l of rat liver cytosol, 10 mM MgCl 2 , 1 mM ATP, 5 mM creatine phosphate, and 0.2 unit of rabbit muscle creatine kinase. The reaction was incubated for 1 h at 37°C and then desalted through a 10-ml column of Sephadex G-25 (Amersham Biosciences) to remove incompatible reagents that may inhibit the binding assay. The fraction containing prenylated Rab2 was collected and concentrated, and the protein concentration was determined by Micro BCA Protein Assay Reagent (Pierce). Routinely, 40 -45% of the total Rab2 and Rab2NЈ⌬19 was prenylated as determined by phase separation in Triton X-114. Triton X-114 partitioning was performed as described previously (8,21,26).
ELISA-ELISA was performed as described previously (27). Briefly, Nunc immunomodules were coated with peptides and purified Rab2 amino-terminal truncated recombinant proteins (1 g/100 l of 50 mM NaHCO 3 , pH 9.6) listed in Table I at 4°C overnight. The wells were washed in TBS, blocked in TBS and 5% fetal bovine serum for 1 h at 37°C, additionally washed in TBS, and then incubated with 5 g of purified recombinant PKC/ for 3 h at 37°C. After each well was washed 3ϫ with TBS, a monoclonal antibody to PKC/ (BD Biosciences) in 50 mM Tris (pH 7.5), 5 mM MgCl 2 , 100 mM NaCl, 10 M GTP␥S was added for 2 h at 37°C, washed, and further incubated with an anti-mouse alkaline phosphatase-conjugated secondary reagent for 1 h at 37°C. The wells were again washed with TBS, developed with Sigma FAST p-nitrophenyl phosphate (Sigma-Aldrich, St. Louis, MO), and then read at 405 nm on a microplate reader.
Quantitative Microsomal-binding Assay-NRK cells were washed 3ϫ with ice-cold phosphate-buffered saline. The cells were scraped off the dish with a rubber policeman into 10 mM Hepes (pH 7.2) and 250 mM mannitol, then broken with 15 passes through a 27-gauge syringe. The broken cells were pelleted at 500 ϫ g for 5 min at 4°C, and the supernatant was removed and re-centrifuged at 20,000 ϫ g for 20 min at 4°C. The resultant pellet containing ER, pre-Golgi, and Golgi membranes was washed with 1 M KCl in 10 mM Hepes (pH 7.2) for 10 min on ice to remove peripherally associated proteins and centrifuged at 20,000 ϫ g for 15 min at 4°C, and the membrane pellet was resuspended in 10 mM Hepes (pH 7.2) and 250 mM mannitol and employed in the binding reaction (9,21). Membranes (30 g of total protein) were added to a reaction mixture that contained 27.5 mM Hepes (pH 7.2), 2.75 mM MgOAc, 65 mM KOAc, 5 mM EGTA, 1.8 mM CaCl 2 , 1 mM ATP, 5 mM creatine phosphate, and 0.2 unit of rabbit muscle creatine kinase. Recombinant Rab2 and Rab2NЈ⌬19 were added at the concentrations indicated under "Results," and the reaction mixture was incubated on ice for 15 min. The reactions were supplemented with rat liver cytosol (50 g) and 2.0 M GTP␥S and then shifted to 37°C and incubated for 10 min. The binding reaction was terminated by transferring the samples to ice and centrifuged at 20,000 ϫ g for 10 min at 4°C to obtain a pellet (P1). The supernatant (20,000 ϫ g) was re-centrifuged at 100,000 ϫ g for 60 min in a micro-ultracentrifuge (Sorval Discovery M120) to recover released vesicles (P2). P1 and P2 were separated by SDS-PAGE and transferred to nitrocellulose in 25 mM Tris (pH 8.3), 192 mM glycine, and 20% methanol. The blot was blocked in TBS, which contained 5% nonfat dry milk and 0.5% Tween 20, incubated with a monoclonal antibody to PKC/ (BD Biosciences), a monoclonal antibody to GAPDH (Chemicon, Temecula, CA), or an affinity-purified polyclonal antibody to the EAGE peptide of ␤-COP (9), washed, further incubated with an HRP-conjugated secondary antibody, developed with ECL (Amersham Biosciences), and then quantified by densitometry.

