Mxi2, a splice variant of p38 stress-activated kinase, is a distal nephron protein regulated with kidney ischemia

Lucia Faccio1, Ang Chen2, Carlo Fusco1, Stefano Martinotti3, Joseph V. Bonventre2, and Antonis S. Zervos1

1 Cutaneous Biology Research Center, Massachusetts General Hospital/Harvard Medical School, 2 Renal Unit, Medical Services, Massachusetts General Hospital and Department of Medicine, Harvard Medical School, Charlestown, Massachusetts 02129, and 3 Department of Experimental Medicine, University of L'Aquila, Italy


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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Mxi2 is one of three known alternative spliced forms of the stress-activated mitogen-activated protein kinase p38 (CSBP). Mxi2 was originally identified as a Max-interacting protein and is the smallest member of the family of stress-activated kinases isolated to date. Mxi2 lacks most of the XI domain found in p38 and instead has a distinct COOH-terminal sequence of 17 amino acids. Here we present the genomic structure of the Mxi2/p38 locus on human chromosome 6q21.2/21.3 and establish the origin of the three spliced forms of p38. Using Mxi2-specific antibodies in mouse organs, we found the Mxi2 protein to be present exclusively in the kidney. Mxi2 is present predominantly in the distal tubule of the nephron and the level of the protein decreased during kidney ischemia-reperfusion. Stress signals or other known activators of the p38 pathway including MAP kinase-kinase 3 and MAP kinase-kinase 6 did not induce the kinase activity of Mxi2 using ATF-2 as a substrate. With the use of hybrid proteins encoding different portions of Mxi2 and p38 polypeptides, the different properties of Mxi2 can be assigned to its unique COOH terminus.

Mxi2 kinase; stress signaling; p38 kinase; distal tubule; Mxi2/p38 genomic locus


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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IN EUKARYOTIC CELLS, extracellular signals are transduced through the activation of different cascades, one of which is the mitogen-activated protein (MAP) kinase cascade. There are three different families of MAP kinases in mammals: extracellular signal-regulated kinase (ERK) (6, 7), c-Jun NH2-terminal kinase or stress-activated kinase (SAPK) (9, 14, 29, 32, 41), and p38 kinase/Mxi2/Hog1/p40/RK/CSBP (11, 16, 18, 26, 40, 49).

The p38 was first identified as a lipopolysaccharide (LPS)-induced phosphoprotein in CD14-transfected murine pre-B cells (17). Human p38 (CSBP2/p38alpha ) was isolated by its ability to bind to the anti-inflammatory drug CSAID (26). Other groups also independently identified p38 (RK, p40) as the kinase which phosphorylates MAP kinase-activated protein kinase 2 (MAPKAPK2) in response to chemical stress and heat shock (11, 40). The p38 sequence shows high homology with HOG1, the osmo-sensing gene of Saccharomyces cerevisiae (16), and expression of p38 can rescue the HOG1 phenotype in yeast (16). The p38 is activated in response to a number of different stress stimuli including LPS, tumor necrosis factor-alpha (TNF-alpha ), interleukin-1 (IL-1), and environmental stress such as heat shock, high osmolarity, ultraviolet radiation, and H2O2 (11, 16, 38, 40). This activation is mediated by several upstream kinases including MAP kinase-kinase 3 (MKK3) and MAP kinase-kinase 6 (MKK6) (8, 9, 39). These kinases phosphorylate p38 at threonine and tyrosine in the TGY motif, resulting in p38 activation.

The p38 targets include the transcription factors ATF-2 (38, 39), MEF2C (15), CHOP (45), MAPKAPK2 (40), and PRAK kinase (28). Phosphorylation of these substrates is presumably part of the cellular response that allows cells to recover from stress. This is of profound clinical importance because p38 activation has been shown to be involved in inflammation and ischemia (26, 47). There are three alternative spliced forms of p38 (CSBP2/p38alpha , CSBP1, and Mxi2) (17, 26, 49) as well as several homologues including p38beta , p38beta 2, p38gamma , and p38delta (8, 10, 12, 18, 24, 25, 27, 31, 44). These homologues are expressed at different levels in human tissues and can be activated by different, although sometimes overlapping, stress stimuli (10, 19).

