Rabbit anti-Rab11a (VU57) antibodies were developed against the amino terminus of human Rab11a and were specific for Rab11a versus Rab11b and Rab25 (Lapierre, unpublished data). The other antibodies used were rat anti-ZO-1 (Chemicon International, Temecula, CA), mouse anti-ZO-1 (Zymed Laboratories, South San Francisco, CA), mouse pan anti-cadherin (Sigma-Aldrich, St. Louis, MO), mouse anti-p120 catenin (BD Biosciences, San Jose, CA), mouse monoclonal anti-ezrin (4A5; Chemicon International), rabbit anti-occludin (8 μg/ml; Zymed Laboratories), anti-green fluorescent protein (GFP) mouse monoclonal (8362–1; BD Biosciences), and anti-GFP rabbit (AB290; Abcam, Cambridge, MA). All secondary antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA). Dr. Roy Zent (Department of Medicine, Vanderbilt University, Nashville, TN) kindly provided mouse monoclonal anti-GP135.
Database Searches and Alignment
Rab11-FIP2 homologues were identified through GenBank searches using the Rab11 binding domain. FlyBase, Joint Genome Institute Xenopus
, and UCSC Genscan were also used to identify homologues. Alignments were performed using ClustalW (http://www.ebi.ac.uk/clustalw/
All site-directed mutagenesis of Rab11-FIP2 was performed using Pfu Turbo polymerase according to the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) with a 16-min extension time. Primers were synthesized (Invitrogen, Carlsbad, CA) with one nucleotide change per oligonucleotide sequence. The TCA encoding for amino acid 227 was changed to GCA for the S227A mutant. All constructs were created in pEGFP-C2 (Clontech, Mountain View, CA) and subsequently recloned into pET-30a (Novagen, Madison, WI) with EcoRI and SalI restriction sites.
For recombinant protein production constructs in pET-30a vectors were retransformed into BL21(DE3)pLysS bacteria. Bacteria were grown to log phase and then induced with 400 ng/ml isopropyl β-d-thiogalactoside for 3 h at 37°C. To harvest protein, bacteria were pelleted at 2000 × g and then resuspended in lysis buffer (50 mM sodium phosphate buffer, pH 8.0, 300 mM NaCl with protease inhibitors [protein buffer], and 10 mM imidazole). Protein was harvested according to the manufacturer’s protocol (Novagen). Briefly, the bacteria were then sonicated four times for 20 s at maximum potency on ice. The lysate was extracted with 0.1% Triton X-100 for 5 min on ice. The extracted lysate was cleared by centrifugation at 15,000 × g, and the resulting supernatant was incubated with nickel-affinity resin at 4°C (His-Bind; Novagen). The beads and protein were washed in protein buffer with 20 mM imidazole. The bound protein was eluted overnight at 4°C with elution buffer (protein buffer with 250 mM imidazole). Recombinat MARK2 was purchased from Upstate Biotechnology (Lake Placid, NY) (14-544).
Rabbit Gastric Tubulovesicle Preparation
Fractions of rabbit gastric mucosal microsomes were prepared as described previously from the fundic mucosa of New Zealand White rabbits (Basson et al., 1991
). The rabbit gastric mucosa tissue was homogenized in 5 volumes of 15 mM HEPES, 300 mM sucrose buffer, pH 7.4, with protease inhibitors (Sigma-Aldrich) with a Potter homogenizer at 1000 rpm. The homogenate was sequentially centrifuged at 500 × g
for 10 min, 5000 × g
for 10 min, 17,000 × g
for 20 min, and 100,000 × g
for 60 min, and the 100,000 × g
pellet was resuspended in the homogenization buffer and frozen at −80°C until use.