Rab2
Interacts Directly with PKC/-Because Rab2 recruits PKC/ to membranes in a dose-dependent manner, we used a combination of in vivo and in vitro approaches to determine whether Rab2-PKC/ physically associate. First, the detergent lysate from HeLa cells was incubated with preimmune serum or with an affinity-purified anti-Rab2 polyclonal antibody. Western blot analysis of the immune complex showed that Rab2 and PKC/ specifically co-immunoprecipitated, whereas no interaction was detected with preimmune serum (Fig. 1A).
To further verify Rab2-PKC/ interaction in vivo, we employed a mammalian two-hybrid assay. This system allows the detection of transient and weak protein-protein interactions. For this assay, Rab2 cDNA was used as bait and cloned into pM to generate a GAL4 DNA-binding domain-Rab2 fusion protein, whereas PKC/ cDNA that serves as prey was cloned into pVP16 to generate a VP16 activation domain-PKC / fusion protein. HeLa cells were co-transfected with these two constructs and with a reporter plasmid that contains the CAT gene. If the two fusion proteins interact in vivo, transcription of HeLa cell lysate was incubated with preimmune serum or an affinitypurified anti-Rab2 polyclonal antibody and Protein G plus/Protein Aagarose for 4 h at RT. The immune complexes were collected by centrifugation, washed, and analyzed as described under "Experimental Procedures." The blot was probed with an anti-PKC/ monoclonal antibody and an affinity-purified Rab2 polyclonal antibody, washed, incubated with HRP-conjugated secondary antibodies, and then developed with ECL. B, mammalian two-hybrid assay. The lysates from HeLa cells transiently transfected with: a, pG5CAT, pM, and pVP16 (basal control); b, pM-Rab2, pVP-16, and pG5CAT (pM control); c, pM, pVP16-PKC/, and pG5CAT (pVP16 control); or d, pVP16-PKC/, pM-Rab2, and pG5CAT (experiment) were separated on SDS-PAGE and transferred to nitrocellulose. The blot was probed with an anti-CAT polyclonal antibody, washed, further incubated with an HRP-conjugated secondary antibody, and then developed with ECL. C, overlay binding assay. Purified recombinant Rab2 (5 g) was separated by SDS-PAGE and transferred to nitrocellulose. The blot was incubated in 50 mM Tris (pH 7.4), 0.1% BSA, 10 M GTP␥S, 200 mM NaCl, 20 g/ml phosphatidylserine, and 10 g/ml purified recombinant PKC/ for 4 h at RT, washed, probed with an anti-PKC/ monoclonal antibody, washed incubated with an HRP-conjugated secondary antibody and developed with ECL. Control was incubated in the absence of PKC /. D, GST-pull-down. GST-PKC/ (5 g) or GST (5 g) were preincubated with 20 l of glutathione-Sepharose 4B for 1 h at room temperature and then washed 3ϫ with TBS 1% Triton X-100 to remove any unbound protein. The beads were resuspended in 50 mM Tris (pH 7.5), 5 mM MgCl 2 , 100 mM NaCl, 10 M GTP␥S, or 10 M GDP, and then 5 g of purified recombinant Rab2 was added, and the mixture was incubated for an additional 2 h at RT. The beads were washed 4ϫ with 1 ml of 50 mM Tris (pH 7.5), 10 mM MgCl 2 , and 100 mM NaCl, and bound proteins were boiled in sample buffer, separated by SDS-PAGE, and then transferred to nitrocellulose. The blot was probed with an affinity-purified Rab2 polyclonal antibody, washed, incubated with an HRP-conjugated secondary antibody, and then developed with ECL. All experiments were performed a minimum of three times. the CAT reporter gene is activated. Three days post-transfection, the cells were lysed in sample buffer, and the lysate was separated on SDS-PAGE and immunoblotted with an anti-CAT polyclonal antibody. Fig. 1B shows that cells co-transfected with pM-Rab2 and pVP16-PKC/ expressed ϳ15-fold higher level of CAT protein compared with controls cells indicating that Rab2 and PKC/ interacted in vivo.
