Phosphorylation-dependent conformation and proteolytic stability of c-Myb.
The c-Myb oncoprotein is a critical regulator of hematopoietic cell proliferation and differentiation. Normal c-Myb is rapidly degraded by the ubiquitin-26S proteasome pathway, and instability determinants have been localized within the negative regulatory domain in the carboxyl terminus. Our recent work has shown that, in myeloid cells, inhibition of cellular Ser/Thr protein phosphatases with okadaic acid (OA) causes a rapid increase in c-Myb phosphorylation and 26S proteasome-dependent breakdown [J. Bies, S. Feikova, D. P. Bottaro, and L. Wolff (2000) Oncogene 19, 2846-2854]. Furthermore, phosphoamino acid analyses revealed that the increase in phosphorylation was mainly on threonine residues. Here we investigated the ability of c-Myb to bind DNA following phosphorylation. Our results suggest that the hyperphosphorylated form of c-Myb binds to DNA with affinity very similar to the hypophosphorylated form. Therefore, the increased proteolytic instability of the former cannot be explained by a difference in DNA-binding capacity. Conformational changes in the carboxyl terminus were proposed previously to be a consequence of phosphorylation because we observed phosphorylation-induced alterations in gel electrophoresis mobilities and alterations in recognition by specific monoclonal antibodies. Further support for this notion has come from this study, in which we have detected new degradation products in electrophoretic mobility shift assays, as well as an increased rate of in vitro proteolysis, following OA treatment. We speculate that these alterations in the conformation of the negative regulatory domain expose epitopes on the surface of c-Myb, which in turn can serve as recognition signal(s) for ubiquitin-26S proteasome proteolytic machinery.
Bies
J
J
Laboratory of Molecular Virology, Slovak Academy of Sciences, Bratislava, 833 92, Slovakia. exonbiex@savba.sk
Feiková
S
S
Markus
J
J
Wolff
L
L
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011487:Protein Conformation; D020598:Proto-Oncogene Proteins c-myb / Q000737:chemistry* / Q000378:metabolism; D016415:Sequence Alignment
D020598:Proto-Oncogene Proteins c-myb