OVEREXPRESSION OF MITOGEN-ACTIVATED PROTEIN-KINASE KINASE (MAPKK) ANDITS MUTANTS IN NIH 3T3 CELLS - EVIDENCE THAT MAPKK INVOLVEMENT IN CELLULAR PROLIFERATION IS REGULATED BY PHOSPHORYLATION OF SERINE RESIDUESIN ITS KINASE SUBDOMAIN-VII AND SUBDOMAIN-VIII

Mitogen-activated protein kinase kinase (MAPKK) is a dual specificity protein kinase that exhibits a high degree of specificity toward its d ownstream target, mitogen-activated protein kinase (MAPK). In this stu dy, we used stable overexpression of MAPKK and its mutants in NIH 3T3 cells to study effects on downstream components of the MAPK signaling cascade and to correlate them to physiological responses. We have muta ted the potential regulatory serine residue 222 to alanine (S222A) or to glutamate (S222E) and serines 212 and 218 together to alanine resid ues (S212A,S218A). Lysine 97 was mutated to alanine (K97A) to provide an inactive enzyme. Overexpression of the wild type MAPKK had no effec t on any of the parameters examined. The K97A and S222A mutants served as dominant negatives by suppressing MAPKK, MAPK, and p90(rsk) activa tion in vivo. S222E enhanced all of these activities, and S212A,S218A had a small inhibitory effect. A similar trend was observed when cellu lar proliferation was examined and the different effects were accompan ied by altered cellular shape. Taken together, our results demonstrate a direct linkage between the MAPK signaling pathway and the control o f cellular proliferation and morphology and also establish that phosph orylation of: serine 222 is essential for MAPKK activation together wi th the phosphorylation of an additional serine(s) (probably serine 218 ).