This potential in vivo interaction was further evaluated in a blot overlay assay that is routinely used to identify PKC-interacting proteins (22,25,28). Rab2 was separated on SDS-PAGE and transferred to nitrocellulose, and the membrane was incubated in overlay buffer supplemented with purified recombinant PKC/ and GTP␥S. Any PKC/ bound to Rab2 was detected after incubation with a monoclonal antibody to the kinase followed by a secondary HRP-conjugated antibody and development with ECL. Consistent with the in vivo results, we detected PKC/ binding to Rab2 (Fig. 1C). This in vitro result was further confirmed in a GST pull-down experiment in which GST-PKC/ was first immobilized on glutathione-Sepharose 4B and then incubated with purified recombinant Rab2 in a buffer containing GTP␥S or GDP. After extensive washing, agarose beads containing GST-PKC/ retained Rab2-GTP, whereas minimal interaction was detected with Rab2-GDP (Fig. 1D). These combined in vivo and in vitro results are highly suggestive that activated Rab2 interacts directly with PKC/.
PKC/ Binds to the Rab2 Amino Terminus-Based on our previous studies, there was evidence to suggest that the Rab2 amino terminus interacted with PKC/ (11). Because a peptide corresponding to the first 7 amino acids of Rab2 following the initiator methionine (AYAYLFK) had minimal effect on membrane trafficking (8), we reasoned that the PKC/ binding domain must include residues downstream from the first seven but before the serine at position 20 that binds a magnesium ion and helps promote the activated conformation of Rab proteins (29). A family of Rab2 amino-terminal peptides and truncation proteins was characterized in an ELISA to explore the potential role of the Rab2 amino terminus in binding to PKC/. As we anticipated, a strong interaction was detected between PKC/ and intact Rab2 (Table I). In contrast, Rab2 truncation mutants missing 1-8 or 1-14 residues exhibited an increasingly lower interaction with PKC/, whereas no interaction was detected between PKC/ and Rab2NЈ⌬19. Conversely, Rab2 peptides encoding for the deleted amino acids (2-7, 2-9, and 2-19) showed enhanced PKC/ interaction with increasing length of the peptide (Table I). We did not detect PKC/ interaction with Rab2 residues 20 -211 nor with an amino-terminal-scrambled peptide. These results are highly suggestive that the first 19 amino acids of Rab2 are required for interaction with PKC/.
The Amino Terminus of Rab2 Is Required to Stimulate PKC/ Recruitment to NRK Microsomes-We reasoned that if Rab2 recruits PKC/ to VTCs and interacts via the amino terminus with PKC/, then a Rab2 amino-terminal truncation mutant should not stimulate PKC/ membrane association. To address this question, purified recombinant Rab2NЈ⌬19 was introduced into a quantitative microsomal binding assay (9,11). For this assay, NRK microsomes were prepared from whole cell homogenates and washed with 1 M KCl to remove peripherally associated proteins. These membranes were preincubated in buffer for 15 min on ice in the absence or presence of recombinant Rab2 or Rab2NЈ⌬19. The reaction was then supplemented with rat liver cytosol and GTP␥S and incubated for 10 min at 32°C to promote binding of soluble molecules. Membranes recovered after centrifugation at 20,000 ϫ g were separated by SDS-PAGE, transferred to nitrocellulose, and then probed with antibodies specific to PKC/ and GAPDH. Although recombinant Rab2 stimulated PKC/ membrane association, addition of Rab2 NЈ⌬19 at either an equivalent or higher concentration had no effect on PKC/ recruitment to NRK microsomes ( Fig. 2A). Based on the previous observation that PKC/ interacts with GAPDH, we determined whether removal of the Rab2 amino terminus also effected GAPDH recruitment to membrane. To address this question, the blot was re-probed with an anti-GAPDH monoclonal antibody. Unlike Rab2 that promoted GAPDH membrane binding, microsomes treated with Rab2NЈ⌬19 contained GAPDH at a level comparable to the control ( Fig. 2A).
