Identification of Protein Instability Determinants in the Carboxy-Terminal Region of c-Myb Removed as a Result of Retroviral Integration in Murine Monocytic Leukemias

Abstract

The c-myb oncogene has been a target of retroviral insertional mutagenesis in murine monocytic leukemias. One mechanism by which c-myb can be activated is through the integration of a retroviral provirus into the central portion of the locus, causing premature termination of c-myb transcription and translation. We had previously shown that a leukemia-specific c-Myb protein, truncated at the site of proviral integration by 248 amino acids, had approximately a fourfold-increased half-life compared to the normal c-Myb protein, due to its ability to escape rapid degradation by the ubiquitin-26S proteasome pathway. Here we provide evidence for the existence of more than one instability determinant in the carboxy-terminal region of the wild-type protein, which appear to act independently of each other. The data were derived from examination of premature termination mutants and deletion mutants of the normal protein, as well as analysis of another carboxy-terminally truncated protein expressed in leukemia. Evidence is provided that one instability determinant is located in the terminal 87 amino acids of the protein and another is located in the vicinity of the internal region that has leucine zipper homology. In leukemias, different degrees of protein stability are attained following proviral integration depending upon how many determinants are removed. Interestingly, although PEST sequences (rich in proline, glutamine, serine, and threonine), often associated with degradation, are found in c-Myb, deletion of PEST-containing regions had no effect on protein turnover. This study provides further insight into how inappropriate expression of c-Myb may contribute to leukemogenesis. In addition, it will facilitate further studies aimed at characterizing the specific role of individual regions of the normal protein in targeting to the 26S proteasome.

Functional Analysis of Carboxy-Terminal Deletion Mutants of c-Myb

“…The biological function of BS69 is not clear at this point, but the protein was first identified as a transcriptional repressor that interacts with the adenoviral E1A protein and contains a number of conserved protein domains found in other transcriptional regulators. Finally, although the BN subregion also contains a predicted PEST domain, this region of c-Myb has not been implicated in the regulation of c-Myb protein stability (6). Consistent with this, our immunoblotting analysis shows no evidence of increased protein levels of CCC-dBN and CCC-dPSBN.…”

Nucleic Acid Therapeutics for Hematologic Malignancies—Theoretical Considerations

“…Our experience with targeting the obligate hematopoietic transcription factor c-myb, whose mRNA and protein have very short half-lives of ∼30-60 min each, 56 and which is required for G1/S transition, as well activation of other critical cellular genes, 53,55,[57][58][59][60][61] would seem to be a good example.…”

Hyperphosphorylation and increased proteolytic breakdown of c-Myb induced by the inhibition of Ser/Thr protein phosphatases

“…To address this question, we compared the half-lives of wild-type (wt) protein with that of mutants where Ser 11, 12 or 528 had been replaced with alanine to prevent phosphorylation (FL-Myb(S11,12A); FL-Myb(S528A)). We also included in this experiment a COOH-terminally truncated form of c-Myb CT6-MybD(266aa) (Bies et al, 1999), which is missing the leucine zipper and negative regulatory domain and resembles the stable oncogenic form of c-Myb expressed in leukemia (Bies and Wol, 1997). For these experiments we used COS7 cells, because the protein can be transiently expressed at high levels and c-Myb is degraded by the 26S proteasome in these cells at a rate similar to that observed in hematopoietic cells (Bies et al, 1999).…”

“…We also included in this experiment a COOH-terminally truncated form of c-Myb CT6-MybD(266aa) (Bies et al, 1999), which is missing the leucine zipper and negative regulatory domain and resembles the stable oncogenic form of c-Myb expressed in leukemia (Bies and Wol, 1997). For these experiments we used COS7 cells, because the protein can be transiently expressed at high levels and c-Myb is degraded by the 26S proteasome in these cells at a rate similar to that observed in hematopoietic cells (Bies et al, 1999). Exponentially growing COS7 cells, transiently transfected with plasmid DNAs containing wt c-Myb and mutants, were metabolically labeled 36 h after transfection and chased in complete medium either in the presence or the absence of OA.…”

“…The steady state level of any protein is the result of the balance between the rates of its production and degradation. We previously showed that c-Myb is degraded by the 26S proteasome in hematopoietic cells, and that the region responsible for its very short halflife is located in the COOH-terminus (Bies and Wol, 1997;Bies et al, 1999). In the present study, we looked at the eect of phosphorylation on the proteolytic processing of the protein by examining its turnover in cells treated with okadaic acid (OA), an inhibitor of cellular Ser/Thr protein phosphatases.…”