Abstract
S6 phosphatase activities, which dephosphorylate the phosphorylated S6 synthetic peptide, RRLSSLRASTSKSESSQK, were purified to near homogeneity from the membrane and cytosolic fractions of the rat parotid gland. Multiple S6 phosphatases were fractionated on Mono Q and gel filtration columns. In the cytosolic fraction, at least three forms of S6 phosphatase, termed peaks I, II, and III, were differentially resolved. The three forms had different sizes and protein compositions. The peak I enzyme, which had an approximately Mr of 68 kDa on gel filtration, appears to represent a dimeric form of the 39 kDa protein. This S6 phosphatase showed the high activity in the presence of EGTA and was completely inhibited by nanomolar concentrations of either okadaic acid or inhibitor 2. The peak II S6 phosphatase enzyme, with an Mr of 35 kDa, was activated by Mn2+. This form could be a proteolytic product of the catalytic subunit of type 1 phosphatase, due to its sensitivities to okadaic acid and inhibitor 2. The peak III enzyme, with an Mr of 55 kDa, is a Mn2+-dependent S6 phosphatase. This S6 phosphatase can be classified as a type 1 phosphatase, due to its sensitivity to okadaic acid, since the IC50 of okadaic acid is 4 nM. However, the molecular mass of this S6 phosphatase differs from that of the type 1 catalytic subunit (37 kDa) and showed less sensitivity to inhibitor 2. On the other hand, the membrane fraction contained one form of the S6 phosphatases, termed peak V (Mr 34 and 28 kDa), which could be classified as a type 1 phosphatase. This S6 phosphatase activity was greatly stimulated by Mn2+.
Similar content being viewed by others
Abbreviations
- PP1-C:
-
catalytic subunit of type 1 protein phosphatase
- SDS:
-
sodium dodecyl sulfate
- Hepes:
-
4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid
- PMSF:
-
phenylmethylsulfonyl fluoride
- Mops:
-
4-morpholine propanesulfonic acid
- EDTA:
-
ethylenediaminetetraacetate
- EGTA:
-
[ethylenbis (oxyethylenenitrilo)]-tetra acetic acid
References
Butcher FR, Putney JW: Regulation of parotid gland function by cyclic nucleotides and calcium. Adv Cyclic Nucl Res 13: 215–249, 1980
Jahn R, Unger C, Söling H-D: Specific protein phosphorylation during stimulation of amylase secretion by β-agonists or dibutyl adenosine 3′,5′-monophosphatase in rat parotid gland. Eur J Biochem 112: 345–352, 1980
Thiel G, Schmidt WE, Meyer HE, Söling H-D: Purification and characterization of a 22 kDa microsomal protein from rat parotid gland which is phosphorylated following stimulation by agonists involving cAMP as second messenger. Eur J Biochem 170: 643–651, 1988
Freedman SD, Jamieson JD: Hormone-induced protein phosphorylation. II. Localization to the ribosomal fraction from rat exocrine pancreas and parotid of a 29,000-dalton protein phosphorylatedin situ in response to secretagogues. J Cell Biol 95: 909–917, 1982
Jahn R, Söling H-D: Phosphorylation of the ribosomal protein S6 in response to secretagogues in the guinea pig exocrine pancreas, parotid and lacrimal gland. FEBS Lett 153: 71–76, 1983
Padel U, Kruppa J, Jahn R, Söling H-D: Phosphopeptide patterns of the ribosomal protein S6 following stimulation of guinea pig parotid glands by secretagogues involving either cAMP or calcium as second messenger. FEBS Lett 159: 112–118, 1983
Thomas G, Martin-Perez J, Siegmann M, Otto AM: The effect of serum, EGF, PGF2α and insulin on S6 phosphorylation and the initiation of protein and DNA synthesis. Cell 30: 235–242, 1982
Duncan R, McConkey E: Preferential utilization of phosphorylated 40-S ribosomal subunits during initiation complex formation. Eur J Biochem 123: 535–538, 1982
Burkhard SJ, Traugh JA: Changes in ribosome function by cAMP-dependent and cAMP-independent phosphorylation of ribosomal protein S6. J Biol Chem 258: 14003–14008, 1983
Palen E, Traugh JA: Phosphorylation of ribosomal protein S6 by c-AMP dependent protein kinase and mitogen-stimulated S6 kinase differentially alters translation of globin mRNA. J Biol Chem 262: 3518–3523, 1987
Freedam SD, Jamieson JD: Hormone-induced protein phosphorylation. III. Regulation of the phosphorylation of the secretagogue-responsive 29,000-dalton protein by both Ca2+ and cAMPin vitro. J Cell Biol 95: 918–923, 1982
Traugh JA, Del Grande RW, Tuazon PT: Site-specific phosphorylation of translational components. Cold Spring Harbor Conf Cell Prolif 8: 999–1012, 1981
Wettenhall REH, Cohen P: Isolation and characterization of cyclic AMP-dependent phosphorylation sites from rat ribosomal protein S6. FEBS Lett 140: 263–269, 1982
