Green N C, Rambaldi I, Teakles J, Featherstone M S
McGill Cancer Centre, McGill University, Montréal, Québec H3G 1Y6, Canada.
J Biol Chem. 1998 May 22;273(21):13273-9. doi: 10.1074/jbc.273.21.13273.
HOX proteins are dependent upon cofactors of the PBX family for specificity of DNA binding. Two regions that have been implicated in HOX/PBX cooperative interactions are the YPWM motif, found N-terminal to the HOX homeodomain, and the GKFQ domain (also known as the Hox cooperativity motif) immediately C-terminal to the PBX homeodomain. Using derivatives of the E2A-PBX oncoprotein, we find that the GKFQ domain is not essential for cooperative interaction with HOXA1 but contributes to the stability of the complex. By contrast, the YPWM motif is strictly required for cooperative interactions in vitro and in vivo, even with mutants of E2A-PBX lacking the GKFQ domain. Using truncated PBX proteins, we show that the YPWM motif contacts the PBX homeodomain. The presence of the GKFQ domain increases monomer binding by the PBX homeodomain 5-fold, and the stability of the HOXA1.E2A-PBX complex 2-fold. These data suggest that the GKFQ domain acts mainly to increase DNA binding by PBX, rather than providing a primary contact site for the YPWM motif of HOXA1. We have identified 2 residues, Glu-301 and Tyr-305, required for GKFQ function and suggest that this is dependent on alpha-helical character.
HOX蛋白的DNA结合特异性依赖于PBX家族的辅助因子。已发现与HOX/PBX协同相互作用有关的两个区域，一个是位于HOX同源结构域N端的YPWM基序，另一个是紧接在PBX同源结构域C端的GKFQ结构域（也称为Hox协同基序）。使用E2A-PBX癌蛋白的衍生物，我们发现GKFQ结构域对于与HOXA1的协同相互作用不是必需的，但有助于复合物的稳定性。相比之下，YPWM基序在体外和体内的协同相互作用中是严格必需的，即使是对于缺乏GKFQ结构域的E2A-PBX突变体也是如此。使用截短的PBX蛋白，我们表明YPWM基序与PBX同源结构域接触。GKFQ结构域的存在使PBX同源结构域的单体结合增加5倍，使HOXA1.E2A-PBX复合物的稳定性增加2倍。这些数据表明，GKFQ结构域主要作用是增加PBX与DNA的结合，而不是为HOXA1的YPWM基序提供主要接触位点。我们已经鉴定出GKFQ功能所需的2个残基，即Glu-301和Tyr-305，并表明这取决于α螺旋特征。
