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Abstract
Recently we have shown that the c-myb proto-oncogene product (c-Myb) is degraded in response to Wnt-1 signaling via the pathway involving TAK1 (transforming growth factor-beta-activated kinase), HIPK2 (homeodomain-interacting protein kinase 2), and NLK (Nemo-like kinase). NLK and HIPK2 bind directly to c-Myb, which results in the phosphorylation of c-Myb at multiple sites, followed by its ubiquitination and proteasome-dependent degradation. The v-myb gene carried by avian myeloblastosis virus has a transforming capacity, but the c-myb proto-oncogene does not. Here, we report that two characteristics of v-Myb make it relatively resistant to Wnt-1-induced protein degradation. First, HIPK2 binds with a lower affinity to the DNA-binding domain of v-Myb than to that of c-Myb. The mutations of three hydrophobic amino acids on the surface of the DNA-binding domain in v-Myb decrease the affinity to HIPK2. Second, a loss of multiple NLK phosphorylation sites by truncation of the C-terminal region of c-Myb increases its stability. Among 15 putative NLK phosphorylation sites in mouse c-Myb, the phosphorylation sites in the C-terminal region are more critical than other sites for Wnt-1-induced protein degradation. The relative resistance of v-Myb to Wnt-1-induced degradation may explain, at least in part, the differential transforming capacity of v-Myb versus c-Myb.
Highlights
We have demonstrated that avian myeloblastosis virus (AMV)-encoded v-myb gene product (v-Myb) is relatively resistant to Wnt-1-induced protein degradation
It is possible that resistance of v-Myb to Wnt-mediated protein degradation partially contributes to leukemogenesis
Mutation of the three hydrophobic amino acids on the surface of repeat 2 of v-Myb reduces the affinity for HIPK2
Summary
Plasmids—The chicken cytoplasmic -actin promoter was used to express various forms of Myb in CV-1 cells. CV-1 cells were transfected with a plasmid to express mouse or chicken c-Myb, or AMV v-Myb, and an internal control plasmid pact--gal with (ϩ or ϩϩ) or without (Ϫ) a Wnt-1 (ϩ, 0.5 g; ϩϩ, 2.0 g) or NLK (ϩ, 0.3 g) expression plasmid. Western Blotting—CV-1 cells were transfected with a mixture of the c-Myb expression plasmid (6 g), various amounts of the plasmids that express components of the Wnt-1-TAK1-HIPK2-NLK pathway CAT Reporter Assays—Using the CaPO4 method, CV-1 cells (4 ϫ 105 cells per 100-mm dish) were transfected with a mixture of the pc-mycCAT reporter containing the human c-myc promoter [8] (4 g), plasmids expressing various forms of Myb (2 g), plasmids expressing various components of the Wnt-1-TAK1-HIPK2-NLK pathway Yeast two-hybrid assays were performed as described [14]
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