The 100,000 × g microsomal pellet from rabbit gastric mucosa was thawed on ice and then extracted for 30 min with 1% Triton X-100. The solubilized microsomes were centrifuged at 100,000 × g for 1 h at 4°C. The supernatant from this spin was diluted 1:10 with buffer A (5 mM sodium phosphate, pH 7.2, and 0.1% Triton X-100) for protein purification. The diluted homogenate was loaded on a HiTrap Q FF column (2 ml; Amersham, Little Chalfont, Buckinghamshire, United Kingdom) preequilibrated in buffer A at 1 ml/min. The Rab11-FIP2 kinase activity, which voided the column, was collected and then further purified over a ceramic hydroxylapatite column (Econo-Pac CHT-I 1-ml cartridge; Bio-Rad, Hercules, CA) preequilibrated in buffer A. The void fraction was collected and the bound protein was eluted in a gradient from 0 to 500 mM sodium phosphate, pH 7.2, 0.1% Triton X-100. The Rab11-FIP2 kinase activity eluted at ~250 mM sodium phosphate. The fractions with the highest activity were pooled, diluted 1:1 in buffer A, and chromatographed over MONO-S resin (5 ml) (GE Healthcare). The bound protein was eluted with a continuous salt gradient from 0 to 1 M NaCl in buffer A. The Rab11-FIP2 kinase activity eluted at 400 mM NaCl. The fractions with the highest activity were pooled and further purified over a Cibachrome blue affinity column (HiTrap Blue, 1 ml; Amersham). The proteins were eluted with a continuous gradient to 2 M NaCl in buffer A. Kinase activity eluted at ~500 mM NaCl. Finally, the fractions with the highest activity were loaded onto a 10 to 40% glycerol gradient and centrifuged for 24 h at 160,000 × g. The Rab11-FIP2 kinase activity peaked at ~17% glycerol. Each step was monitored by the in vitro kinase activity assay.
The fraction from the glycerol gradient that contained the greatest amount of kinase activity was subjected to trypsin digestion, and the resulting peptides were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) for protein identification. Before trypsin digestion, the samples were cleaned up using a 10-kDa Ultrafree MC regenerated cellulose filter (Millipore, Billerica, MA), and the proteins were subsequently digested directly off of the filter as described previously (Manza et al., 2005
LC-MS analysis of the resulting peptides was performed using a Thermo Finnigan (Waltham, MA) LTQ ion trap mass spectrometer equipped with a Thermo MicroAS autosampler and Thermo Surveyor HPLC pump, Nanospray source, and Xcalibur 1.4 instrument control. The peptides were separated on a packed capillary tip, 100 μm × 11 cm, with C18 resin (Monitor C18, 5 μm, 100 Å; Column Engineering, Ontario, CA) using an inline solid phase extraction column that was 100 μm × 6 cm packed with the same C18 resin (using a frit generated with liquid silicate Kasil 1; Cortes et al., 1987
) similar to that described previously (Licklider et al., 2002
), except the flow from the HPLC pump was split before the injection valve. The flow rate during the solid phase extraction phase of the gradient was 1 μl/min and during the separation phase was 700 nl/min. Mobile phase A was 0.1% formic acid, mobile phase B was acetonitrile with 0.1% formic acid. A 95-min gradient was performed with a 15-min washing period (100% A for the first 10 min followed by a gradient to 98% A at 15 min) to allow for solid phase extraction and removal of any residual salts. After the initial washing period, a 60-min gradient was performed where the first 35 min was a slow, linear gradient from 98% A to 75% A, followed by a faster gradient to 10% A at 65 min and an isocratic phase at 10% A to 75 min. The MS/MS spectra of the peptides was performed using data-dependent scanning in which one full MS spectra, using a full mass range of 400-2000 amu, was followed by three MS/MS spectra. Proteins were identified using the SEQUEST algorithm (Yates et al., 1995
) and the SEQUEST Browser software in the BioWorks 3.1 software package (Thermo Electron, San Jose, CA), using the nonredundant database from National Center for Biotechnology Information (Bethesda, MD).
For phosphorylation mapping experiments, the Rab11-FIP2 band was excised from a one-dimensional-SDS-PAGE gel and either trypsin or chymotrysin digestion was performed in-gel. The samples were then analyzed using data-dependent analysis similar to that described above with the addition of a neutral loss scan to scan for neutral loss of phosphoric acid (loss of 98) in the top three ions. If a neutral loss ion was found, it was fragmented and an MS/MS/MS spectrum was collected. In addition to using the SEQUEST algorithm to search for phosphorylations on serines, threonines or tyrosines, the data were searched for modifications using the PMOD algorithm (Hansen et al., 2005
In Vitro Kinase Activity Assay during Purification
The chromatographic fractions were added to the substrate [Rab11-FIP2(190-383)] in a 50 mM Tris buffer containing 5 mM MgCl2, 1 mM EGTA, protease inhibitors (1:100), and 25 μM dithiothreitol on ice. [γ-32P]ATP was added, and the reaction was incubated at 35°C for 10 min. The reactions were terminated with the addition of SDS buffer (final concentrations, 300 mM Tris, pH 7.5, 1% SDS, 20 mM EDTA, and 17.5 mM sucrose) and incubated at 70°C for 10 min. The samples were resolved on 12% SDS-PAGE gels, stained with colloidal Coomassie (GelCode blue; Pierce Chemical, Rockford, IL) for protein detection, dried under vacuum, and analyzed with phosphorimaging (Molecular Dynamics) for [32P]phosphate incorporation.