Although the p38 kinases have been well studied, little is known about the role of Mxi2 kinase in stress signaling. Mxi2 is an alternative spliced form of p38, isolated via its interaction with Max in a yeast two-hybrid screen (49). Max is a bHLH-Zip protein that forms specific heterodimers with Myc as well as a network of bHLH-Zip proteins (50). Comparison of the primary sequence of Mxi2 and p38 shows identical sequence from amino acids 1-280 (49). Mxi2 has 17 COOH-terminal amino acids not found in p38, whereas p38 is longer in its COOH-terminal sequence by 80 residues (49).

Here we report the genomic structure of the p38 locus and establish the origin of the three alternative spliced forms of p38, including Mxi2. With the use of Mxi2-specific antibodies, we found the protein to be expressed predominantly in the distal tubules of mouse kidneys and not in any other mouse organs. To investigate the potential function of Mxi2 in the kidney, we used a mouse ischemia-reperfusion model to monitor the level of Mxi2 as well as its distribution at different times during kidney stress and recovery.

We also investigated the properties of Mxi2 in a side-by-side comparison with p38 kinase. Our data suggest that Mxi2 has unique properties, a very restricted tissue distribution, and is regulated during kidney ischemia-reperfusion.


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Organization of p38 genomic locus located on human chromosome 6. The nonredundant database maintained by the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov) was searched using the GAPPED BLAST computer program (http://www.ncbi.nlm.nih.gov/cgi-bin/BLAST/nph-newblast?Jform=1) (1). The genomic structure of p38/Mxi2 was predicted by comparing the cDNA sequence of p38, Mxi2, and CSBP1 with the PAC genomic clone 179N16, which encompasses the 6p21.1-21.33 chromosomal region (GenBank accession no. z95152; Chromosome 6 Project Group, Sanger Center, UK), using the BLAST 2 sequences computer program on the NCBI server (http://www.ncbi.nlm.nih.gov/gorf/bl2.html) (1). The donor-acceptor sequences flanking the exons were identified using the Gene Finder programs (http://dot.imgen.bcm.tmc.edu:9331/gene-finder/gf.html) (43).

Tissue distribution of Mxi2 protein. Single mouse organs were resuspended in 5 ml of lysis buffer (20 mM HEPES pH 7.4, 2 mM EGTA, 1 mM EDTA, Trasylol, beta -glycerophosphate 50 mM, 1% Triton X-100, and 1 mM Na3VO4) and protease inhibitor cocktail (Boehringer Mannheim). Organs were kept on ice and were homogenized using a Brinkmann homogenizer (setting 6, three times for 6 s each) and were then sonicated for 10 s. Debris was collected by slow-spin centrifugation (3,000 rpm for 10 min) and the supernatants were further clarified by spinning them at 38,000 rpm for 1 h. The protein concentration of lysates was estimated using the Bradford assay (Bio-Rad). From each kidney lysate, 100 µg of protein extract were resolved on an SDS-PAGE gel and processed for Western blot analysis as described (49).

Antibodies used included N-20 p38 antibody (Santa Cruz) or an Mxi2-specific polyclonal antibody that was raised against a synthetic peptide corresponding to the 17 unique COOH-terminal amino acids of Mxi2. The Mxi2-specific antiserum was affinity purified on a column that carried the synthetic peptide (Research Genetics).

Mxi2 levels are regulated by kidney ischemia-reperfusion. BALB/c male mice (20-25 g), anesthetized with pentobarbital sodium (65 mg/kg ip), underwent uninephrectomy of the left kidney followed 24 h later by ischemia of the right kidney imposed by placement of a microaneurysm clamp on the renal artery and vein for 30 min followed by reperfusion (35). Kidneys were harvested 1, 24, and 48 h after reperfusion. In bilateral ischemia experiments, ischemia was performed for 30 min followed by reperfusion (20). Kidneys were harvested at the same time points as with nephrectomy/unilateral ischemia. Equal amounts of protein extract from control and postischemic kidneys were resolved by SDS-PAGE. The level of Mxi2 and p38 proteins was determined on Western blots using the N-20 p38 antibody or the C-17 Mxi2-specific antibody.

