Differential Sensitivity of v-Myb and c-Myb to Wnt-1-induced Protein Degradation

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 (TGF- b 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 (AMV) 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 (DBD) of v-Myb than to that of c-Myb. The mutations of three hydrophobic amino acids on the surface of the DBD in v-Myb decrease the affinity to HIPK2. Secondly, a loss of multiple NLK phosphorylation sites by truncation of the C-terminal region of c-Myb increases its stability. Among fifteen 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.


INTRODUCTION
The v-myb oncogene, which is carried by the avian myeloblastosis virus (AMV), and causes acute myeloblastic leukemia, is derived from the c-myb protooncogene (1). Studies of c-myb-deficient mice showed that c-myb is essential for the proliferation of immature hematopoietic cells and early T cell development (2,3). The c-myb gene product (c-Myb) is a transcriptional activator that recognizes a specific DNA sequence (4)(5)(6)(7). Multiple c-Myb target genes, including c-myc, have been identified, and they are involved in cell cycle control, lineage commitment during differentiation, and blockage of apoptosis (8)(9)(10)(11)(12). Three functional domains were identified in c-Myb that are responsible for DNA binding, transcriptional activation, and negative regulation (5). The DNA-binding domain (DBD) in the N-terminal region of c-Myb consists of three imperfect tandem repeats of 51-52 amino acids, each containing a helix-turn-helix variation motif (13). The transcriptional coactivator cAMP responsive element binding protein (CREB) binding protein (CBP) binds to the transcriptional activation domain to mediate trans-activation (14).
The v-myb gene product (v-Myb) encoded by AMV is an amino (N)-and carboxy (C)-terminally truncated version of chicken c-Myb (1) and has a strong transformation activity in hematopoietic cells, whereas c-Myb has not. This difference is at least partly due to a deletion of the negative regulatory domain (NRD) located in subdomains, and the deletion of any of these increases both the trans-activation and transformation capacity of c-Myb (18,19). The v-Myb encoded by AMV lacks the Cproximal region of the NRD. Mutations of the leucine-rich region in the NRD are sufficient for oncogenic activation of c-myb. Although the mechanism by which c-Myb is regulated by its NRD has not been completely clarified, several possibilities have been suggested. First, deletion or mutations of the NRD reduces the affinity for corepressors, leading to increased c-Myb activity (20,21). Two corepressors, BS69 and TIF1β, bind directly to the NRD and negatively regulate c-Myb-mediated transactivation. Since other multiple corepressors, including Ski, N-CoR, and mSin3A, also bind to the DBD together with TIF1β, a deletion of NRD also reduces interactions with these corepressors. Further, three point mutations in the DBD of v-Myb reduce the affinity for these corepressors. Second, the C-truncated form of c-Myb is more resistant to proteasome-dependent protein degradation compared to c-Myb (22). However, the mechanism by which c-Myb is stabilized by its C-terminal truncation remains unknown.
signaling controls differentiation or apoptosis in many cell types, including hematopoietic cells (24,25), the Wnt-induced c-Myb degradation may play some role in the proliferation and differentiation of hematopoietic cells. Here, we report that v-Myb is relatively resistant to Wnt-1-induced protein degradation.

MATERIALS AND METHODS
Plasmids ---The chicken cytoplasmic β-actin promoter was used to express various forms of Myb in CV-1 cells. N-and C-terminally truncated forms of Myb (NT2-V, V-CTV, and NT2-CTV) with FLAG tag at their C-termini were expressed from the chicken cytoplasmic β-actin promoter. The pact-v-Myb-FLAG plasmid to express v-Myb-FLAG, in which the FLAG tag is linked to the C-terminus of v-Myb, was constructed. Plasmids to express various components of the Wnt-1-TAK-HIPK2-NLK pathway have been described previously (23). GST Pull-down Assays and Yeast Two-hybrid Assay ---GST pull-down assays were performed as described (23). To increase the solubility of GST fusion proteins expressed in bacteria, the thioredoxin coexpression system (26)  were only partially degraded by coexpression of Wnt-1 (Fig. 3B). In addition, NT2-CTV, which was generated by truncation of both N-and C-terminal regions of c-Myb, was also apparently resistant to Wnt-1-induced degradation (Fig. 3B). Consistent with these results, Wnt-1 did not inhibit trans-activation of the c-myc promoter mediated by NT2-V, V-CTV, or NT2-CTV (Fig. 3C). Thus, truncation of either the N-or Cterminal region of c-Myb confers resistance to Wnt-1-induced protein degradation.

