Three forms of phosphatase type 1 in Swiss 3T3 fibroblasts. Free catalytic subunit appears to mediate s6 dephosphorylation.
The major 40 S ribosomal protein S6 phosphatase in Swiss mouse 3T3 fibroblasts is a type 1 enzyme (Olivier, A. R., Ballou, L. M., and Thomas, G. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 4720-4724). Polyclonal antibodies were raised against a synthetic peptide containing the carboxyl-terminal 14 amino acids of the catalytic subunit of phosphatase 1 (PP-1C). Results from Western blot analysis and immunoprecipitation show that the peptide antiserum specifically recognizes PP-1C in cell extracts. Anion-exchange chromatography of cell extracts and Western blot analysis revealed three peaks of PP-1C termed A, B, and C. Peaks A and C are associated with the major type 1 S6 phosphatase activities, but peak B exhibits little activity. The phosphatase in peak A (Mr 39,000) appears to represent the free catalytic subunit, whereas the enzymes in peaks B and C display sizes of 68,000-140,000. Peak B contains two additional proteins of Mr 26,000 and 48,000 that co-immunoprecipitate with PP-1C, while peak C has a single additional protein of Mr 100,000. Fifteen min after serum withdrawal there is a 2-fold stimulation of S6 phosphatase activity in peak A that can be accounted for by an increase in the amount of PP-1C. The amount of PP-1C in the inactive peak B fraction also increases during this time and this increase is associated with changes in the phosphorylation state of the Mr 26,000 and 48,000 proteins. The results are discussed in relation to regulatory mechanisms which are thought to modulate the activity of type 1 phosphatase.
Olivier
A R
AR
Friedrich Miescher-Institut, Basel, Switzerland.
Thomas
G
G
D000818:Animals; D000936:Antigen-Antibody Complex; D002460:Cell Line; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000201:enzymology; D015151:Immunoblotting; D007074:Immunoglobulin G / Q000302:isolation & purification; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D008970:Molecular Weight; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D038601:Ribosomal Protein S6; D012269:Ribosomal Proteins / Q000378:metabolism*
D000936:Antigen-Antibody Complex; D007074:Immunoglobulin G; D007527:Isoenzymes; D046911:Macromolecular Substances; D038601:Ribosomal Protein S6; D012269:Ribosomal Proteins; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1