Immunohistochemistry. Kidneys from control or postischemic mice were briefly perfused in situ via the heart with PBS, followed by 2% paraformaldehyde, 70 mM lysine, and 10 mM sodium periodate and fixed as described in Ref. 5. Kidney slices were immersed in 30% sucrose and then frozen in liquid nitrogen. Five-micrometer sections were cut using a Reichert Frigocut microtome. Sections were rehydrated in PBS and then incubated in 1% BSA to block nonspecific binding. An antibody recognizing GP330 (21), which specifically stains the brush-border membranes of proximal tubule cells, was used at a dilution of 1:100. The Mxi2-specific C-17 antibody was used at 1:200 dilution. Tissue sections were incubated with primary antibodies for 1 and 2 h, respectively, at room temperature, followed by two washes in high-salt PBS (2.7% NaCl) and a final wash in PBS. Goat anti-mouse IgG, conjugated with FITC, and donkey anti-rabbit IgG, conjugated with Cy3, were applied for 1 h at room temperature and rinsed the same as the primary antibodies. Staining with anti-GP330 was performed first, followed by staining with Mxi2 antibodies on the same kidney sections, to define the relative localization of these proteins. Sections were mounted with Vectorshield and visualized under a Nikon FXA photomicroscope. Images were captured and stored on a JAZ disk using IP Lab Spectrum software (Scanalytics, Vienna, VA).

Cell culture and transfection. HEK-293 cells were maintained in DMEM (Sigma) containing 10% (vol/vol) FCS (Sigma). All transient transfections were done on 100-mm dishes using the calcium phosphate method (36). Briefly, the transfection cocktail was prepared by resuspending 10 µg of DNA in 500 µl of 250 mM CaCl2 and left at room temperature for 30 min. The CaCl2 mix was then added to 500 µl of HEPES buffer pH 7.2 while bubbling air through it. The mix was left at room temperature for 20 min and then added to the cells. To evaluate the effect of anisomycin, a known p38 activator, cells were treated 3 days after transfection with 10 µg/ml of anisomycin (Calbiochem) for 30 min.

Immunoprecipitation. Cells were washed briefly with PBS and lysed using 400 µl of lysis buffer (25 mM Tris · HCl pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 2 mM EGTA, 1 mM EDTA, 15 mM NaF, 1 mM Na3VO4, 1 mM phenyl phosphate, 40 mM p-nitrophenyl phosphate) and protease inhibitor cocktail.

Lysates were incubated on ice for 30 min and then centrifuged for 15 min at 14,000 rpm to remove insoluble debris. Total protein in cell lysates was quantified by the Bradford assay. The anti-HA monoclonal antibody 12CA5 (Boehringer Mannheim) was prebound to protein G-agarose beads at a concentration of 0.1 mg of antibody/ml of beads. Cell lysate (100 µg total protein lysate) prepared as described above was added to 50 µl of 50% suspension of beads and gently shaken for 2 h at 4°C. The beads were washed four times with 1× STN buffer (5× STN: 50 mM Tris · HCl pH 7.5, 750 mM NaCl, and 5% Nonidet P-40). The identity of the epitope-tagged protein in the immunoprecipitates was confirmed by Western blot analysis with rabbit N-20 p38 antibody (Santa Cruz).

Maltose fusion proteins. The full-length coding sequence for Mxi2 or p38 was PCR amplified, DNA digested with BamH I and Xba I restriction enzymes, and cloned in frame into pMal (New England Biolabs) vector. The DNA sequence of the PCR products was confirmed by sequencing both strands with a commercially available kit (US Biochemicals). Production of the recombinant Mal-Mxi2 (74.5 kDa) or Mal-p38 (80 kDa) was monitored by SDS-PAGE. Fusion proteins were purified by affinity chromatography according to the manufacturer's recommended protocol (New England Biolabs).

Kinase assays. Immunoprecipitated complexes containing HA-p38, HA-Mxi2, or HA-hybrid polypeptides were isolated as described above. Beads were washed four times with 1× STN buffer and once with kinase buffer (25 mM HEPES pH 7.4, 25 mM beta -glycerophosphate, 25 mM MgCl2, 0.1 mM Na3VO4, and 25 mM dithiothreitol). Kinase reactions were performed in a final volume of 40 µl kinase buffer in the presence of 25 mM cold ATP, 10 µCi [gamma -32P]ATP, and 0.2 mg GST-ATF-2. The reactions were incubated for 30 min at 30°C, stopped by addition of 2× SDS loading buffer, and analyzed by SDS-PAGE and autoradiography.

