A CONSERVED C-TERMINAL DOMAIN IN PBX INCREASES DNA-BINDING BY THE PBXHOMEODOMAIN AND IS NOT A PRIMARY SITE OF CONTACT FOR THE YPWM MOTIF OF HOXA1

HOX proteins are dependent upon cofactors of the PBX family for specif icity 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 cooperativ ity motif) immediately C-terminal to the PBX homeodomain. Using deriva tives of the E2A-PBX oncoprotein, we find that the GKFQ domain is not essential for cooperative interaction with HOXA1 but contributes to th e stability of the complex. By contrast, the YPWM motif is strictly re quired for cooperative interactions in vitro and in vivo, even with mu tants of E2A-PBX lacking the GKFQ domain. Using truncated PBX proteins , we show that the YPWM motif contacts the PBX homeodomain, The presen ce 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 moti f of HOXA1. We have identified 2 residues, Glu-301 and Tyr-305, requir ed for GKFQ function and suggest that this is dependent on cw-helical character.