M. Arsura et al., VARIANT MAX PROTEIN, DERIVED BY ALTERNATIVE SPLICING, ASSOCIATES WITHC-MYC IN-VIVO AND INHIBITS TRANSACTIVATION, Molecular and cellular biology, 15(12), 1995, pp. 6702-6709
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.