Construction of p38/Mxi2 hybrid polypeptides. HA-p38 and HA-Mxi2 clones were prepared as described in Ref. 49. The four COOH-terminal truncated forms of Mxi2, Mxi2 (1-291 amino acids), (1-287 amino acids), (1-283 amino acids), (1-280 amino acids), and the COOH-terminal deleted form of p38 (1-297 amino acids) were PCR amplified and the DNA digested with Mfe I/Xba I and cloned in frame into pMT3 vector (13). The chimeric forms Mxi2+PSD and p38+MSD were generated by PCR. Mxi2+PSD is a fusion of Mxi2 full length with the p38 COOH-terminal amino acid sequence 297-360. p38+MSD consisted of the full-length p38 with the Mxi2-specific COOH-terminal amino acid sequence 280-297. Both PCR products were digested with Mfe I/Xba I and cloned in frame in the pMT3 vector (49).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Characterization of p38 genomic locus. The cDNA sequence of human p38 was used to search for significant homologies with genomic clones present in the nonredundant database. One such clone, generated from chromosome 6q21.1/21.33 (PAC genomic clone 179N16, accession no. z95152), represented a genomic DNA sequence of 172,048 nucleotides and included the complete cDNA sequence of p38/CSBP2, Mxi2, and CSBP1. This is in accord with a previous report that placed the location of the p38/CSBP gene on human chromosome 6q21.2/21.3 (30). Figure 1A is a schematic diagram of the genomic structure of p38/Mxi2 locus. Comparing the cDNAs of the three spliced forms of p38 (CSBP1, CSBP2, and Mxi2) to the PAC genomic clone 179N16 derived from chromosome 6p21.1-21.33 shows 13 different exons that account for all three spliced forms (Fig. 1A). The 3' terminal coding exon (exon 11') for the Mxi2 variant is simply an extension of exon 11, indicating that p38 or Mxi2 is controlled by a splice/don't splice mechanism at this donor site (Fig. 1B). The exon 11 extension (11') includes a sequence that encodes the COOH-terminal Mxi2-specific 17 amino acids, a short 3' untranslated region followed by an AAATAAA polyadenylation signal.


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Fig. 1.   Structure of p38 locus on human chromosome 6q21.2/21.3. A: linear order of exons for the three different splice variants of p38: Mxi2, p38/CSBP2, and CSBP1. Exon-intron structure spans ~90 kb. Schematic representation shown is not in scale. B: sequence flanking exon 11-11' of human p38/CSBP2 gene. Common coding sequence for p38 and Mxi2 is shown in uppercase letters. Splice donor and splice acceptor are underlined. Sequence belonging to exon 11' is shown in bold. Corresponding 17 amino acid sequence unique to COOH terminus of Mxi2 is shown in capital letters.

Mxi2 expression. Mxi2 was originally cloned from a HeLa cDNA library, and Northern blot analysis showed that Mxi2 mRNA is expressed ubiquitously (49). Further analysis showed that the Mxi2 protein was not detectable in several cell lines tested, including HeLa cells (results not shown). With the use of both an antibody that recognizes the NH2-terminal common portion of Mxi2 and p38, and a specific polyclonal antibody against the unique COOH-terminal sequence of Mxi2, Mxi2 protein was detected in mouse kidneys (Fig. 2, A and B). Under the same conditions, Mxi2 protein was not detected in mouse brain, heart, liver, lung, spleen, or skeletal muscle (Fig. 2, A and B). Immunohistochemical staining of frozen mouse kidney sections localized Mxi2 to the distal tubules of nephrons. Figure 2C shows Mxi2 staining of two such representative sections of normal mouse kidneys. Sections were stained with anti-GP330 antibody to distinguish the proximal from the distal tubules of nephrons, as GP330 is localized at the brush border of proximal tubules only (21, 22) (Fig. 2C, I). In general, there was no colocalization of GP330 and Mxi2 when both images were merged (Fig. 2C, III). Mxi2 was found almost exclusively at the distal tubules (Fig. 2C, II), but occasionally some weak proximal tubule and interstitial staining was also observed (results not shown). By contrast, p38 staining was uniformly distributed throughout the mouse kidney sections.


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Fig. 2.   Western blot analysis of protein extracts from several mouse tissues using N-20 antibody (A) or C-17 antibody (B). Blot was initially probed with C-17 antibody (which is Mxi2 specific), then stripped and reprobed with N-20 antibody, which recognizes both Mxi2 and p38 proteins. Mxi2 protein is expressed only in kidney. C: immunohistochemical staining for Mxi2 in mouse kidneys. Normal kidney proximal tubules were identified by apical staining with anti-GP330 (green, I). II shows Mxi2 (red) staining; no colocalization of GP330 and Mxi2 was observed when both images were merged (III). Top, a transverse section; bottom, a sagittal section.