In Situ Phosphorylation
We used [32P]orthophosphate incorporation to assess phosphorylation in situ. MDCK cells were plated on 60-mm Transwell filters (Corning Life Sciences, Acton, MA) and allowed to polarize at confluence for 3 d. MDCK cells stably expressing enhanced green fluorescent protein (EGFP)-Rab11-FIP2 or EGFP-Rab11-FIP2(S227A) constructs were incubated with [32P]orthophosphate in phosphate-free DMEM supplemented with 2 mg/ml bovine serum albumin for 2 h. The cells were solubilized in lysis buffer [30 mm Tris, pH 8.5, 150 mM NaCl, 20 mM magnesium acetate, 1% 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate (CHAPS) with protease inhibitors, and phosphatase inhibitors], extracted on ice for 20 min, and centrifuged for 20 min at 15,000 × g to pellet the insoluble material.
For immunoprecipitation, anti-rabbit IgG Dynabeads (Dynal Biotech, Lake Success, NY) were loaded with either 5 μl of anti-GFP antibody AB290 serum (Abcam) or control rabbit serum for 2 h at 4°C. Beads were washed three times with Tris-buffered saline (TBS). Lysate was diluted in immunoprecipitation buffer (final concentration 30 mM Tris, pH 7.5, 150 mM, NaCl, 20 mM magnesium acetate, protease inhibitors, and phosphatase inhibitors) and incubated with the beads overnight at 4°C. The beads were washed twice for 20 min with immunoprecipitation buffer with 0.1% CHAPS and then once with 30 mM Tris, pH 7.5. The beads were eluted in 1% SDS buffer, and proteins were resolved on 10% SDS-PAGE gels, which were either dried and visualized by a phosphorimaging screen (Molecular Dynamics) for 32P-phosphoproteins or transferred to nitrocellulose for anti-GFP immunoblotting.
Protein samples were resolved on 10% SDS-polyacrylamide gels following a standard Laemmli protocol (Laemmli, 1970
). All incubations were performed at room temperature. Proteins were transferred to nitrocellulose. Blots were blocked for 1 h with 5% dry milk powder (DMP)/TBS and 0.05% Tween 20 (TBS-T). The blots were incubated with primary antibody diluted in 2.5% DMP/TBS-T for 1.5 h (mouse monoclonal anti-GFP 1:2000), washed three times for 10 min in TBS-T, and incubated for 1 h with horseradish peroxidase-labeled anti-mouse secondary; Jackson ImmunoResearch Laboratories) diluted in 1% DMP/TBS-T. The blots were then washed three times with TBS-T followed by one time with TBS, and specific labeling was detected by enhanced chemiluminescence (SuperSignal; Pierce Chemical) with autoradiography using Kodak BioMax ML film (Eastman Kodak, Rochester, NY).
Parental T23 MDCKs (Barth et al., 1997
) as well as the stably transfected cell lines were grown in DMEM supplemented with 10% fetal bovine serum (FBS) (Invitrogen), penicillin-streptomycin, 2 mM l
-glutamine, and 0.1 mM minimal essential medium (MEM) nonessential amino acids (Invitrogen). Media for cell lines also contained 0.5 mg/ml G418 sulfate (Cellgro; Mediatech, Herndon, VA), and 0.25 ng/ml hygromycin (Invitrogen). In the stable cell lines, expression of the EGFP chimeras was inhibited with 20 ng/ml doxycycline (Calbiochem, San Diego, CA). To examine EGFP protein expression, cells were grown on 0.4-μm Transwell filters (Corning Life Sciences) without doxycycline in tetracycline screened FBS (Hyclone Laboratories, Logan, Utah) media for 2–4 d.
GFP Constructs and Transfections
Doxycycline-inhibitable expression vectors were generated by excising the EGFP-Rab11-FIP2 wild-type and mutant sequences from pEGFP-vectors with NheI and SmaI and ligating into a pTRE2hyg vector (Clontech) cut with NheI and EcoRV. Transfection was performed using Effectene (QIAGEN, Valenica, CA) following the manufacturer’s protocol. One microgram of vector was transfected into a 60-mm plate of T23 MDCK cells in normal media. The following day, the cells were trypsinized and replated in serial dilutions, including 0.25 ng/ml hygromycin for selection and 20 ng/ml doxycycline for suppression of EGFP expression. Multiple colonies were selected, expanded for 10 d and then screened for EGFP expression in media with tetracycline-screened serum. Multiple clones were initially characterized, all of which showed the same expression pattern and level of the EGFP construct. One clone was selected for each construct to use as the tetracycline-repressible stable cell lines [expressing EGFP-Rab11-FIP2 wild type or EGFP-Rab11-FIP2(S227A)].
