Multimerization via its myosin domain facilitates nuclear localization and inhibition of core binding factor (CBF) activities by the CBFbeta-smooth muscle myosin heavy chain myeloid leukemia oncoprotein.
In CBFbeta-SMMHC, core binding factor beta (CBFbeta) is fused to the alpha-helical rod domain of smooth muscle myosin heavy chain (SMMHC). We generated Ba/F3 hematopoietic cells expressing a CBFbeta-SMMHC variant lacking 28 amino acids homologous to the assembly competence domain (ACD) required for multimerization of skeletal muscle myosin. CBFbeta-SMMHC(DeltaACD) multimerized less effectively than either wild-type protein or a variant lacking a different 28-residue segment. In contrast to the control proteins, the DeltaACD mutant did not inhibit CBF DNA binding, AML1-mediated reporter activation, or G(1) to S cell cycle progression, the last being dependent upon activation of CBF-regulated genes. We also linked the CBFbeta domain to 149 or 83 C-terminal CBFbeta-SMMHC residues, retaining 86 or 20 amino acids N-terminal to the ACD. CBFbeta-SMMHC(149C) multimerized and slowed Ba/F3 proliferation, whereas CBFbeta-SMMHC(83C) did not. The majority of CBFbeta-SMMHC and CBFbeta-SMMHC(149C) was detected in the nucleus, whereas the DeltaACD and 83C variants were predominantly cytoplasmic, indicating that multimerization facilitates nuclear retention of CBFbeta-SMMHC. When linked to the simian virus 40 nuclear localization signal (NLS), a significant fraction of CBFbeta-SMMHC(DeltaACD) entered the nucleus but only mildly inhibited CBF activities. As NLS-CBFbeta-SMMHC(83C) remained cytoplasmic, we directed the ACD to CBF target genes by linking it to the AML1 DNA binding domain or to full-length AML1. These AML1-ACD fusion proteins did not affect Ba/F3 proliferation, in contrast to AML1-ETO, which markedly slowed G(1) to S progression dependent upon the integrity of its DNA-binding domain. Thus, the ACD facilitates inhibition of CBF by mediating multimerization of CBFbeta-SMMHC in the nucleus. Therapeutics targeting the ACD may be effective in acute myeloid leukemia cases associated with CBFbeta-SMMHC expression.
Kummalue
Tanawan
T
Division of Pediatric Oncology, Johns Hopkins University, Baltimore, Maryland.
Lou
Jianrong
J
Friedman
Alan D
AD
D021581:Active Transport, Cell Nucleus / Q000502:physiology*; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter; D006801:Humans; D007951:Leukemia, Myeloid / Q000378:metabolism*; D018995:Myosin Heavy Chains / Q000235:genetics / Q000378:metabolism*; D019913:Nuclear Localization Signals / Q000235:genetics / Q000378:metabolism; D011108:Polymers; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D024745:Smooth Muscle Myosins / Q000235:genetics / Q000378:metabolism*; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D019913:Nuclear Localization Signals; D011108:Polymers; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D050656:Transcription Factor AP-2; D014157:Transcription Factors; D024745:Smooth Muscle Myosins; D018995:Myosin Heavy Chains