Protein phosphatase 2A inactivates the mitogen-stimulated S6 kinase from Swiss mouse 3T3 cells.
Treatment of quiescent 3T3 cells with sodium orthovanadate induces a 10-fold stimulation of a kinase that phosphorylates ribosomal protein S6. The kinase in crude extracts is extremely labile and rapidly loses activity when incubated at 37 degrees C. This reaction is blocked by phosphatase inhibitors such as p-nitrophenyl phosphate and beta-glycerophosphate, suggesting that dephosphorylation of the kinase leads to its inactivation (Novak-Hofer, I., and Thomas, G. (1985) J. Biol. Chem. 260, 10314-10319). After three steps of purification the kinase can be separated from greater than 99% of the cellular phosphorylase a phosphatases. At this stage the kinase preparation is almost completely stable but can be inactivated by readdition of specific column fractions that contain both phosphorylase phosphatase and protease activity. However, employing a number of specific inhibitors it is shown that the inactivating agent in these fractions is a protein phosphatase. Furthermore, the physical and enzymatic properties of the kinase inactivator argue that it can be classified as a type 2A phosphatase. These results are consistent with the finding that the purified catalytic subunits of phosphatase type 1 and type 2A also inactivate the kinase. At equivalent phosphorylase a phosphatase activities, the type 2A catalytic subunit is 3 times more potent than the type 1 enzyme in carrying out this reaction. These data indicate that the major S6 kinase inactivator in 3T3 cell extracts is a type 2A phosphatase, supporting the hypothesis that the orthovanadate-stimulated S6 kinase is regulated in vivo by a phosphorylation-dephosphorylation mechanism.
Ballou
L M
LM
Friedrich Miescher-Institut, Basel, Switzerland.
Jenö
P
P
Thomas
G
G
D000818:Animals; D002460:Cell Line; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004533:Egtazic Acid / Q000494:pharmacology; D005347:Fibroblasts / Q000201:enzymology; D051379:Mice; D008970:Molecular Weight; D010749:Phosphoprotein Phosphatases / Q000494:pharmacology*; D010765:Phosphorylase Phosphatase / Q000302:isolation & purification; D047428:Protein Kinase Inhibitors; D054648:Protein Phosphatase 2; D019893:Ribosomal Protein S6 Kinases; D012969:Sodium Fluoride / Q000494:pharmacology; D013997:Time Factors
D047428:Protein Kinase Inhibitors; D004533:Egtazic Acid; D012969:Sodium Fluoride; D019893:Ribosomal Protein S6 Kinases; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D010765:Phosphorylase Phosphatase