Variant Max Protein, Derived by Alternative Splicing, Associates with c-Myc In Vivo and Inhibits Transactivation

Abstract

Max (Myc-associated factor X) was first identified as a protein belonging to the basic helix-loop-helix/leucine zipper (bHLH/LZ) family of transcription factors that can associate with c-Myc in vitro and in vivo to form heterodimeric complexes with specific DNA binding activity (9, 10). Max exists in two forms as a result of alternative splicing of an exon, termed a, which encodes a nine-amino-acid insertion in the basic region (9). Both Max products have been shown either to heterodimerize with c-Myc or to form unphosphorylated homodimers and to specifically bind to the consensus sequence CACGTG (EMS [E-box Myc site]) (1,2,7,9,14,20,29,35,36). On the other hand, Myc forms homodimers only poorly under conditions that are physiologically relevant in vivo (29). Mutational analysis of Max indicated that the helix 1, helix 2, and leucine zipper (LZ) domains are critical for EMS DNA binding of homodimers, whereas the LZ domain is critical for binding of c-Myc-Max heterodimers (36). Cotransfection of Myc and Max expression vectors indicate that Max facilitates E-box-mediated transactivation by Myc at low doses, presumably via stimulation of c-Myc binding (2, 3, 15, 23). Conversely, overexpression of Max is inhibitory, suggesting that Max homodimers, which cannot transactivate, can compete for binding. More recently, two other members of the bHLH/LZ family of transcription factors, Mad and Mxi, have been shown to dimerize with Max product, but not with c-Myc, and bind efficiently to the E-box consensus sequence (4,5,42).Several other alternatively spliced forms of Max mRNAs have been reported (9,22,33,40,41). Makela and coworkers have identified an alternatively spliced Max mRNA containing an in-frame stop codon which would yield a putative protein (termed ⌬Max) truncated at the COOH terminus (33). Following in vitro transcription and translation, ⌬Max was able to associate with c-Myc and bind DNA in a sequence-specific manner. However no protein product was detected in the cell lines found to express ⌬max mRNA (33). Unlike Max, ⌬Max enhanced Myc transformation activity in a Ras cotransformation assay. Subsequently, Vastrik et al. (41) identified additional alternatively spliced max mRNAs of 1.9, 2.4, 3.0, and 3.5 kb, in addition to the predominant wild-type (wt) 2.3-kb species. These species displayed tissue-and cell line-specific expression in Northern (RNA) blot analysis (41). The 3.5-kb mRNA encoded a protein with biological activity similar to that of ⌬Max, but again the authors were unable to detect the putative protein product of this alternatively spliced max mRNA. Furthermore, in Xenopus laevis, the Max homologs XMax1 and XMax2 (resulting from alternative use of exon a) and two alternatively spliced mRNAs, termed XMax3 and XMax4, with an additional 81-base insertion in the C-terminal region of the coding sequence, termed exon b, have been identified (22,40). While these max mRNAs were found to be ubiquitously expressed during early embryonic development, and both in vitro-translated XMax2 and XMax4 proteins we...

A general method to design dominant negatives to B-HLHZip proteins that abolish DNA binding

“…Previously, D-Ns have been built to Max that consist of a deletion within the basic region (28,29). Our work indicates that replacing the basic region with an acidic region produced a more potent D-N. Because naturally occurring deletions of the basic region and helix 1 have D-N properties, a more thorough biophysical characterization of these molecules is needed (30). The ability of the described D-Ns to inhibit DNA binding of B-HLHZip proteins in a dimerization specific manner can be used to explore several biological issues.…”

The basic region/helix – loop – helix/leucine zipper domain of Myc proto-oncoproteins: Function and regulation

“…Since Max does not seem to contain a transactivation domain, Myc is the part of the heterodimer responsible for activation of transcription through its TAD. Various forms of Max proteins have been identi®ed due to alternative splicing with p21 and p22 (or Max and Max9, respectively, di ering by a nine amino acid insertion in p22) being the predominant forms (Blackwood and Eisenman, 1991;MaÈ kelaÈ et al, 1992;VaÈ strik et al, 1993;Arsura et al, 1995). In contrast to Myc, Max proteins are ubiquitously expressed (for review see Henriksson and LuÈ scher, 1996).…”

Inhibition of c-myc Expression Induces Apoptosis of WEHI 231 Murine B Cells

“…Nuclear proteins were isolated by using radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate [SDS], 1% sodium sarcosyl) as described previously (4). The protease inhibitors leupeptin (10 g/ml), phenylmethylsulfonyl fluoride (0.5 mM), and dithiothreitol (1 mM), which were prepared as fresh stocks, were added to buffers just prior to use.…”