| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2000: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1999: ¥2,000,000 (Direct Cost: ¥2,000,000)
|
|---|
| Research Abstract |
Members of the MAP kinase superfamily play important roles in a variety of signal transduction pathways. We report molecular cloning and characterization of a novel member of the MAP kinase superfamily. We isolated mouse and human cDNAs that encode complete open reading frames of a novel protein kinase, termed MOK.MOK contains the protein serine/threonine kinase consensus motifs and shows modest similarity to members of the MAP kinase superfamily and MAK and MAK-related kinase (MRK). In addition, MOK possesses a Thr-Glu-Tyr (TEY) motif in the activation loop domain, like classical MAP kinases. MOK is able to phosphorylate several known MAP kinase substrates and to undergo autophosphorylation. A mutation in the TEY motif to AEF abolished the kinase activity of MOK, and treatment of cells with a phosphatase inhibitor okadaic acid enhanced the kinase activity of MOK.Phorbol ester TPA was found to stimulate the kinase activity of MOK.These results indicate that MOK is distantly related to members of known subfamilies of the MAP kinase superfamily and therefore can be classified as a novel member.
Next, we searched for cellular proteins that specifically associate with MOK.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.Closely related protein kinases MAK and MRK were also found to associate with HSP90 while conventional MAP kinases (ERK, p38, and SAPK/JNK) were not associated with HSP90. In addition, we found that other molecular chaperones including Cdc37, HSC70, HSP70, and HSP60, were detected specifically in the MOK-HSP90 immunocomplexes.
|
