A mitogen-activated protein (MAP) kinase activating factor in mammalian mitogen-stimulated cells is homologous to Xenopus M phase MAP kinase activator.

K Shirakabe,Y Gotoh,E Nishida

doi:10.1016/s0021-9258(18)42056-x

Abstract

The mitogen-activated protein (MAP) kinases, a family of 40-45-kDa kinases whose activation requires both tyrosine and threonine/serine phosphorylations, are suggested to play key roles in various phosphorylation cascades. A previous study of Krebs and co-workers (Ahn, N. G., Seger, R., Bratlien, R. L., Diltz, C. D., Tonks, N. K., and Krebs, E. G. (1991) J. Biol. Chem. 266, 4220-4227) detected an activity in epidermal growth factor (EGF)-stimulated 3T3 cells that can stimulate inactive MAP kinases. We observed this activity in rat 3Y1 cells treated with various mitogenic factors and in PC12 cells treated with nerve growth factor (NGF). Its kinetics of activation and deactivation following EGF or NGF stimulation roughly paralleled that of MAP kinase. The MAP kinase activator required the presence of ATP and a divalent cation such as Mn2+ and Mg2+ and was inactivated by phosphatase 2A treatment in vitro. This activator has been isolated from EGF-stimulated 3Y1 cells by sequential chromatography and identified as a 45-kDa monomeric protein. It was able to activate mammalian and Xenopus MAP kinases in vitro and was very similar to Xenopus M phase MAP kinase activating factor, which was purified previously from mature oocytes (Matsuda, S., Kosako, H., Takenaka, K., Moriyama, K., Sakai, H., Akiyama, T., Gotoh, Y., and Nishida, E. (1992) EMBO J. 11, 973-982), in terms of its functional, immunological, and physicochemical properties. Thus, the same or a similar upstream activating factor may function in mitogen-induced and M phase-promoting factor-induced MAP kinase activation pathways.

Highlights

A Mitogen-activated Protein (MAP) Kinase Activating Factorin Mammalian Mitogen-stimulatedCells Is Homologous to Xenopus M Phase mitogen-activated protein (MAP) Kinase Activator*
We observed to be activated in M phase duringoocyte maturation (Gotoh this activity in rat 3 Y 1 cells treated with variousmi- et al, 1991a; Posada et al, 1991;Ferrell et al, 1991)under the togenic factors and in PC12 cells treated with nerve control of M phase-promoting factor (MPF) (Gotoh et al, growth factor (NGF)
The MAP kinase activator required the presence of ATP and a divalent cation such as Mn2+and Mg2+and was inactivated by phosphatase 2A treatment in vitro.This activator has been isolated from epidermal growth factor (EGF)-stimulated 3 Y 1 cells by sequential chromatography and identified as a 45-kDa monomeric protein

Summary

A MAP Kinase Activating Activity in Mitogen-stimulated

Separation of the MAP Kinase Activator from MAP Kinase-Cell Fibroblastic Cells-We recently identified in mature Xenopus extracts (-300 pl) were loaded onto aDEAE-cellulose(500pl) column oocytes aprotein factor that is capable of inducing phosequilibrated with buffer A (20 mM Tris-C1, pH 7.5, 25 mM @-glycerophosphate, 2 mM EGTA, 1 mM PMSF, 20 pg/ml aprotinin, 2 mM DTT, 1mM vanadate) containing 100 mM NaC1. The active MBP kinase activity of the MAP kinase, increased very fractions, which had the activity to phosphorylate and activate recombinant MAP kinase, eluting at -100 mM NaCl were pooledand loaded onto a hydroxylapatite (2 ml) column equilibrated with buffer C (10 mM potassium phosphate, pH 7.0,12.5 mM P-glycerophosphate, 0.2 mM EGTA, 1 mM PMSF, 20 pg/ml aprotinin, 2 mM DTT, 1 mM vanadate). Cell Extraction-Quiescent 3Y1 cells or PC12 cells were treated with each of factors at 37 'C for indicated times and washed with iceing 12-O-tetradecanoylphorbol-13-acetatep,latelet-derived growth factor, insulin, okadaic acid, and fresh fetal calf serum (Fig. 2) These results suggest that the activity observed here may function as anupstream activator of MAP kinase cold Hepes-buffered saline.

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DISCUSSION