Delgrande RW, Traugh JA: Phosphorylation of 40-S ribosomal subunits by cAMP-dependent, cGMP-dependent and protease-activated protein kinases. Eur J Biochem 123: 421–428, 1982
Erikson E, Maller JL: A protein kinase from Xenopus eggs specific for ribosomal protein S6. Proc Natl Acad Sci USA 82: 742–746, 1985
Tabarini D, Heinrich J, Rosen OM: Activation of S6 kinase activity in 3T3-L1 cells by insulin and phorbol ester. Proc Natl Acad Sci USA 82: 4369–4373, 1985
Cobb MH: An insulin-stimulated ribosomal protein S6 kinase in 3T3-L1 cells. J Biol Chem 261: 12994–12999, 1986
Gorelick FS, Cohen JA, Freedman SD, Delahunt WG, Gershoni JM, Jamieson JD: Calmodulin-stimulated protein kinase activity from rat pancreas. J Cell Biol 97: 1294–1298, 1983
Ballou LM, Jeno P, Thomas G: Protein phosphatase 2A inactivates the mitogen-stimulated S6 kinase from Swiss mouse 3T3 cells. J Biol Chem 263: 1188–1194, 1988
Padel U, Söling H-D: Phosphorylation of the ribosomal protein S6 during agonist-induced exocytosis in exocrine glands is catalyzed by calcium-phospholipid dependent protein kinase (protein kinase C). Eur J Biochem 151: 1–10, 1985
Hara-Yokoyama M, Sugiya H, Furuyama S, Wang JH, Yokoyama N: Dephosphorylation of ribosomal protein S6 phosphorylated via the cAMP-mediated signaling pathway in rat parotid gland: Effect of okadaic acid and Zn2+ Biochem and Mol Biol Int 34: 1177–1187, 1994
Tonks NK, Cohen P: The protein phosphatases involved in cellular regulation. Identification of the inhibitor-2 phosphatases in rabbit skeletal muscle. Eur J Biochem 145: 65–70, 1984
Cheng H-C, Litwin CME, Hwang DM, Wang JH: Structual basis of specific and efficient phosphorylation of peptides derived from p34cdc2 by a pp60src-related protein tyrosine kinase. J Biol Chem 266: 17919–17925, 1991
Stewart AA, Hemmings BA, Cohen P: The MgATP-dependent protein phosphatase and protein phosphatase 1 have identical substrate specificities. Eur J Biochem 115: 197–205, 1981
Laemmli UK: Cleavage of strucural proteins during the assembly of the head of bacteriophage T4 Nature 227: 680–685, 1970
Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248–254, 1976
Ingebritsen TS, Cohen P: The protein phosphatases involved in cellular regulation. Classification and substrate specificities. Eur J Biochem 132: 255–261, 1983
Brautigan D, Shriner CL: Methods to distinguish various types of protein phosphatase activity. Methods Enzymol 159: 339–346, 1988
Andres J, Johansen JW, Maller JL: Identification of protein phosphatases 1 and 2B as ribosomal protein S6 phosphatasesin vitro andin vivo. J Biol Chem 262: 14389–14393, 1987
Brautigan D, Shriner CL, Gruppuso PA: Phosphorylase phosphatase catalytic subunit. J Biol Chem 260: 4295–4302, 1985
Khatra BS: Subunit structure and properties of the glycogen-bound phosphoprotein phosphatase from skeletal muscle. J Biol Chem 261: 8944–8952, 1986
Wollny E, Watkins K, Kramer G, Hardesty B: Purification to homogeneity and partial characterization of a 56,000-dalton phosphoprotein phosphatase from rabbit reticulocytes. J Biol Chem 259: 2484–2492, 1984
Tipper J, Wollny E, Fullilove S, Kramer G, Hardesty B: Interaction of the 56,000-dalton protein phosphatase from reticulocytes with regulin and inhibitor 2. J Biol Chem 261: 7144–7150, 1986
Berndt N, Campbell DG, Caudwell FB, Cohen P, da Cruz e Silvia OB, Cohen PT: Isolation and sequence analysis of a cDNA clone encoding a type-1 protein phosphatase catalytic subunit: homology with protein phosphatase 2A. FEBS Lett 223: 340–346, 1987
Cohen PT: Two isoforms of protein phosphatase 1 may be produced from the same gene. FEBS Lett 232: 17–23, 1988
Sasaki K, Shima H, Kitagawa Y, Irino S, Sugimura T, Nagao M: Identification of members of the protein phosphatase 1 gene family in the rat and enhanced expression of protein phosphatase 1α gene in rat hepatocellular carcinomas. Jpn J Cancer Res 81: 1272–1280, 1990
Andres JL, Maller JL: Purification and characterization of a novel protein phosphatase highly specific for ribosomal protein S6. J Biol Chem 264: 151–156, 1989
Olivier AR, Ballou LM, Thomas G: Differential regulation of S6 phosphorylation by insulin and epidermal growth factor in Swiss mouse 3T3 cells: Insulin activation of type 1 phosphatase. Proc Natl Acad Sci USA 85: 4720–4724, 1988
Olivier AR, Thomas G: Three forms of phosphatase type 1 in Swiss 3T3 fibroblasts. J Biol Chem 265: 22460–22466, 1990
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Yokoyama, N. Purification and characterization of multiple S6 phosphatases from the rat parotid gland. Mol Cell Biochem 148, 123–132 (1995). https://doi.org/10.1007/BF00928149
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00928149