POSSIBLE INVOLVEMENT OF DIFFERENTIAL SPLICING IN REGULATION OF THE ACTIVITY OF ARABIDOPSIS ANP1 THAT IS RELATED TO MITOGEN-ACTIVATED PROTEIN-KINASE KINASE KINASES (MAPKKKS)

Three types of Arabidopsis cDNA (cANP1, cANP2 and cANP3) have been iso lated that encode putative protein kinases, designated ANP1, ANP2 and ANP3. These kinases exhibit a high degree of homology to NPK1, a tobac co protein that is a member of the family of mitogen-activated protein kinase kinase kinases (MAPKKKs), which appears to function in the pro liferation of tobacco cells. The predicted amino acid sequences of the kinase domains in the amino-terminal halves of the ANPs were more tha n 80% identical to that of NPK1, while the kinase-unrelated regions in the carboxy-terminal halves exhibited relatively low homology. Two sp ecies of cANP1 were identified, ANP1L cDNA (cANP1L) and ANP1S cDNA (cA NP1S), which were derived from a single ANP1 gene: the former had an i ntron-like sequence in the coding region for the kinase-unrelated regi on, while the latter did not include such an intron-like sequence. cAN P1L encoded a putative protein with both kinase and kinase-unrelated d omains, resembling NPK1, whereas cANP1S encoded only the aimino-termin al kinase domain because the intron-like sequence was absent, with res ulting elimination of most of the kinase-unrelated region. Genetic ana lysis with mutant yeast cells showed that over-expression of cANP1L or of cANP1S activated the mating pheromone-responsive signal pathway wh ich is mediated by a MAP kinase cascade. Moreover, the extent of such activation by cANP1S was greater than that by cANP1L. These results pr edict that differential splicing of the intron-like sequence in the AN P1 transcript might be at least one of the molecular mechanisms involv ed in the generation of active ANP1 protein kinase.