Overexpression of mitogen-activated protein kinase kinase (MAPKK) and its mutants in NIH 3T3 cells: Evidence that MAPKK involvement in cellular proliferation is regulated by phosphorylation of serine residues in its kinase subdomains VII and VIII

Rony Seger, Dalia Seger, Alfred A. Reszka, Erlynda S. Munar, Hagit Eldar-Finkelman, Grazyna Dobrowolska, Amy M. Jensen, Jean S. Campbell, Edmond H. Fischer, Edwin G. Krebs*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

204 Scopus citations

Abstract

Mitogen-activated protein kinase kinase (MAPKK) is a dual specificity protein kinase that exhibits a high degree of specificity toward its downstream target, mitogen-activated protein kinase (MAPK). In this study, 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 mutated the potential regulatory serine residue 222 to alanine (S222A) or to glutamate (S222E) and serines 212 and 218 together to alanine residues (S212A,S218A). Lysine 97 was mutated to alanine (K97A) to provide an inactive enzyme. Overexpression of the wild type MAPKK had no effect on any of the parameters examined. The K97A and S222A mutants served as dominant negatives by suppressing MAPKK, MAPK, and p90rsk activation in vivo. S222E enhanced all of these activities, and S212A,S218A had a small inhibitory effect. A similar trend was observed when cellular proliferation was examined and the different effects were accompanied by altered cellular shape. Taken together, our results demonstrate a direct linkage between the MAPK signaling pathway and the control of cellular proliferation and morphology and also establish that phosphorylation of serine 222 is essential for MAPKK activation together with the phosphorylation of an additional serine(s) (probably serine 218).

Original languageEnglish
Pages (from-to)25699-25709
Number of pages11
JournalJournal of Biological Chemistry
Volume269
Issue number41
StatePublished - 14 Oct 1994
Externally publishedYes

Funding

FundersFunder number
National Institute of General Medical SciencesP01GM042508
National Institute of Diabetes and Digestive and Kidney DiseasesR01DK042528

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