Oncogenic activation of c-Myb by carboxyl-terminal truncation leads to decreased proteolysis by the ubiquitin-26S proteasome pathway

Abstract

c-myb activation by insertional mutagenesis in murine myeloid leukemias can lead to amino (NH 2 )-terminal or carboxyl (COOH)-terminal truncation of its protein product. We observed that in these leukemias, the steady state level of the protein truncated at the COOH terminus was remarkably higher than that of the protein truncated at the NH 2 -terminus or full length wild-type protein. To examine the rate of proteolysis of di erent forms of Myb in a uniform cellular background, the proteins were constitutively expressed in the myeloblast cell line M1, using the retrovirus vector LXSN. In pulse chase experiments, using metabolically 35 S-labeled proteins, it was determined that COOH-terminal truncation of c-Myb by 248 aa (CT-c-Myb) substantially increases protein stability, resulting in a t 1/2 of about 140 min, as compared to 50 min for full length c-Myb (FL-c-Myb). In an investigation of the mechanism involved in the in vivo degradation of this short lived transcription factor, inhibitors of the lysosomal (chloroquine), proteasomal (ALLM, ALLN, lactacystin) and calpains (EGTA, E64d, BAPTA/AM) pathways were utilized. Results of this experiment identi®ed the 26S proteasome as a major pathway responsible for rapid breakdown of the protein in hematopoietic cells. Further experiments carried out in vitro demonstrated that c-Myb can be ubiquitinated, suggesting that this process may be involved in the targeting of wild-type c-Myb to degradation by the 26S proteasome. In addition, it was demonstrated that CT-cMyb was less e ciently ubiquitinated than wild-type protein indicating that defects in modi®cation account for its escape from rapid turnover. We speculate that the increased half-life of c-Myb resulting from truncation could contribute to its transforming potential.

c-Myb acetylation at the carboxyl-terminal conserved domain by transcriptional co-activator p300

“…Several phosphorylation sites have been demonstrated in the N-and C-termini of c-Myb, and some of these sites seem to be functionally important (Aziz et al, 1995;Miglarese et al, 1996). Furthermore, the deletion of the C-terminal region including PEST sequences increased the protein stability (Bies and Wol , 1997). These ®ndings suggest that posttranslational modi®ca-tion of the C-terminus, such as phosphorylation or degradation, plays a critical role in the regulation of cMyb function.…”

Mutations in Multiple Domains of c-Myb Disrupt Interaction with CBP/p300 and Abrogate Myeloid Transforming Ability

“…16,21). Although the mechanism of negative regulation by the NRD is not fully understood, it is likely to act through multiple mechanisms including interaction with other proteins (22)(23)(24), intramolecular interactions (25), and effects on protein stability (26).…”

MYB IS Required for B-Selection During Thymopoises

“…c-Myb is also regulated by post-transcriptional modifications including sumoylation, phosphorylation, ubiquitination, and acetylation [177][178][179][180][181][182][183]. These modifications lead to changes in c-Myb stability, DNA-binding activity and transcriptional transactivating activity.…”

“…In addition, it was recently reported that phosphorylation of Thr354, Thr486, Ser556, and Thr572 in the NRD by p38MAPK in response to stress led to proteasomal degradation of c-Myb [183]. The proteasome is targeted to the NRD, through ubiquitination which occurs at an undefined lysine residue(s) in the NRD [180,185]. Thr572 is also targeted for phosphorylation by glycogen synthase kinase 3 (GSK3) which recruits E3 ubiquitin ligase Fbw7 leading to degradation of c-Myb [186].…”

“…Two recent reports demonstrated that Myb mRNA is a target of microRNA miR-150 [220], which is expressed in mature resting lymphocytes [221], and miR-15a [222]. The c-Myb protein also has a short half-life of approximately 20-30 minutes in vivo [180,185,223]. As discussed in the previous section, c-Myb is a target for many forms of post-translational protein modifications including phosphorylation, sumoylation and ubiquitination which target it for proteosomal degradation [177,[180][181][182][183][184]189].…”