THE MOUSE P44 MITOGEN-ACTIVATED PROTEIN-KINASE (EXTRACELLULAR SIGNAL-REGULATED KINASE-1) GENE - GENOMIC ORGANIZATION AND STRUCTURE OF THE 5'-FLANKING REGULATORY REGION

Mitogen-activated protein kinase (MAPK) or extracellular signal-regula ted kinase are ubiquitous kinases conserved from fungi to mammals. The ir activity is regulated by phosphorylation on both threonine and tyro sine, and they play a crucial role in the regulation of proliferation and differentiation. We report here the cloning of the murine p44 MAP kinase (extracellular signal-regulated kinase 1) gene, the determinati on of its intron/exon boundaries, and the characterization of its prom oter. The gene spans approximately eight kilo-bases promoter. bases (k b) and can be divided into nine exons and eight introns, each coding r egion exon containing from one to three of the highly conserved protei n kinase domains. Primer extension analysis reveals the existence of t wo major start sites of transcription located at -183 and -186 base pa irs (bp) as well as four discrete start sites for transcription locate d at -178, -192, -273, and -292 bp of the initiation of translation. H owever, the start site region lacks TATA-like sequences but does conta in initiator-like sequences proximal to the major start sites obtained by primer extension. 1 kb of the promoter region has been sequenced. It contains three putative TATA boxes far upstream of the main start s ites region, one AP-1 box, one AP-2 box, one Malt box, one GAGA box, o ne half serum-responsive element, and putative binding sites for Spl ( five), GC-rich binding factor (five), CTF-NF1 (one), Myb (one), p53 (t wo), Ets-1 (one), NF-IL6 (two), MyoD (two), Zeste (one), and hepatocyt e nuclear factor-5 (one). To determine the sites critical for the func tion of the p44 MAPK promoter, we constructed a series of chimeric gen es containing variable regions of the 5'-flanking sequence of p44 MAPK gene and the coding region for luciferase. Activity of the promoter, measured by its capacity to direct expression of a luciferase reporter gene, is strong, being comparable with the activity of the Rous sarco ma virus promoter. Progressive deletions of the similar to 1 kb (-1200 /-78) promoter region allowed us to define a minimal region of 186 bp (-284/ -78) that has maximal promoter activity. Within this context, d eletion of the AP-2 binding site reduces by 30-40% the activity of the promoter. Further deletion of this minimal promoter that removes the major start sites (-167/-78) surprisingly preserves promoter activity, This result implicates a major role of this region that contains the Spl sites. Finally, removal of the major start sites of transcription as well as the Spl sites reveals additional promoter activity at the u pstream transcription minor start sites (-240/-167), an activity that is enhanced by the upstream cis-acting elements, In summary, our findi ngs reveal a complex pattern of transcriptional of the mouse p44 MAPK promoter.