Point Mutations in the DBD of v-Myb Weaken Wnt-1-induced Negative
Regulation ---In addition to the truncation, we also found that point mutations in v-Myb contribute to the relative resistance to Wnt-1-induced degradation. Three hydrophobic amino acids on the surface of repeat 2 in the DBD of c-Myb, which are changed to non-hydrophobic amino acids in v-Myb ( Fig. 4A) (27,28), are critical for interactions with the transcription factor C/EBPβ on the target promoter (29) and with corepressors (21). Therefore, we investigated whether the change of these three amino acids has any effect on Wnt-1-induced protein degradation. The c-Myb-3M mutant, in which these three amino acids were mutated to non-hydrophobic amino acids as in v-Myb, were relatively resistant to Wnt-1-induced protein degradation (Fig. 4B) http://www.jbc.org/ Downloaded from gave rise to higher levels of lacZ than the mutant R23 fusion (Fig. 5B). The expression levels of LexA-wild-type R23 was about half that of the mutant R23 fusion (Fig. 5C,   left). Therefore, the affinity of one molecule of mutant R23 with HIPK2 was estimated to be about one fourth that of wild-type R23 (Fig. 5C, right). Thus, the three point mutations in the DBD of v-Myb decrease the affinity for HIPK2.

The C-proximal NLK Phosphorylation Sites of c-Myb Are Critical for NLK-
induced Protein Degradation ---The results of Figure 3 suggested that a truncation of the N-or C-terminal region of c-Myb confers resistance to Wnt-1-induced protein degradation. The C-terminal half of c-Myb contains the multiple NLK phosphorylation sites, whereas the N-terminal region does not, suggesting that the removal of multiple NLK phosphorylation sites by a truncation of the C-terminal region may be responsible for the reduced sensitivity to Wnt-1 induced protein degradation. Mouse c-Myb contains fifteen putative NLK phosphorylation sites, and replacement of all fifteen of these Ser or Thr residues by Ala (15A mutant) completely blocked Wnt-1-induced protein degradation (23). To examine the role of NLK phosphorylation sites in the Cterminal region, we introduced Ala mutations in these sites (Fig. 6A). Ala mutations at the C-terminal two sites alone (C2A) did not confer complete resistance to Wnt-1induced protein degradation, although this mutant was slightly resistant compared to wild-type c-Myb. However, mutants in which three or nine sites in the C-terminal  (Fig. 6AB).
There are 12 Ser/Thr residues that are conserved between chicken and mouse c-Myb, but three residues (208T, 227S, and 233S) are changed in chicken c-Myb (Fig.   6A). We speculated that loss of these three NLK phosphorylation sites in chicken c-Myb may affect its sensitivity to Wnt-1-induced protein degradation. To investigate this, we generated a mouse c-Myb mutant in which the three residues at 208, 227, and 233 were replaced by Ala (N3A). However, N3A was degraded by Wnt-1 and NLK (Fig. 6B). This is consistent with the results that chicken and mouse c-Myb have similar sensitivities to Wnt-1-induced protein degradation (Fig. 1A). Introducing Ala mutations into the C-terminal three or nine sites of the N3A mutant (N3A+C3A or N3A+C9A) conferred resistance to Wnt-1 and NLK-induced protein degradation.
We also examined the role of the N-proximal NLK phosphorylation sites.
Introducing Ala mutations into the N-terminal five or seven sites (N5A and N7A) changed the sensitivity to Wnt-1-induced protein degradation, but not to the NLKinduced protein degradation (Fig. 6B). Thus, the NLK phosphorylation sites in the Cterminal region are more important for Wnt-1-induced protein degradation than the Nproximal phosphorylation sites. corepressor Ski and C/EBPβ (21,29). HIPK2 and Ski negatively regulate the c-Mybdependent trans-activation of the c-myc promoter by inducing protein degradation and by recruiting the HDAC complex, respectively (23,21). These data indicate that v-Myb can escape both of these types of negative regulation because it possesses these point mutations. c-Myb and C/EBPβ , through its interaction with the three hydrophobic amino acids in repeat 2 of the DBD of c-Myb, synergistically activates transcription of mim-1, which encodes one of the hematopoietic differentiation marker (31,29). v-Myb cannot activate the mim-1 gene due to mutation of these amino acids, suggesting that the expression of differentiation markers, but not proliferation control genes, may be