We considered two possibilities to account for the lack of PKC/ and GAPDH recruitment by Rab2NЈ⌬19: 1) the truncated protein could not be in vitro prenylated and therefore unable to associate with membranes and stimulate PKC/ and GAPDH binding and 2) Rab2NЈ⌬19 had a profound effect on vesicle formation, which would give the appearance that there was no enhanced recruitment by the truncated mutant protein; that is, because the membranes analyzed are recovered at insufficient centrifugal force to pellet release vesicles. To address the first possibility, we determined whether Rab2NЈ⌬19 was prenylated in both in vitro and in vivo assays. First, the cell lysate from HeLa cells transfected transiently with pCR3.1-Rab2 was partitioned with Triton X-114, and the distribution of endogenous Rab2 and Rab2NЈ⌬19 in the two phases was analyzed by SDS-PAGE and immunoblotting (26). The endogenous pool of Rab2 is predominantly membraneassociated (ϳ75%) and found in the detergent-rich phase (Fig.  2B). The phase separation of transiently expressed Rab2NЈ⌬19 showed that ϳ50% of the mutant distributed with the detergent phase (Fig. 2B). The increase in the distribution of the truncation mutant to the cytosolic pool may simply reflect saturation of the prenylation enzyme (geranylgeranyltransferase II) and limited cofactors required for the modification. Rab2NЈ⌬19 was further characterized in an in vitro prenylation reaction that employs [ 3 H]geranylgeranyl pyrophosphate. Fig. 2C shows that purified recombinant Rab2NЈ⌬19 can be prenylated as evidenced by the incorporation of radiolabeled lipid. Despite the finding that Rab2NЈ⌬19 was prenylated in vivo and in vitro, there was the slight possibility that the mutant protein could not bind to membranes used in the binding assay. To address this question, the assay was performed with increasing concentrations of purified recombinant Rab2NЈ⌬19. The truncated mutant protein bound to membranes in a concentration-dependent manner (Fig. 2D). It appears that Rab2NЈ⌬19 binds to NRK microsomes and is posttranslationally modified at its carboxyl terminus.
We then performed experiments to determine whether the truncated protein could generate vesicles containing ␤-COP. In this case, the microsomal binding assay was supplemented with 300 ng of Rab2 or Rab2NЈ⌬19. This concentration of Rab2 effectively stimulates retrograde-vesicle formation (21). The membranes (P1) from the binding reaction were collected by centrifugation at 20,000 ϫ g, and the low speed supernatant was re-centrifuged at 100,000 ϫ g to recover any released vesicles (P2). Western blot analysis of P1 and P2 from the binding assay supplemented with Rab2 showed that Rab2 stimulated the release of vesicles containing ␤-COP (ϳ76% of total ␤-COP signal) (Fig. 2E). In contrast, ϳ34% of the total ␤-COP signal was detected in the P2 fraction from membranes treated with Rab2NЈ⌬19. Although Rab2NЈ⌬19 associates with NRK microsomes, the truncated mutant does not stimulate vesicle formation. Therefore, the failure of Rab2NЈ⌬19 to recruit PKC/ and GAPDH to NRK microsomes is most likely due to missing residues in the Rab2 amino terminus that interact with the kinase. Rab2 Binds to the PKC/ Regulatory Domain and Inhibits PKC/-dependent GAPDH Phosphorylation-To map the Rab2 FIG. 2. Rab2 amino terminus is essential to recruit PKC/ to NRK microsomes. A, salt-washed NRK microsomes prepared as described under "Experimental Procedures" were preincubated with Rab2 or Rab2NЈ⌬19 for 15 min on ice. Cytosol and GTP␥S were then added, and the membranes were incubated for 10 min at 32°C. To terminate the reaction, the membranes were collected by centrifugation, and the membrane pellet was separated by SDS-PAGE and transferred to nitrocellulose. The blot was probed with a monoclonal antibody to PKC/ and a monoclonal antibody to GAPDH, washed, further incubated with HRP-conjugated secondary antibody, and then developed with ECL. The amount of recruited PKC/ and GAPDH was quantitated by densitometry. A representative Western blot is shown for each protein. The results are the mean Ϯ S.D. of three independent experiments performed in duplicate. B, the lysate from HeLa cells transiently transfected with pcR3.1-Rab2NЈ⌬19 was partitioned with Triton X-114 into hydrophobic (D) and hydrophilic (A) fractions, acetone-precipitated, separated by SDS-PAGE, and then transferred to nitrocellulose. The blot was probed with an affinity-purified anti-Rab2 polyclonal antibody, washed, incubated with an HRPconjugated secondary antibody, and then developed with ECL. C, purified recombinant Rab2NЈ⌬19 was subjected to in vitro prenylation (Ϫ) without or (ϩ) with cytosol, which