|Budget Amount *help
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2002: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2001: ¥2,800,000 (Direct Cost: ¥2,800,000)
The purpose of this research is to elucidate the intracellular signaling mechanisms regulated by VHL tumor suppressor protein (pVHL). pVHL forms a protein complex (VCB-Cul2) with elongin C, elongin B, and Cul2, which functions as a ubiquitin ligase. Hypoxia inducible factor (HIF)-1 has been identified as a target for the VCB-Cul2 ubiquitin ligase and its participation in the regulation of VEGF expression has been reported. However, a variety of cellular defects caused by the depletion of pVHL, explained solely by the ubiquitin-mediated degradation of HIF. We show here that a member of the atypical protein kinase C (PKC) group, PKCλ, is ubiquitinated by the pVHL-containing E3 enzyme. An active PKCλ mutant is ubiquitinated more extensively than wild-type PKCλ in HEK293 cells, and the ubiquitination is further enhanced by the overexpression of pVHL. Activation of wild-type PKCλ by serum stimulation of cells enhances the ubiquitination of the protein. The existence of regulatory domain of PKCλ is necessary for this ubiquitination. In addition, expression of mutant pVHL, which lacks the C-terminal elongin binding domain, dominant negatively represses the ubiquitination of the constitutively active PKCλ mutant and the ubiquitination of wild-type PKCλ activated by serum stimulation. These results support the notion that active PKCλ is preferentially ubiquitinated by VCB-Cul2 ubiquitin ligase. Taken together with known function of aPKC in the ligase. Taken together with known function of aPKC in the regulation of cell polarity and cell growth, our results suggest that loss of function of pVHL to control aPKC activity may result in tumorigenesis of epithelial cells.