Ischemia and reperfusion. A series of studies were performed to investigate whether stress induced by kidney ischemia has any effect on the level and/or distribution of Mxi2 protein. Mxi2 protein levels were significantly decreased at 24 and 48 h after 30 min of transient ischemia in both uninephrectomized and bilateral ischemia models (Fig. 3). In contrast, the level of p38 remained unchanged compared with contralateral or sham-operated controls (Fig. 4). Immunostained postischemic kidneys showed a similar pattern but lower intensity of distal tubule distribution for Mxi2 than that observed in control kidneys (results not shown). The subcellular localization of Mxi2 remained cytoplasmic before and after ischemia-reperfusion (results not shown).


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Fig. 3.   Western blot analysis of extracts from mouse kidneys after kidney ischemia-reperfusion. In uninephrectomy model for kidney ischemia, contralateral kidneys were taken out 24 h before a 30 min clamping was performed on remaining kidney. Ischemic kidneys were harvested 1, 24, and 48 h after ischemia. Contralateral kidneys were used as controls. In bilateral kidney ischemia model, both renal pedicles were clamped for 30 min; ischemic kidneys were harvested 1, 24, and 48 h after ischemia. Kidneys from sham-operated animals were used as controls. Mxi2 protein levels were significantly decreased after ischemia in both uninephrectomy and bilateral ischemia models. This is in contrast to the p38 protein levels that remained unchanged. Antibodies used were C-17 Mxi2-specific antibody and N-20 p38 antibody. C, contralateral control; I, ischemic; and S, sham-operated control.



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Fig. 4.   Activation of p38 but not Mxi2 by different stress stimuli. A: HEK-293 cells transiently transfected with HA-p38 or HA-Mxi2 were treated with ultraviolet (UV) radiation (40 J/cm2), interleukin-1 (IL-1; 80 µg/ml), or anisomycin (10 µg/ml), followed by 30 min incubation at 37°C. Kinase activity of HA-p38 or HA-Mxi2 isolated by immunoprecipitation using anti-HA antibodies was monitored using GST-ATF-2 (amino acids 1-109) as a substrate in an in vitro kinase reaction. B: Western blot analysis using a phosphotyrosine antibody that recognizes phosphorylated threonine and tyrosine in TGY motif. C: Western blot analysis using N-20 p38 antibody that recognizes both Mxi2 and p38. D: kinase activity of p38 or Mxi2 isolated from HEK-293 cells treated with various stress signals. 293 Cells were transiently transfected with HA-Mxi2 or HA-p38 encoding constructs. After 48 h, cells were treated with one of the following different stress stimuli: UV radiation (40 J/cm2 followed by incubation at 37°C for 20 min), NaCl (0.5 M for 30 min), hydrogen peroxide (500 µM for 20 min), sodium vanadate (1 mM for 20 min), anisomycin (10 µg/ml for 30 min), epidermal growth factor (EGF; 20 ng/ml for 10 min), IL-1beta (100 ng/ml for 20 min), TNF-alpha (tumor necrosis factor-alpha ; 50 ng/ml for 20 min), sorbitol (0.4 M for 20 min), heat shock (43°C for 30 min), phorbol 12-myristate 13-acetate (PMA; 100 nM for 20 min), and arsenite (200 µM for 20 min) (18, 44). Activity of Mxi2 and p38 isolated by immunoprecipitation using HA monoclonal antibody was monitored using a phosphospecific antibody, and kinase activity was measured using GST-ATF-2 as a substrate in an in vitro kinase reaction.

Mxi2 kinase activity. The p38 family of kinases can be activated by a variety of different stress stimuli and inflammatory cytokines. To investigate how Mxi2 might be regulated by different stress signals, we transiently transfected HEK-293 cells with vectors expressing HA-Mxi2 or HA-p38. After 48 h, the cells were treated with different stress stimuli, and HA-Mxi2 or HA-p38 proteins were isolated by immunoprecipitation using anti-HA antibodies. The kinase activity of these proteins was tested using GST-ATF-2 (amino acids 1-109) as a substrate. The p38 was activated strongly by ultraviolet radiation and anisomycin and moderately by IL-1 (Fig. 4A). The kinase activity of p38 was specifically inhibited by SB-203580.