supplies geranylgeranyltransferase II transferase and 30 pmol of [ 3 H]geranylgeranyl pyrophosphate as described under "Experimental Procedures." The reaction was analyzed by SDS-PAGE and fluorography. Shown are representative results of three independent experiments. D, salt-washed NRK microsomes were preincubated with increasing concentrations of purified recombinant Rab2NЈ⌬19 for 15 min on ice. Cytosol and GTP␥S were then added, and the membranes were incubated for 10 min at 32°C. The reaction was terminated and analyzed as above. The blot was probed with an affinity-purified anti-Rab2 polyclonal antibody, washed, further incubated with an HRP-conjugated secondary antibody, and then developed with ECL. Shown are representative results of three independent experiments. E, salt-washed NRK microsomes were preincubated with 300 ng of purified recombinant Rab2 or Rab2NЈ⌬19 for 10 min on ice. Cytosol and GTP␥S were then added, and the binding reaction was incubated for 10 min at 32°C. Microsomes were collected by centrifugation (20,000 ϫ g for 10 min) to obtain a pellet (P1). The supernatant was re-centrifuged at 100,000 ϫ g for 30 min, the resultant pellet (P2) and P1 were separated by SDS-PAGE, immunoblotted with an anti-␤-COP polyclonal antibody, and the blot was developed with ECL. The results are the mean Ϯ S.D. of three independent experiments. binding domain in PKC/ we created by PCR two GST-PKC fusion proteins that encode for the regulatory (reg) domain (residues 1-245) and the catalytic (cat) domain (residues 246 -587). These two fragments were screened for interaction with Rab2 by GST-pull down experiments. As we anticipated, Rab2 bound to the PKC/ regulatory domain (Fig. 3A). To date, all proteins that have been reported to bind to aPKCs do so via this domain. Because PKC-interacting proteins can serve as substrates and or regulate kinase activity (30), we determined whether Rab2 had any effect on PKC/-mediated GAPDH phosphorylation. Although Rab2 interacts directly with PKC/, we knew from our earlier studies that Rab2 is not phosphorylated by the kinase (22). An in vitro kinase assay was performed with purified recombinant PKC/ in the absence or presence of Rab2. As we previously observed, PKC/ efficiently phosphorylated GAPDH (Fig. 3B). However, when the assay was supplemented with Rab2, PKC/-dependent GAPDH phosphorylation was nearly inhibited. Given that Rab2 interacts with PKC/ via its amino terminus, we determined whether Rab2 (13-mer) had an effect on PKC/ enzymatic activity by adding the peptide to the kinase assay. Similar to Rab2, the Rab2 (13-mer) inhibited GAPDH phosphorylation suggesting that the Rab2 amino terminus plays a role in reg-ulating PKC/ kinase activity. In further support of this interpretation, we found that PKC/ phosphorylated GAPDH when the kinase reaction was performed in the presence of Rab2NЈ⌬19. These results indicate that Rab2-PKC/ interaction interferes with the ability of PKC/ to phosphorylate GAPDH. DISCUSSION All Rab proteins interact with a multitude of effectors that are diverse in structure and function. These effectors are routinely part of a large protein complex. The identification of these effectors and determination of how they associate with a Rab protein provides insight into Rab function. From our previous studies we learned that Rab2 recruits PKC/ to NRK microsomes in a dose-dependent manner (11). Because specific binding proteins mediate PKC localization by placing the isozyme in proximity of its substrate, we performed in vivo and in vitro assays to demonstrate that PKC/ interacted directly with Rab2-GTP. PKC/ binding to Rab2 would explain why this particular isozyme was recruited to the VTC and would ensure that a signaling molecule is associated with Rab2 to regulate a transport-related event through phosphorylation.
To map the PKC/ binding domain in Rab2, we focused our attention on the Rab2 amino terminus based on the previous observations that: 1) the Rab2 (13-mer) stimulated PKC/ membrane association; 2) truncation of the Rab2 (N119I) amino terminus attenuated its inhibitory property; and 3) the amino terminus of several Rab proteins is required for their function and interaction with effector molecules. When a family of Rab2 peptides and Rab2 amino-terminal truncation proteins were evaluated for their ability to bind PKC/, only reagents containing residues between 1 and 19 interacted with the kinase. Most likely, the PKC/ binding domain in Rab2 resides within those amino acids. Interestingly, this segment includes one of the five Rab-specific sequence motifs that are predicted to mediate interaction with different effectors (7,31).
