Fbw7 promotes ubiquitin-dependent degradation of c-Myb: involvement of GSK3-mediated phosphorylation of Thr-572 in mouse c-Myb

Abstract

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 g-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 sitedirected 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.

Adenovirus E1A Inhibits SCFFbw7 Ubiquitin Ligase

“…However, the ubiquitin ligase that targets E1A for ubiquitin-dependent degradation has not been identified. We noticed that there was a possible Cdc4 phosphodegron sequence (11,19) within the 13S E1A gene product but not within the 12S product (aa residues 183-191 of 13S E1A). Fbw7/Cdc4 recognizes the Cdc4 phosphodegron and binds to the substrates via a WD40 repeat domain.…”

“…Plasmids-Mammalian expression plasmids for full-length E1A, full-length Fbw7, and its deletion mutants, Skp2, Fbw1, c-Myb, ubiquitin, cyclin E, c-Myc, GSK3␤, IB␣, and IKK were described in previous reports (9,11,(32)(33)(34). Expression plasmids for Roc1/Rbx1 and its deletion mutants, CUL1-5, and CUL1 deletion mutants were kindly provided by Drs.…”

“…Thus, we supposed that the ubiquitylation activity of the SCF Fbw7 complex is affected by E1A. To test this hypothesis, we assessed the effect of E1A on c-Myb ubiquitylation, which is mediated by the SCF Fbw7 complex and then followed by proteasomal degradation (11,12) in mammalian cells (in vivo ubiquitylation assay). Myc epitope-tagged c-Myb (c-Myb-Myc), FLAG-ubiquitin, and HA-Fbw7 expression plasmids were cotransfected into HEK293 cells with or without a His 6 -Xpresstagged E1A (hereafter referred to as Xpress-E1A) expression plasmid.…”

“…Fbw7 (F-box and WD repeat domain-containing 7, also known as Fbxw7, Sel-10, hCdc4, or hAgo) is an F-box protein that binds to and ubiquitylates some key regulators of cell growth, including cyclin E (8), c-Myc (9, 10), c-Myb (11,12), c-Jun (13), and Notch (14). Given that Fbw7 is responsible for degradation of several protooncogene products, Fbw7 can be considered to be a tumor suppressor.…”

MYB IS Required for B-Selection During Thymopoises

“…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].…”