| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2002: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2001: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
We have recently identified and cloned a novel member of MAP kinase superfamily protein, MOK. To address its regulatory mechanisms, we searched for cellular proteins that specifically associate with MOK by co-immunoprecipitation experiments. Several cellular proteins including a major 90-kDa molecular chaperone HSP90 were found associated with MOK. Treatment of cells with geldanamycin, an HSP90-specific inhibitor, rapidly decreased the protein level of MOK, and the decrease was attributed to enhanced degradation of MOK through proteasome-dependent pathways. Our data suggest that the association with HSP90 may regulate intracellular protein stability and solubility of MOK. Experiments with a series of deletion mutants of MOK indicated that the region encompassing the protein kinase catalytic subdomain I IV is required for HSP90 binding. In addition, we found that other molecular chaperones including Cdc37, HSC70, and HSP70 were detected specifically in the MOK-HSP90 immunocomplexes. The
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se results taken together suggest a role of a specific set of molecular chaperones in the stability of signal transducing protein kinases. We also examined the association of molecular chaperones with other closely related kinases including ERK, p38, JNK/SAPK, MAK, MRK, HIPK1, HIPK2, Dyrk1A, and Dyrk2. Only MAK and MRK among them were associated with HSP90 and Cdc37, suggesting that HSP90-Cdc37 molecular chaperones recognize and stabilize a specific subset of protein kinases with similar catalytic domains
To analyze physiological role of MOK, we have started making a MOK-knockout mouse strain. A fragment of MOK genomic DNA was isolated by screening mouse genomic library with MOK full-length cDNA as a probe. The coding region of MOK with 12 exons locates within an 80kbp region in 14q32. The obtained 14kbp genomic fragment contains exon 2-4 of MOK. Restriction enzyme mapping was performed and a targeting vector with TK/neo selection markers was constructed. We are currently trying to introduce the targeting vector into ES cells by using homologous recombination technique Less
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