The requirement for the Rab2 amino terminus to promote interaction with specific components involved in ER to Golgi transport is in agreement with results reported for Rab5 in which its amino terminus was essential for early endosome fusion (5,6). Recently, Li and Liang (32) have shown that the Rab5 phosphate-binding loop (P-loop, residues 24 -36) is necessary for interaction with specific factors on endosomal membranes. The P-loop also contains a Rab complementarity-determining region (CDR). The combination of the variable Rab CDR and the conserved switch mechanism enables Rab proteins to interact with a wide variety of effectors in a specific and activation state-dependent manner (7). For example, Rabphilin-3A interacts with residues 19 -22 of the Rab3A CDR. Deletion of those residues abolishes binding to Rabphilin-3A (7). It is also noteworthy that the Rabphilin-3A effector domain possesses two conserved zinc binding motifs and that the cysteines are sensitive to mutation. Similar motifs are also found in other Rab effectors, such as Rim and early-endosomal autoantigen 1 (33,34). Likewise, PKC/ contains a zinc finger.
To determine the Rab2 binding site in the kinase, we generated two GST-PKC fusion proteins that encode for the regulatory and catalytic domain. We expected that Rab2 would bind to the PKC/ regulatory domain based on the observations reported for other proteins that interact with the aPKCs. This part of the kinase not only contains motifs involved in binding of phospholipid cofactors and calcium, but also participates in protein-protein interactions. For example, the novel protein modulators lambda-interacting protein and Par-4 have been reported to interact with the regulatory domain of PKC (35,36). Additionally, Ras binds to the PKC regulatory domain (37). Because these proteins regulate aPKC kinase activity, FIG. 3. Rab2 binds to the regulatory domain of PKC/ and inhibits kinase activity. A, the PKC/ regulatory domain (residues 1-147) and the catalytic domain (residues 248 -588) were expressed as GST-tagged fusion proteins and immobilized on glutathione-Sepharose 4B. Purified recombinant Rab2 (5 g) in 50 mM Tris (pH 7.5), 5 mM MgCl 2 , 100 mM NaCl, 10 M GTP␥S was added, and the mixture was incubated for an additional 2 h at RT. The beads were washed 4ϫ with 1 ml of 50 mM Tris (pH 7.5), 10 mM MgCl 2 , and 100 mM NaCl, boiled in sample buffer, separated by SDS-PAGE, and then transferred to nitrocellulose. The blot was probed with an affinity-purified Rab2 polyclonal antibody, washed, incubated with an HRP-conjugated secondary antibody, and then developed with ECL. Shown are representative results of three independent experiments. B, rabbit muscle GAPDH (1 g) was incubated with purified PKC/ in the absence or presence of 50 ng of purified recombinant Rab2, or 75 M Rab2 (13-mer), or 50 ng of purified recombinant Rab2NЈ⌬19 in a kinase buffer supplemented with phosphatidylserine (100 g/ml as sonicated vesicles) and 10 Ci of [ 32 P]ATP, and then incubated for 20 min at 32°C. Reactions were separated by SDS-PAGE and the gel processed for autoradiography. Phospho-GAPDH was quantified by using a PhosphorImager. The results are the mean Ϯ S.D. of five independent experiments.
we evaluated the effect of Rab2 on PKC/-mediated GAPDH phosphorylation.
Contrary to our prediction that Rab2-PKC/ interaction would promote GAPDH phosphorylation, no phosphorylated product was detected. The inhibition was reversed when the assay was performed in the presence of Rab2NЈ⌬19, which lacks the PKC/ binding domain. This finding implies that the interaction between the Rab2 amino terminus and the PKC/ regulatory domain modulates enzyme activity by either directly interfering with kinase function or blocking access to GAPDH. Because GAPDH is phosphorylated by PKC/ on NRK microsomes incubated with Rab2, one or more unknown effectors must be recruited to VTCs that relieve the inhibition imposed by Rab2-PKC/ interaction allowing GAPDH phosphorylation. This would ensure that GAPDH was not prematurely phosphorylated prior to recruitment of components required for vesicle formation. Although GAPDH is required for transport in the early secretory pathway, membrane-associated GAPDH is not a critical component of the budding machinery. Instead, phospho-GAPDH plays an essential role in promoting MT nucleation at the VTC. Studies are in progress to identify the downstream regulator of PKC/ kinase activity.