A NOVEL NF-KAPPA-B COMPLEX CONTAINING P65 HOMODIMERS - IMPLICATIONS FOR TRANSCRIPTIONAL CONTROL AT THE LEVEL OF SUBUNIT DIMERIZATION

The predominant inducible form of the NF-kappaB transcription factor is a heteromeric complex containing two Rel-related DNA-binding subunits, termed p65 and p50. Prior transfection studies have shown that when these p65 and p50 subunits are expressed independently as stable homodimers, p65 stimulates kappaB-directed transcription, whereas p50 functions as a kappaB-specific repressor. While authentic p50 homodimers (previously termed KBF1) have been detected in nuclear extracts from nontransfected cells, experimental evidence supporting the existence of p65 homodimers in vivo was lacking. We now provide direct biochemical evidence for the presence of an endogenous pool of inducible p65 homodimers in intact human T cells. As with the prototypical NF-kappaB p50-p65 heterodimer, this novel p65 homodimeric form of NF-kappaB is functionally sequestered in the cytoplasm but rapidly appears in the nuclear compartment following cellular stimulation. Site-directed mutagenesis studies indicate that the homodimerization function of p65 is dependent upon the presence of cysteine 216 and a conserved recognition motif for protein kinase A (RR-PS; amino acids 273 to 276), both of which reside within a 91-amino-acid segment of the Rel homology domain that mediates self-association. In contrast, mutations at these two sites do not affect heterodimerization of p65 with p50 or its functional interaction with IkappaBalpha These later findings indicate that neither homo- nor heterodimer formation is an absolute prerequisite for IkappaBalpha recognition of p65. Taken together with prior in vivo transcription studies, these results suggest that the biological activities of p65 and p50 homodimers are independently regulated, thereby providing an integrated and flexible control mechanism for the rapid activation and repression of NF-kappaB/Rel-directed gene expression.