A-myb is expressed in bovine vascular smooth muscle cells during the late G1-to-S phase transition and cooperates with c-myc to mediate progression to S phase.
The Myb family of transcription factors is defined by homology within the DNA binding domain and includes c-Myb, A-Myb, and B-Myb. The protein products of the myb genes all bind the Myb-binding site (MBS) [YG(A/G)C(A/C/G)GTT(G/A)]. A-myb has been found to display a limited pattern of expression. Here we report that bovine aortic smooth muscle cells (SMCs) express A-myb. Sequence analysis of isolated bovine A-myb cDNA clones spanning the entire coding region indicated extensive homology with the human gene, including the putative transactivation domain. Expression of A-myb was cell cycle dependent; levels of A-myb RNA increased in the late G1-to-S phase transition following serum stimulation of serum-deprived quiescent SMC cultures and peaked in S phase. Nuclear run-on analysis revealed that an increased rate of transcription can account for most of the increase in A-myb RNA levels. Treatment of SMC cultures with 5,6-dichlorobenzimidazole riboside, a selective inhibitor of RNA polymerase II, indicated an approximate 4-h half-life for A-myb mRNA during the S phase of the cell cycle. Expression of A-myb by SMCs was stimulated by basic fibroblast growth factor, in a cell density-dependent fashion. Cotransfection of a human A-myb expression vector activated a multimerized MBS element-driven reporter construct approximately 30-fold in SMCs. The activity of c-myb and c-myc promoters, which both contain multiple MBS elements, were similarly transactivated, approximately 30- and 50-fold, respectively, upon cotransfection with human A-myb. Lastly, A-myb RNA levels could be increased by a combination of phorbol ester plus insulin-like growth factor 1. To test the role of myb family members in progression through the cell cycle, we comicroinjected c-myc and myb expression vectors into serum-deprived quiescent SMCs. The combination of c-myc and either A-myb or c-myb but not B-myb synergistically led to entry into S phase, whereas microinjection of any vector alone had little effect on S phase entry. Thus, these results suggest that A-myb is a potent transactivator in bovine SMCs and that its expression induces progression into S phase of the cell cycle.
Marhamati
D J
DJ
Department of Biochemistry, Boston University School of Medicine, Massachusetts 02118, USA.
Bellas
R E
RE
Arsura
M
M
Kypreos
K E
KE
Sonenshein
G E
GE
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D018076:DNA, Complementary / Q000737:chemistry / Q000302:isolation & purification; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D016193:G1 Phase; D006801:Humans; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D016196:S Phase; D015534:Trans-Activators / Q000378:metabolism*
D018076:DNA, Complementary; C088831:MYBL1 protein, human; D011518:Proto-Oncogene Proteins; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D015534:Trans-Activators; D016222:Fibroblast Growth Factor 2