VARIANT MAX PROTEIN, DERIVED BY ALTERNATIVE SPLICING, ASSOCIATES WITHC-MYC IN-VIVO AND INHIBITS TRANSACTIVATION

Max (Myc-associated factor X) is a basic helix-loop-helix/leucine zipp er protein that has been shown to play a central role in the functiona l activity of c-Myc as a transcriptional activator. Max potentiates th e binding of Myc-Max heterodimers through its basic region to its spec ific E-box Myc site (EMS), enabling c-Myc to transactivate effectively . In addition to the alternatively spliced exon a, several naturally o ccurring forms of alternatively spliced max mRNAs have been reported, but variant protein products from these transcripts have not been dete cted. Using Western blot (immunoblot) and immunoprecipitation analysis , we have identified a variant form of Max protein (16 to 17 kDa), ter med dMax, in detergent nuclear extracts of murine B-lymphoma cells, no rmal B lymphocytes, and NIH 3T3 fibroblasts. Cloning and sequencing re vealed that dMax contains a deletion spanning the basic region and hel ix 1 and the loop of the helix-loop-helix region, presumably as a resu lt of alternative splicing of max RNA. S1 nuclease analysis confirmed the presence of the mRNA for dMax in cells, The dMax protein, prepared via in vitro transcription and translation, associated with bacterial ly synthesized Myc-glutathione S-transferase. Coimmunoprecipitation of dMax and c-Myc indicated their intracellular association. In vitro-sy nthesized dMax failed to bind EMS DNA, presumably because of the absen ce of the basic region. Coexpression of dMax inhibited EMS-mediated tr ansactivation by c-Myc. Thus dMax, which can interact with c-Myc, appe ars to function as a dominant negative regulator, providing an additio nal level of regulation to the transactivation potential of c-Myc.