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Molecular and Cellular Biology, December 2002, p. 8278-8291, Vol. 22, No. 23
0270-7306/02/$04.00+0     DOI: 10.1128/MCB.22.23.8278-8291.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Multimerization via Its Myosin Domain Facilitates Nuclear Localization and Inhibition of Core Binding Factor (CBF) Activities by the CBFß-Smooth Muscle Myosin Heavy Chain Myeloid Leukemia Oncoprotein

Division of Pediatric Oncology, Johns Hopkins University, Baltimore, Maryland

Received 30 May 2002/ Returned for modification 19 August 2002/ Accepted 26 August 2002

In CBFß-SMMHC, core binding factor beta (CBFß) is fused to the -helical rod domain of smooth muscle myosin heavy chain (SMMHC). We generated Ba/F3 hematopoietic cells expressing a CBFß-SMMHC variant lacking 28 amino acids homologous to the assembly competence domain (ACD) required for multimerization of skeletal muscle myosin. CBFß-SMMHC(ACD) multimerized less effectively than either wild-type protein or a variant lacking a different 28-residue segment. In contrast to the control proteins, the ACD mutant did not inhibit CBF DNA binding, AML1-mediated reporter activation, or G₁ to S cell cycle progression, the last being dependent upon activation of CBF-regulated genes. We also linked the CBFß domain to 149 or 83 C-terminal CBFß-SMMHC residues, retaining 86 or 20 amino acids N-terminal to the ACD. CBFß-SMMHC(149C) multimerized and slowed Ba/F3 proliferation, whereas CBFß-SMMHC(83C) did not. The majority of CBFß-SMMHC and CBFß-SMMHC(149C) was detected in the nucleus, whereas the ACD and 83C variants were predominantly cytoplasmic, indicating that multimerization facilitates nuclear retention of CBFß-SMMHC. When linked to the simian virus 40 nuclear localization signal (NLS), a significant fraction of CBFß-SMMHC(ACD) entered the nucleus but only mildly inhibited CBF activities. As NLS-CBFß-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 G₁ to S progression dependent upon the integrity of its DNA-binding domain. Thus, the ACD facilitates inhibition of CBF by mediating multimerization of CBFß-SMMHC in the nucleus. Therapeutics targeting the ACD may be effective in acute myeloid leukemia cases associated with CBFß-SMMHC expression.

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