Identification of protein instability determinants in the carboxy-terminalregion of c-myb removed as a result of retroviral integration in murine monocytic leukemias

The c-myb oncogene has been a target of retroviral insertional mutagenesis in murine monocytic leukemias. One mechanism by which c-myb can be activate d is through the integration of a retroviral provirus into the central port ion 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 app roximately a fourfold-increased half-life compared to the normal c-Myb prot ein, due to its ability to escape rapid degradation by the ubiquitin-26S pr oteasome pathway. Here we provide evidence for the existence of more than o ne instability determinant in the carboxy-terminal region of the wild-type protein, which appear to act independently of each other. The data were der ived from examination of premature termination mutants and deletion mutants of the normal protein, as well as analysis of another carboxy-terminally t runcated protein expressed in leukemia. Evidence is provided that one insta bility 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 leu cine zipper homology, In leukemias, different degrees of protein stability are attained following proviral integration depending upon how many determi nants 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 protei n turnover. This study provides further insight into how inappropriate expr ession of c-Myb may contribute to leukemogenesis. In addition, it will faci litate further studies aimed at characterizing the specific role of individ ual regions of the normal protein in targeting to the 26S proteasome.