Isolation of the mouse gene for MK5.
Two pairs of oligonucleotide PCR primers derived from MK5 cDNA (AF039840) were used to screen a 129SvJ P1 genomic library (Genome Systems, St. Louis, Mo.) from which one positive clone was obtained. The clone contained a part (about 13 kb) of the MK5 gene encoding exons 3 (homologous to genome locations 8162573 to 8162670 of the minus strand of the C57BL/6J strain) to 11 (genome locations 8149536 to 8149666 of the minus strand of C57BL/6J strain; NCBI Mouse Genome Resources) of the MK5 gene.
Construction of the targeting vector and transfection of ES cells.
SacI fragments of the P1 clone were subcloned into pBluescript II KS(+). The neomycin cassette was inserted between a 2.0-kb SacI fragment containing exon 5 and a 9.0-kb SpeI/SacI fragment containing exons 7 to 11 (Fig. ), replacing parts of introns 5 and 6 and the entirety of exon 6. The targeting vector was linearized by digestion with ClaI. E14-1 embryonic stem (ES) cells which were derived from the 129/Ola substrain were a generous gift from Thomas Müller (MDC, Berlin, Germany). Trypsinized ES cells in suspension with the linearized targeting vector were electroporated using a Bio-Rad Gene Pulser II apparatus at 500 μF and 240 V/cm and subsequently seeded on neomycin-resistant mouse embryonic fibroblast (MEF) feeder cells. Selection of transfected ES cells in cell medium containing 385 μg of G-418/ml proceeded for 7 days. Subsequently, clones were picked with 50-μl pipette tips and placed into individual wells of a 96-well plate. After 5 days of cultivation in selection medium, half of the cells were frozen and the other half were allowed to grow to confluence on gelatin-coated 48-well plates for 5 to 7 days before Southern blot analysis was performed.
FIG. 1. Generation of MK5-deficient mice by homologous recombination. (A) Schematic structure of the MK5 gene, the targeting vector, and the targeted locus. Restriction enzymes are indicated as follows: B, BamHI; E, EcoRV; N, NotI; S, SacI; Spe, SpeI. The neomycin (more ...) Southern blot analysis.
Genomic DNA was prepared from ES cells or mouse tail, digested by BamHI, separated by agarose gel electrophoresis, blotted onto nitrocellulose, and hybridized using the external probe P1 (BamHI-SacI fragment; Fig. ). Homologous recombination was detected by the appearance of an additional fragment of about 4.4 kb (Fig. ).
Generation of chimeras.
Two ES cell clones (60 and 149) were used for injection into C57BL/6 blastocysts at the EMBL Transgenic Service, where all further steps for generation of chimeras were also carried out by Kristina Vintersten. Coat-color chimera mice with chimerism levels between 80 to 100% were mated to C57BL/6 mice.
Mouse tail genomic DNA was used for genotyping by PCR using primers 5′-cgtaacactagccacagttgtaactga and 5′-catatacttgtaagcacagctctgagtt. A 970-bp fragment was characteristic for the wild-type allele, while a 1.2-kb fragment represented the targeted allele (Fig. ).
Total RNA of 5 × 106 macrophages was isolated by using a peqGold RNA Pure kit (PEQLAB), separated electrophoretically in 1.25% agarose-formaldehyde gels, and transferred to nitrocellulose. MK5 mRNA was detected by hybridization to 32P-labeled MK5 cDNA fragments (bp 707 to 2128 of AF039840).
Reverse transcription (RT) was performed with a 20-μl reaction mixture consisting of 1× display THERMO-RT buffer (Qiagen), 0.5 mM each deoxynucleoside triphosphate, 0.8 μg of total RNA, 1 μM T25 primer, and 2 μl of display THERMO-RT terminator mix. The reaction was carried out at 42°C for 40 min and stopped by exposure to 65°C for 10 min. RT solution (1 μl) was used for PCR in a 25-μl reaction mixture containing 1× PCR buffer (Qiagen), 0.2 mM each deoxynucleoside triphosphate, 0.2 μM primers atgtcggaggacagcgacatggagaaag and ctactggggctcgtggggaagggtctgc, and 2 U of HotStarTag polymerase (Qiagen), with thermal cycling as follows: first, 96°C for 15 min; then, 35 cycles of 96°C for 1 min, 50°C for 1 min, and 72°C for 1 min; and a final elongation step at 72°C for 5 min. Products are analyzed using 1.5% agarose gel electrophoresis. PCR products were extracted from the gel, cloned into TOPO vector, and sequenced.
Mouse tissue preparation.
During mouse autopsy, all tissues were fixated in neutral-buffered formalin, embedded in paraffin through graded series of alcohol, sectioned at 5-μm intervals, and stained with hematoxylin and eosin.