We used a phosphospecific antibody (New England Biolabs) that recognizes phosphorylated threonine and tyrosine in the TGY motif of p38 or Mxi2 when these kinases are activated. This antibody clearly recognized p38 but did not recognize Mxi2 (Fig. 4B). Figure 4C shows the amount of protein of HA-Mxi2 and HA-p38 that was immunoprecipitated and used in this kinase experiment. A number of known p38 stress activators including hydrogen peroxide, sodium vanadate, epidermal growth factor, IL-1, TNF-alpha , sorbitol, heat shock, phorbol 12-myristate 13-acetate, and arsenite (16, 38) each stimulated the kinase activity of p38 to various degrees, but none of these stimuli was able to activate Mxi2 (Fig. 4D).

Regulation of Mxi2 by MKKs. MAP kinases are activated through direct phosphorylation by upstream MKKs kinases (8, 9, 39). MKK3 and MKK6 are known upstream activators of p38 (9, 34). To determine whether MKK3 or MKK6 could activate Mxi2, we cotransfected HEK-293 cells with expression vectors of HA-Mxi2 or HA-p38 together with HA-MKK3 or an inactive form of HA-MKK3KR (kindly provided by A. Molmar, Massachusetts General Hospital) of which the invariant lysine at the ATP-binding site was substituted with an arginine (33). The p38 isolated from cells cotransfected with MKK3 showed high-kinase activity against GST-ATF-2 (amino acids 1-109). When the inactive MKK3KR was used, no activity was detected. On the contrary, Mxi2 showed no detectable activity in the presence of MKK3 as monitored by the absence of GST-ATF-2 phosphorylation (Fig. 5A). MKK3 or MKK3KR alone were not able to phosphorylate GST-ATF-2.


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Fig. 5.   Regulation of p38 but not Mxi2 kinase activity by MKK3. A: HEK-293 cells were cotransfected with HA-tagged Mxi2 or p38 and an expression vector encoding MKK3 or a mutant inactive form, MKK3KR. HA-p38 and HA-Mxi2 were precipitated using anti-HA monoclonal antibody, and their activity was tested in an in vitro kinase assay using GST-ATF-2 (amino acids 1-109) as a substrate. MKK3 is a strong activator of p38 kinase activity (lane 3). Activation of p38 was specific because an inactive form of MKK3 (MKK3KR) had no detectable effect (lane 4). The p38 had a very weak activity due to autophosphorylation (lane 8). MKK3 and MKK3KR alone had no activity against GST-ATF-2 substrate (lanes 6 and 7). Mxi2 had no detectable kinase activity in presence or absence of MKK3 (lanes 1 and 2). B: Western blot analysis using a phosphotyrosine antibody that recognizes phosphorylated threonine and tyrosine in TGY motif. C: relative levels of p38 and Mxi2 proteins used in this assay. Same blot used in A was reprobed with N-20 p38 antibody, which recognizes both Mxi2 and p38. Equal amounts of p38 and Mxi2 proteins were used in assay.

The same blot was probed with the phosphospecific antibody, and whereas p38 was clearly activated by MKK3, no phosphorylation was seen on Mxi2 (Fig. 5B). Figure 5C shows that similar amounts of HA-Mxi2 and HA-p38 proteins were immunoprecipitated and used in this kinase experiment.

To investigate whether MKK6 might be an activator of Mxi2, we used bacterially made Mal-Mxi2 or Mal-p38 and GST-MKK6 (kindly provided by J. Kyriakis, Massachusetts General Hospital) in an in vitro kinase reaction. Figure 6A shows that MKK6 was able to phosphorylate and activate p38 and that this activation led to the phosphorylation of GST-ATF-2. On the contrary, Mxi2 was not activated by MKK6 and was unable to phosphorylate GST-ATF-2. Both proteins (Mal-Mxi2 and Mal-p38) were present in similar amounts in the kinase reactions, as determined with the anti-p38 antibody (Santa Cruz), which recognizes the first 20 NH2-terminal amino acids of both Mxi2 and p38 (Fig. 6B).


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Fig. 6.   Activation of p38 but not Mxi2 by MKK6. A: in vitro kinase assay in presence or absence of 10 µM SB-203580 was performed using GST-MKK6 purified protein as an activator of p38/Mxi2, Mal-Mxi2, or Mal-p38 proteins and GST-ATF-2 (amino acids 1-109) as a specific substrate. Kinase reactions were stopped by adding SDS-sample buffer and analyzed by SDS-PAGE. Substrate phosphorylation was detected by autoradiography. B: same blot was also probed using N-20 p38 antibody, which recognizes both Mxi2 and p38. It shows that Mal-p38 and Mal-Mxi2 proteins were present in equal amounts.

