Elimination of protein kinase MK5/PRAK activity by targeted homologous recombination

Abstract

MK5 (mitogen-activated protein kinase [MAPK]-activated protein kinase 5), also designated PRAK (p38-regulated and -activated kinase), was deleted from mice by homologous recombination. Although no MK5 full-length protein and kinase activity was detected in the MK5 knockout mice, the animals were viable and fertile and did not display abnormalities in tissue morphology or behavior. In addition, these mice did not show increased resistance to endotoxic shock or decreased lipopolysaccharide-induced cytokine production. Hence, MK5 deletion resulted in a phenotype very different from the complex inflammation-impaired phenotype of mice deficient in MK2, although MK2 and MK5 exhibit evolutional, structural, and apparent extensive functional similarities. To explain this discrepancy, we used wild-type cells and embryonic fibroblasts from both MK2 and MK5 knockout mice as controls to reexamine the mechanism of activation, the interaction with endogenous p38 MAPK, and the substrate specificity of both enzymes. In contrast to MK2, which shows interaction with and chaperoning properties for p38 MAPK and which is activated by extracellular stresses such as arsenite or sorbitol treatment, endogenous MK5 did not show these properties. Furthermore, endogenous MK5 is not able to phosphorylate Hsp27 in vitro and in vivo. We conclude that the differences between the phenotypes of MK5- and MK2-deficient mice result from clearly different functional properties of both enzymes.

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 cassette (neo) was cloned between SacI and SpeI sites, deleting exon 6 of MK5, which codes for part of kinase subdomains VIa and VIb. (B) Southern blot and PCR analysis of the F₁ generation from the chimeric mice generated. For Southern hybridization after BamHI digestion, the external probe indicated in panel A was used. The targeted allele is represented by a 4.4-kb fragment, while the wild-type allele is represented by a 6.5-kb fragment. PCR was carried out using primers at both sides flanking the neo-cassette insertion and the same DNA as for Southern analysis. Insertion was detected by amplification of a 1.2-kb fragment, while experiments using the wild-type allele resulted in amplification of a 0.97-kb fragment. (C) Detection of MK5 mRNA isolated from macrophages of wild-type (+/+) and MK5^−/− mice. As an equal loading control, actin mRNA was detected. (D) RT-PCR using macrophage total RNA (as analyzed in panel C) as the template. The fragment amplified from wild-type (+/+) macrophage mRNA was about 1.4 kb, while the fragment from MK5-deficient mRNA was about 100 bp shorter. (E and F) Western blot detection (using a polyclonal antiserum against recombinant MK5 [catalog no. 06-960; Upstate Biotechnology]) of MK5 protein from macrophages (E) and immortalized MEFs (F) derived from MK5-deficient (MK5^−/−) mice and, as controls, from wild-type (wt) and MK2-deficient (MK2^−/−) animals. The specific immunoreactive band of MK5 migrated with an apparent molecular mass of about 54 kDa. C, positive control (A431 cell lysate) (catalog no. 12-301; Upstate Biotechnology).

FIG. 2.

Comparison of morphologies of selected tissues (heart muscle, skeletal muscle, and pancreas tissue [including an island]) from MK5-deficient (MK5^−/−), MK2-deficient (MK2^−/−), and wild-type (wt) mice. Tissues were stained with hematoxylin and eosin. Bars, 100 μm.

FIG. 3.

Comparison of the phenotype of MK5 deficiency with the MK2-deficient inflammatory phenotype. (A) Survival of LPS-galactosamine-induced endotoxic shock. wt, wild type. (B) LPS-induced cytokine (TNF, IL, and IFN) production of spleen cell cultures from MK5- and MK2-deficient mice plotted as percentages of cytokine production of wild-type spleen cells. Results are shown as means ± standard errors of the means (n = 24 in each group).

FIG. 4.

Analysis of interaction of MK5 with endogenous p38 MAPK. (A) Western blot detection of p38 MAPK levels in MK5-deficient mouse tissues and, as controls, in MK2-deficient and wild-type (wt) mouse tissues. Numbers below the bands represent the results of band quantification using channel 700 of an Odyssey Infrared Imager and Odyssey 1.0 software (Li Cor). (B) Western blot detection of endogenous p38 MAPK after TAP of proteins interacting with MK5 or MK2 from 293 cells. V, vector control without kinase fusion; C, control without transfection of TAP construct. (C) Expression control for TAP fusion proteins and p38 MAPK in 293 cell lysates before affinity purification. Lanes correspond to those shown in panel B.

FIG. 5.

Analysis of stress-activated stimulation of MK5 and MK2 in MEFs by combined IP-kinase assays. Wild-type (wt) and MK2- and MK5-deficient MEFs were stimulated for 60 min with 250 μM arsenite (lanes A) or 300 mM sorbitol (lanes S) or left untreated (lanes C). Cells were lysed, and kinases were immunoprecipitated by MK2 (A)- and MK5 (B)-specific antibodies. Kinase activity in the IP was determined using [γ-³³P]ATP and the peptide KKLRRTLSVA as the substrate. Incorporation of radioactive phosphate into the peptide was measured after binding to phosphocellulose filters was performed.

FIG. 6.

Hsp25 phosphorylation in vitro and in vivo. (A) Cell lysates from wild-type (wt) and MK2- and MK5-deficient MEFs which were left nonstimulated (lanes C) or were stimulated by arsenite treatment (250 μM for 45 min) (lanes Ars) were incubated in vitro with recombinant Hsp25 and [γ-³²P]ATP, and Hsp25 phosphorylating activity was detected by phosphorimaging. (B) 2D phosphoproteomics of wild-type and MK2- and MK5-deficient cells before and after stimulation by arsenite. The spot for phospho-Hsp25 was identified by mass spectroscopy and is indicated by an arrowhead. The autoradiograms shown are representative of the results of four separate experiments.

FIG. 7.

IP-in-gel kinase assay (using different antibodies for IP of protein kinases) of reactivity with Hsp27. Wild-type (wt), MK2- and MK5-deficient MEFs were stimulated for 45 min with 250 μM arsenite (lanes A) or 100 nM phorbol myristate acetate (lanes P) or left untreated (lanes C). Cells were lysed, kinases were immunoprecipitated by (A) MK5-specific antibodies (8), (B) MK2-specific antibodies (3), or (C) anti-PRAK antibodies, and Hsp27 kinase activity was monitored by an in-gel kinase assay (10, 27).