To immortalize primary MEFs from MK5−/−
mice, cells were cotransfected with pSV40Tag encoding simian virus 40 large T antigen and pREP8 plasmid (Invitrogen) in a 10:1 mixture; colonies were selected with 3 mM histidinol (Sigma). MK2−/−
and wild-type MEFs were obtained as described previously (10
For detection of MK5, proteins were extracted from 2 × 105 macrophages or MEFs, separated in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (12% acrylamide), transferred to nitrocellulose, and analyzed by Western blotting using anti-PRAK antibodies (catalog no. 06-960; Upstate Biotechnology) and a secondary rabbit anti-sheep horseradish peroxidase-conjugated immunoglobulin G (IgG) antibody which was detected by enhanced chemiluminescence. As a positive control, A431 cell lysate (catalog no. 12-301; Upstate Biotechnology) was used. For determination of p38 MAPK levels, 50 μg of tissue lysate was separated for each lane by SDS-polyacrylamide gel electrophoresis and blotted onto nitrocellulose. p38 MAPK was detected using an affinity-purified polyclonal rabbit pan-p38 MAPK antiserum (catalog no. 9212; Cell Signaling Technology) diluted 1:1,000 in Odyssey blocking buffer (Li Cor). As a secondary antibody, Alexa Fluor 680 goat anti-rabbit IgG (Molecular Probes) was used in a dilution of 1:2,000. Blots were scanned and quantified using channel 700 of an Odyssey Infrared Imager and Odyssey 1.0 software (Li Cor).
Endotoxic shock and cytokine enzyme-linked immunosorbent assays.
LPS from Escherichia coli
O26:B6 (catalog no. L8274; Sigma) and d-
-Gal) (Sigma) were diluted in pyrogen-free saline and injected intraperitoneally in a combination of LPS at 50 μg/kg of body weight and d
-Gal at 1 g/kg. Spleen cells (107
cells/ml) from zymosan (Sigma)-primed animals were stimulated in vitro with 5 μg of LPS/ml. Release of cytokines was measured in the culture supernatant by enzyme-linked immunosorbent assays, as described previously (10
The cDNAs for mouse MK2 and MK5 were cloned into pcDNA3.1/NT-GFP-TOPO-TAP (Cellzome, Heidelberg) coding for eukaryotic expression of green fluorescent protein (GFP)--calmodulin-binding protein (CBP)-tobacco etch virus-protease cleavage site-protein A fusions. 293 cells were transiently transfected with the constructs and lysed at 48 h after transfection, and GFP A-CBP fusion protein interacting partner complexes were purified by subsequent binding to rabbit IgG beads (Sigma), TEV protease (Qiagen) cleavage, and binding to calmodulin affinity resin (Stratagene). Endogenous p38 MAPK bound to fusion protein was detected by Western blotting using pan-p38 MAPK antibodies (Cell Signaling Technology). Expression of equal amounts of endogenous p38 MAPK and tandem affinity purification (TAP) fusion protein in each cell lysate was demonstrated by Western blotting using the p38 MAPK antibody and the specific binding of the secondary antibody to the protein A domain of the fusion protein, respectively.
MK5 and MK2 kinase assay.
MEFs (2 × 106
) were washed with ice-cold phosphate-buffered saline (PBS), lysed in 500 μl of lysis buffer (3
), vortexed for 10 s, and put on ice for 5 min. After centrifugation at 10,000 × g
at 4°C for 10 min, the supernatant was transferred to a new tube and the protein concentrations were determined using a Bio-Rad protein determination kit. Where indicated, kinase was immunoprecipitated from the lysate containing 1 mg of total protein by using 2 μg of PRAK antibody (catalog no. 06-960; Upstate Biotechnology) or 5 μl of a MK2-specific antiserum (3
); otherwise, lysate (1 μl) was directly used in the kinase assay. For immunoprecipitations (IP), after 1 h of mixing at 4°C, 30 μl of protein G-agarose (Pharmacia) (a 50% slurry preequilibrated in lysis buffer) was added and the combination was mixed for another hour. Agarose was washed three times with lysis buffer containing 0.5 M NaCl and two times with 50 mM Tris-HCl (pH 7.5). Then, 40 μl of substrate buffer (0.5 mM EGTA, 0.5 mg of bovine serum albumin/ml, 30 μM PRAK substrate peptide, 50 mM Tris-HCl, pH 7.5) was added. After preheating at 30°C for 3 min, 10 μl of hot buffer (75 mM MgCl2
, 0.5 mM ATP, 2 μl of [γ-33
P]ATP) was added and a sample was incubated at 30°C for 20 min. A 20-μl aliquot was spotted on Whatman p81 paper and washed extensively in 1% phosphoric acid before radioactivity was measured. Detection of kinase activity in cell lysates was carried out using Hsp25 as substrate as described previously (5
In vivo phosphate labeling, two-dimensional (2D) electrophoresis, phosphorimaging, and detection of phospho-Hsp25.