Polymeric Immunoglobin A (pIgA) Trafficking in MDCK Cells
Labeling of pIgA and trafficking experiments were done as described previously (Hales et al., 2002
) except that we used the cell culture media described above. Cells were loaded from the apical side and fixed at time 0 after a 30-min loading.
Analysis of 125I-IgA Postendocytotic Fate
I-IgA was iodinated using the ICl method to a specific activity of 1.0–2.0 times] 107 cpm/μg (Breitfeld et al., 1989
). The postendocytotic fate of a preinternalized cohort of 125
I-IgA (at 5–10 μg/ml) was analyzed as previously described (Apodaca et al., 1994
). In brief, filter-grown MDCK cells expressing the various FIP2 constructs and wild-type pIgR were cultured in the presence or absence of doxycycline, and 125
I-IgA internalized from the basolateral cell surface of the cells for 10 min at 37°C. The basal surface of the cells was rapidly washed three times, the apical and basolateral medium was aspirated and replaced with fresh medium. The cells were then incubated for 3 min at 37°C. This wash procedure takes 5 min at 37°C. Fresh medium was added to the cells, and they were chased for up to 2 h at 37°C. At the designated time points, the apical and basolateral media (0.5 ml) were collected and replaced with fresh media. After the final time point, filters were cut out of the insert, and the amount of 125
I-IgA quantified with a gamma counter. The media samples were precipitated with 10% trichloroacetic acid (TCA) for 30 min on ice and then centrifuged in a microfuge for 15 min at 4°C. The amount of 125
I-IgA in the TCA-soluble (degraded) and insoluble fractions (intact) was quantified with a gamma counter.
Cells were grown to confluence on filters in regular media with or without doxycycline. Cells were washed with low calcium media (MEM [Cellgro 15–015-CV], 10% dialyzed FBS, penicillin-streptomycin, 2 mM l-glutamine, and 0.1 mM MEM nonessential amino acids; Invitrogen). The cells were incubated in low calcium media overnight with or without doxycycline. Calcium was added to the top and bottom of the filter to a final concentration of 1.8 mM. The cells were collected at the indicated time points.
Laminin Replating Assay
Cells were trypsinized and replated on 24-well plate 0.45-μm laminin-coated filters (BD Biosciences). The cells were collected at the indicated time points.
Immunofluorescence for Calcium Switch and Replating Experiments
Cells were washed one time with phosphate-buffered saline (PBS) and then preextracted on ice in 0.2% Triton X-100 in PBS. Cells were fixed for 30 min in 3% paraformaldehyde at room temperature. Cells were permeabilized with 0.05% Triton X-100 in PBS for 5 min on ice. Cells were incubated with anti-p120 and ZO-1 antibodies in PBS for 1 h on ice. Cells were washed one time with PBS. Cells were incubated with species-specific Cy3-anti mouse IgG and Cy5-anti-rat IgG in PBS for 30 min at room temperature. Cells were washed first with PBS and then with 50 mM sodium phosphate. Finally, cells were stained with 4,6-diamidino-2-phenylindole (DAPI) in sodium phosphate. Filters were cut out of the transwells and mounted with Prolong Anitfade solution (Molecular Probes). Cells were imaged on a Zeiss LSM510 confocal microscope using a 100× lens. The z-sections were 0.3 μm.
Cells were washed three times with PBS and then fixed in 4% paraformaldehyde for 15 min at room temperature. The cells were washed twice with PBS and stored at 4° in PBS until staining. Cells were blocked with extraction buffer (10% normal donkey serum, 150 mM NaCl, 20 mM sodium phosphate, pH 7.4, and 0.3% Triton X-10) for 20 min. Primary antibody was immediately added in antibody buffer (10% normal donkey serum, 150 mM NaCl, 20 mM sodium phosphate, pH 7.4, and 0.05% Tween 20) for 2 h. The cells were washed with PBS three times. Secondary species-specific Cy dye-labeled anti-IgGs were added for 1 h in antibody buffer. After washing with PBS two times, the cells were washed with 50 mM sodium phosphate once and then stained with DAPI in sodium phosphate. Filters were cut out of the transwells and mounted with Prolong Antifade solution (Molecular Probes). Cells were imaged on a Zeiss LSM510 confocal microscope (Carl Zeiss, Thornwood, NY) using a 100× lens. Z-sections were 0.5 μm.