Mxi2/p38 hybrid proteins. The inability of Mxi2 to be activated by stress signals was surprising because Mxi2 has both an intact ATP-binding domain as well as the substrate-recognition domain (46, 49). Furthermore, Mxi2 has the TGY motif (49) that in p38 is phosphorylated by MKK3 and MKK6 (8, 9, 39). To investigate whether the difference between Mxi2 and p38 was due to the unique COOH terminus of each protein, we constructed several hybrid polypeptides between Mxi2 and p38 (Fig. 7). These constructs were cloned into a derivative of the pMT3 expression vector that adds an HA-epitope tag at the NH2 terminus of the recombinant protein (2).


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Fig. 7.   Construction of p38-Mxi2 hybrid polypeptides. Black box, unique COOH sequence of Mxi2, which consists of 17 amino acids; MSD, Mxi2-specific domain. Gray box, 80 specific amino acids present at p38 COOH terminus; PSD, p38-specific domain. T-loop is surface loop where TGY motif that is phosphorylated by upstream activating kinases is localized. ATP, active site of kinases where ATP molecule is bound.

Kinase activity of Mxi2 hybrid proteins. Constructs encoding the recombinant proteins were transiently transfected into HEK-293 cells. Forty-eight hours later, cells were treated with 10 µg/ml anisomycin for 30 min and the hybrid polypeptides were precipitated from cell extracts using HA antibodies. The kinase activity of the immunoprecipitated proteins was tested using GST-ATF-2 as a substrate.

Figure 8A shows that Mxi2 is an inactive kinase using GST-ATF-2 as a substrate (lane 1) whereas p38 was able to phosphorylate GST-ATF-2 (lane 2). When the length of p38 was reduced to 297 amino acids, the exact size of Mxi2, the kinase activity of p38 (1-297) was lost (lane 3). When we added the 17 Mxi2-specific COOH-terminal sequence (MSD) to the full-length p38, the MSD slightly decreased the activity of p38 (lane 4). Furthermore, Mxi2 showed an increased, albeit weak, kinase activity when the p38-specific COOH-terminal (PSD) domain was added to its COOH terminus (lane 5). Weak kinase activity was also detected for Mxi2 when amino acids from its COOH terminus were successively deleted from 297 down to 280 (lanes 6-9). Mxi2 showed some kinase activity after the removal of even the last six COOH-terminal amino acids (lane 6). Figure 8B shows the relative amount of each of the recombinant proteins precipitated from transiently transfected HEK-293 cells and the approximate mol wt for each of the recombinant polypeptides.


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Fig. 8.   Regulation of different p38/Mxi2 hybrid polypeptides. A: HEK-293 cells transiently transfected with HA-Mxi2 or HA p38, HA-p38 (1-297), HA-p38+MSD, HA-Mxi2+PSD, HA-Mxi2 (1-291), HA-Mxi2 (1-287), HA-Mxi2 (1-283), and HA-Mxi2 (1-280) were treated with anisomycin (10 µg/ml) followed by 30 min incubation at 37°C. Kinase activity of HA-fusion protein isolated by immunoprecipitation using anti-HA antibodies was monitored using GST-ATF-2 (amino acids 1-109) as a substrate in an in vitro kinase reaction. B: same blot was then probed using N-20 p38 antibody, which recognizes both Mxi2 and p38. It shows that equal amounts of proteins were immunoprecipitated.

To verify that the kinase activity detected was due to p38 or Mxi2 recombinant proteins and not to a contaminating kinase, the specific p38 inhibitor SB-203580 was used. Immunoprecipitated proteins from transiently transfected HEK-293 cells were tested for kinase activity using GST-ATF-2 in the presence or absence of SB-203580. Figure 9A shows that the kinase activity of the different forms of p38 and Mxi2 hybrids were specifically inhibited by SB-203580. The relative levels of the proteins immunoprecipitated and used in this assay are shown in Fig. 9B.