Wild-type MEF, MK2−/−, and MK5−/− cells were grown to 90% confluence on 10-cm-diameter dishes in 90% Dulbecco's minimal Eagle's medium supplemented with 10% fetal calf serum and antibiotic-antimycotic in a humidified atmosphere of 95% air and 5% CO2 at 37°C. The medium was changed to phosphate-free Dulbecco's minimal Eagle's medium (Sigma), and cells were incubated with 500 μCi of HCl-free [32P]orthophosphate (DuPont) for 2 h at 37°C. Cells were stimulated with 100 μM sodium arsenite for 45 min, washed with PBS, and lysed in 350 μl of lysis buffer containing 7 M urea, 2 M thiourea, 4% (wt/vol) CHAPS, 15 mM dithiothreitol (electrophoresis grade), 0.5% carrier ampholytes (pH 3 to 10), protease inhibitors (Roche), and 10 nM calyculin A (Calbiochem). The homogenate (about 350 μg) was solubilized by sonication on ice for 15 min followed by 20 min of centrifugation at 14,000 × g. Isoelectric focusing for 2D gel electrophoresis was performed using a Protean isoelectric focusing cell from Bio-Rad according to the instructions of the manufacturer. Supernatant was loaded on a 17-cm-long immobilized pH gradient strip (pH 3 to 10) and reswollen overnight at 50 V. Focusing was carried out for 1 h at 250 V, 1 h at 500 V, and 15 h at 7,000 V.
After equilibration in 50 mM Tris (pH 8.9), 6 M urea, 30% (wt/vol) glycerol, and 2% (wt/vol) SDS, gels were immediately applied to a vertical 12% (wt/vol) SDS gel without a stacking gel. Electrophoresis was carried out at 8°C with a constant current of 40 mA per gel. The gels of radioactively labeled MEF proteins were fixed in 30% ethanol-10% acetic acid and exposed. Radioactive spots visualized by autoradiography were excised. Gel pieces were washed sequentially for 10 min in tryptic digestion buffer (10 mM NH4HCO3) and digestion buffer-acetonitrile (1:1). These steps were repeated three times and led to a shrinking of the gel. It was reswollen with 2 μl of protease solution (Promega) (trypsin at 0.05 μg/μl) in digestion buffer and incubated overnight at 37°C.
Analysis of the tryptic peptides was carried out using an electrospray ion-trap mass spectrometer (MS) (LCQ Classic; Thermo Finnigan) directly coupled to a Nano-high-pressure liquid chromatography (HPLC) system (LC Packings; Dionex). The peptides were automatically transferred from the autosampler (Famos; Dionex) to the preconcentration column (C18 PepMap Nano Precolumn [0.3-mm i.d. by 1 mm long]). After preconcentration and washing for 10 min (40 μl/min in 0.1% trifluoroacetic acid), the peptides were automatically injected into a C18 PepMap Nano-HPLC column (LC Packings; Dionex) (75-μm i.d. by 250 mm long; 300-Å pore size; 5-μm-diameter particle size) using the Switchos system (LC Packings; Dionex). The inert HPLC pump (Ultimate; Dionex) was driven with a flow rate of 160 nl/min. The gradient (solution A, 0.1% formic acid-84% acetonitrile; solution B, 0.1% formic acid-84% acetonitrile) started at 5% of solution B and rose to 50% of solution B in 90 min. A dual-channel UV detector (LC-Packings; Dionex) with a 3-nl flow cell (LC Packings; Dionex) was used at wavelengths of 215 nm (peptide bond) and 295 nm (tryptophan side chains). Separated peptides were directly transferred to the MS via a heated capillary and a metal-backed coated glass needle (PicoTip [catalog no. FS360-20-10]; New Objective Incorporated). The following electrospray ion-trap parameters were used: spray voltage, 1.8 to 2.15 kV; capillary temperature, 200°C; capillary voltage, 42 V; tube lens offset, 30 V; and electron multiplier, −950 V. The collision time was set automatically, depending on the mass of the parent ion. The trapping time was set to 200 ms, and the automatic gain control was set to 107. The data were collected in centroid mode, with one full-MS experiment followed by three experiments investigating the MS-MS spectra of the three most intensive ions (intensity, at least 3 × 105). Dynamic exclusion was used for the data collection, with an exclusion duration of 5 min and an exclusion mass of ± 1.5 Da.
Hsp27 in-gel kinase assay.
For in-gel kinase assays 107
cells were treated with arsenite (150 μM for 45 min) or phorbol myristate acetate (100 nM for 45 min) in the presence of freshly prepared pervanadate solution (final concentration, 40 mM), washed with PBS, and lysed, and MK2 or MK5 was immunoprecipitated using immunoaffinity-purified non-cross-reacting MK5-specific antibodies (catalog no. 06-960; Upstate Biotechnology) (8
), MK5 phosphorylation-specific antibodies (kind gift of Sir P. Cohen, Dundee, Scotland), anti-PRAK-antibodies (kind gift from J. Han, La Jolla, Calif.), and anti-MK2 antibodies (3
) as described above. IPs were separated by electrophoresis in SDS-polyacrylamide gels containing 0.5 mg of recombinant Hsp27/ml or 0.5 mg of bovine serum albumin/ml as a control. After an in-gel kinase assay (27
) was performed, phosphorylated proteins were visualized with a Fuji 1500 BAS phosphorimager and the signal was quantified by the use of TINA 2.09 software.