Fbw7 promotes ubiquitin-dependent degradation of c-Myb: involvement of GSK3-mediated phosphorylation of Thr-572 in mouse c-Myb.
Expression of oncoprotein c-Myb oscillates during hematopoiesis and hematological malignancies. Its quantity is not only regulated through transcriptional control but also through the ubiquitin-proteasome pathway, accompanied by phosphorylation, although the mechanisms are poorly understood. In this report, we tried to identify an E3 ubiquitin ligase, which targets c-Myb for ubiquitin-dependent degradation. We found that an F-box protein, Fbw7, interacted with c-Myb, which is mutated in numerous cancers. Fbw7 facilitated ubiquitylation and degradation of c-Myb in intact cells. Moreover, depletion of Fbw7 by RNA interference delayed turnover and increased the abundance of c-Myb in myeloid leukemia cells concomitantly, and suppressed the transcriptional level of gamma-globin, which receives transcriptional repression from c-Myb. In addition, we analysed sites required for both ubiquitylation and degradation of c-Myb. We found that Thr-572 is critical for Fbw7-mediated ubiquitylation in mouse c-Myb using site-directed mutagenesis. Fbw7 recognized the phosphorylation of Thr-572, which was mediated by glycogen synthase kinase 3 (GSK3). In consequence, the c-Myb protein was markedly stabilized by the substitution of Thr-572 to Ala. These observations suggest that SCF(Fbw7) ubiquitin ligase regulates phosphorylation-dependent degradation of c-Myb protein.
Kitagawa
K
K
Department of Biochemistry 1, Hamamatsu University School of Medicine, Higashi-ku, Hamamatsu, Shizuoka, Japan. kitamasa@hama-med.ac.jp
Hiramatsu
Y
Y
Uchida
C
C
Isobe
T
T
Hattori
T
T
Oda
T
T
Shibata
K
K
Nakamura
S
S
Kikuchi
A
A
Kitagawa
M
M
D000818:Animals; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D044783:F-Box Proteins / Q000235:genetics / Q000378:metabolism*; D000073979:F-Box-WD Repeat-Containing Protein 7; D038362:Glycogen Synthase Kinase 3 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D020598:Proto-Oncogene Proteins c-myb / Q000378:metabolism*; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051881:Receptor, Notch1 / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013912:Threonine / Q000235:genetics / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
D018797:Cell Cycle Proteins; D044783:F-Box Proteins; D000073979:F-Box-WD Repeat-Containing Protein 7; C513273:FBXW7 protein, human; C489428:Myc protein, mouse; C497762:Notch1 protein, mouse; D020598:Proto-Oncogene Proteins c-myb; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D051881:Receptor, Notch1; D025801:Ubiquitin; D013912:Threonine; D044767:Ubiquitin-Protein Ligases; D038362:Glycogen Synthase Kinase 3