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Fig. 9.   Regulation of kinase activity of different p38/Mxi2 hybrid proteins by SB-203580. A: in vitro kinase assay in presence or absence of 10 µM SB-203580 was performed using HA-fusion proteins p38, p38+MSD, Mxi2+PSD, Mxi2 (1-291), Mxi2 (1-287), Mxi2 (1-283), and Mxi2 (1-280) immunoprecipitated from transiently transfected HEK-293 cells stimulated with anisomycin (10 µg/ml). Phosphorylation of GST-ATF-2 substrate was detected by autoradiography. B: to monitor amount of protein immunoprecipitated and used in A, same blot was probed using N-20 p38 antibody, which recognizes both Mxi2 and p38.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The p38 MAP kinase family includes three different alternative spliced forms: p38, Mxi2, and CSBP1 (17, 26, 49). Whereas CSBP1 is derived from an internal splicing, Mxi2 lacks exons 12 and 13 and has instead exon 11' (contiguous to exon 11) as its terminal coding exon. Exons 12 and 13 correspond to the last 80 amino acids of p38, whereas exon 11' corresponds to the unique COOH-terminal amino acid sequence 280-297 of Mxi2. Mxi2 is the smallest member of the p38 family of stress-activated kinases. It lacks most of domain XI, which is found in all other members of the p38 family of kinases. Domain XI, downstream of loop L16, has recently been reported to be involved in the ability of kinases to homodimerize and translocate to the nucleus (23). Besides the unique COOH terminus, Mxi2 is identical to p38, has an intact ATP-binding domain, a substrate recognition domain, and the TGY motif that is phosphorylated in p38 by upstream kinases upon activation by stress.

Mxi2-specific antiserum localized the Mxi2 polypeptide exclusively in the mouse kidney. These results were further confirmed using a p38 antibody that recognizes both Mxi2 and p38. Immunohistochemical studies localized the Mxi2 protein to the distal tubule cells and showed a distinct cytoplasmic staining. Because Mxi2 is a member of the family of stress-activated kinases, it may have a function during kidney stress or injury induced by ischemia or toxic insult. There are several reports showing SAPK and p38 kinase activation during ischemia-reperfusion or osmotic induction (3, 37, 42, 48). Mxi2 protein levels were substantially reduced as early as 1 h after ischemia-reperfusion, whereas the p38 protein levels remained unchanged. The reduction in the Mxi2 protein level could be caused either by switching the splicing mechanism favoring p38 production or by selective degradation of Mxi2.

The specific localization of Mxi2 protein in the distal tubule, which is quite resilient to ischemic injury, suggests a possible function of Mxi2 in limiting cell injury or initiating cellular recovery. Much of the damage in the kidney after ischemia-reperfusion occurs on the proximal tubule (4), an area that we have shown to have an undetectable amount of Mxi2 protein.

Mxi2, unlike p38, was not activated by any of a number of stress signals we tested. Furthermore, MKK3 and MKK6, known activators of p38, were not able to induce Mxi2 kinase activity when ATF-2 was used as a substrate either in vivo or in vitro. Because the only difference between p38 and Mxi2 is at the COOH terminus, we constructed a number of hybrid proteins using the unique Mxi2 or p38 COOH-terminal sequence.

The results of these experiments clearly showed that the differences between p38 and Mxi2 were due to their different COOH termini. The size of the Mxi2 kinase was of major importance for its activity. Decreasing the length of p38 to the exact size of Mxi2 converted p38 into an inactive kinase. Deleting 6 to 17 amino acids from the COOH terminus of the native Mxi2 also creates an active kinase. The unique COOH-terminal 17 amino acids of Mxi2, when crafted onto p38, also decreased its kinase activity. Furthemore, Mxi2-PSD showed weak kinase activity and was not as active as p38. Similarly, the kinase activity observed in all the Mxi2-p38 hybrid proteins was much lower than the activity observed with active p38. The activity of all hybrid proteins was inhibited by the pyridinyl imidazole compound SB-205380.

Recently, we have isolated a novel stress-modulated protease (Omi) that interacts with the unique COOH terminus of Mxi2 via a PDZ domain (10a). Our data suggest that part of the function of Mxi2 protein, at least in kidney, might be the specific inactivation of Omi during normal conditions. Mxi2 binds to the substrate-binding domain of Omi forming an inactive complex. After ischemia, Mxi2 is specifically removed, allowing Omi to perform its normal function.

In summary, we show that Mxi2 is an alternative spliced form of p38 kinase and has unique properties. It is localized exclusively in the distal tubules of the kidney and is regulated during ischemia-reperfusion.


    ACKNOWLEDGEMENTS

We are grateful to A. Alessandrini and T. Gulick for comments, suggestions, and critical reading of the manuscript.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01 DK-55734-01 (to A. S. Zervos).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: A. S. Zervos, Cutaneous Biology Research Center, Massachusetts General Hospital/Harvard Medical School, 149 13th St., Charlestown, MA 02129 (E-mail: zervos{at}frodo.mgh.harvard.edu).

Received 17 August 1999; accepted in final form 4 November 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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