Abstract
Backgroundc-Myb is expressed at high levels in immature progenitors of all the hematopoietic lineages. It is associated with the regulation of proliferation, differentiation and survival of erythroid, myeloid and lymphoid cells, but decreases during the terminal differentiation to mature blood cells. The cellular level of c-Myb is controlled by not only transcriptional regulation but also ubiquitin-dependent proteolysis. We recently reported that mouse c-Myb protein is controlled by ubiquitin-dependent degradation by SCF-Fbw7 E3 ligase via glycogen synthase kinase 3 (GSK3)-mediated phosphorylation of Thr-572 in a Cdc4 phosphodegron (CPD)-dependent manner. However, this critical threonine residue is not conserved in human c-Myb. In this study, we investigated whether GSK3 is involved in the regulatory mechanism for human c-Myb expression.ResultsHuman c-Myb was degraded by ubiquitin-dependent degradation via SCF-Fbw7. Human Fbw7 ubiquitylated not only human c-Myb but also mouse c-Myb, whereas mouse Fbw7 ubiquitylated mouse c-Myb but not human c-Myb. Human Fbw7 mutants with mutations of arginine residues important for recognition of the CPD still ubiquitylated human c-Myb. These data strongly suggest that human Fbw7 ubiquitylates human c-Myb in a CPD-independent manner. Mutations of the putative GSK3 phosphorylation sites in human c-Myb did not affect the Fbw7-dependent ubiquitylation of human c-Myb. Neither chemical inhibitors nor a siRNA for GSK3β affected the stability of human c-Myb. However, depletion of GSK3β upregulated the transcription of human c-Myb, resulting in transcriptional suppression of γ-globin, one of the c-Myb target genes.ConclusionsThe present observations suggest that human Fbw7 ubiquitylates human c-Myb in a CPD-independent manner, whereas mouse Fbw7 ubiquitylates human c-Myb in a CPD-dependent manner. Moreover, GSK3 negatively regulates the transcriptional expression of human c-Myb but does not promote Fbw7-dependent degradation of human c-Myb protein. Inactivation of GSK3 as well as mutations of Fbw7 may be causes of the enhanced c-Myb expression observed in leukemia cells. We conclude that expression levels of human and mouse c-Myb are regulated via different mechanisms.
Highlights
The leucine zipper transcription factor c-Myb is expressed at high levels in immature progenitors of all the hematopoietic lineages, and is essential for fetal liver hematopoiesis, erythroid and myeloid bone marrow colony formation, and T- and B-cell development [1,2,3,4].elevated c-Myb expression is associated with hematological malignancies and has been reported in many cases of acute myeloblastic and lymphoblasticDepartment of Biochemistry 1, Hamamatsu University School of Medicine, Hamamatsu, JapanFull list of author information is available at the end of the article leukemias [1,5,6,7]
glycogen synthase kinase 3 (GSK3) negatively regulates the transcriptional expression of human c-Myb but does not promote Fbw7-dependent degradation of human c-Myb protein
Inactivation of GSK3 as well as mutations of Fbw7 may be causes of the enhanced c-Myb expression observed in leukemia cells
Summary
The leucine zipper transcription factor c-Myb is expressed at high levels in immature progenitors of all the hematopoietic lineages, and is essential for fetal liver hematopoiesis, erythroid and myeloid bone marrow colony formation, and T- and B-cell development [1,2,3,4].elevated c-Myb expression is associated with hematological malignancies and has been reported in many cases of acute myeloblastic and lymphoblasticFull list of author information is available at the end of the article leukemias [1,5,6,7]. The keys to the control of c-Myb protein function are post-transcriptional modifications. The c-Myb protein is phosphorylated by several kinases such as MAPK, Nemo-like kinase (NLK) and glycogen synthase kinase 3 (GSK3) [8,9,10]. It has been reported that phosphorylation influences the activity and stability of the c-Myb protein [11,12,13,14,15,16,17]. The stabilities of many kinds of cellular proteins are often controlled by the ubiquitin proteasome system, a rapid and selective degradation mechanism in cells [18]. A previous study indicated that the stability of c-Myb protein is regulated by this system. SCF-type E3 ubiquitin ligases target various important cellular proteins
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