Search tips
Search criteria 


Logo of molcellbPermissionsJournals.ASM.orgJournalMCB ArticleJournal InfoAuthorsReviewers
Mol Cell Biol. 1994 July; 14(7): 4398–4407.
PMCID: PMC358811

Expression of a peptide inhibitor of protein phosphatase 1 increases phosphorylation and activity of CREB in NIH 3T3 fibroblasts.


We have examined the activity and phosphorylation state of the cyclic AMP (cAMP) response element binding factor (CREB) in intact NIH 3T3 cells following microinjection of expression plasmids encoding regulatory proteins of type 1 (PP1) and 2A (PP2A) serine/threonine-specific protein phosphatases. Changes in CREB phosphorylation in the injected cells were monitored by indirect immunofluorescence using an affinity-purified antiserum (Ab5322) which specifically recognizes CREB phosphorylated at Ser-133, and changes in transcriptional activity of CREB were monitored by expression of a reporter gene regulated by cAMP. cAMP-stimulated phosphorylation in NIH 3T3 cells is normally transient, and as expected, after stimulation of cells with cell-permeable cAMP analogs, the level of phosphorylated CREB was found to initially increase and then return to a basal level within 4 h. Microinjection of an expression vector encoding a constitutively active form of inhibitor 1 (I-1), a PP1-specific inhibitor, by itself resulted in an apparent increase in phosphorylated CREB in unstimulated cells. Moreover, injection of the I-1 vector resulted in the prolonged appearance of phosphorylated CREB in cells after cAMP stimulation. In contrast, injection of a plasmid encoding simian virus 40 small t antigen, which interacts with PP2A to inhibit its activity towards several phosphoprotein substrates, had no effect on the phosphorylation state of CREB in stimulated or unstimulated NIH 3T3 cells. Consistent with these results, injection of the I-1 expression vector activated expression from a coinjected CRE-lacZ reporter plasmid, indicating that the increased phosphorylation of CREB also activated its transcriptional activity. These results provide further evidence for a role of a PP1 as the primary protein (Ser/Thr) phosphatase regulating the dephosphorylation of Ser-133 and thereby limiting the transcriptional activity of CREB.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (2.6M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Aitken A, Cohen P. Isolation and characterisation of active fragments of protein phosphatase inhibitor-1 from rabbit skeletal muscle. FEBS Lett. 1982 Oct 4;147(1):54–58. [PubMed]
  • Alberts AS, Arias J, Hagiwara M, Montminy MR, Feramisco JR. Recombinant cyclic AMP response element binding protein (CREB) phosphorylated on Ser-133 is transcriptionally active upon its introduction into fibroblast nuclei. J Biol Chem. 1994 Mar 11;269(10):7623–7630. [PubMed]
  • Alberts AS, Deng T, Lin A, Meinkoth JL, Schönthal A, Mumby MC, Karin M, Feramisco JR. Protein phosphatase 2A potentiates activity of promoters containing AP-1-binding elements. Mol Cell Biol. 1993 Apr;13(4):2104–2112. [PMC free article] [PubMed]
  • Alberts AS, Frost JA, Thorburn AM. Rapid transcriptional assay for the expression of two distinct reporter genes by microinjection. DNA Cell Biol. 1993 Dec;12(10):935–943. [PubMed]
  • Alberts AS, Thorburn AM, Shenolikar S, Mumby MC, Feramisco JR. Regulation of cell cycle progression and nuclear affinity of the retinoblastoma protein by protein phosphatases. Proc Natl Acad Sci U S A. 1993 Jan 15;90(2):388–392. [PubMed]
  • Andersson S, Davis DL, Dahlbäck H, Jörnvall H, Russell DW. Cloning, structure, and expression of the mitochondrial cytochrome P-450 sterol 26-hydroxylase, a bile acid biosynthetic enzyme. J Biol Chem. 1989 May 15;264(14):8222–8229. [PubMed]
  • Beullens M, Van Eynde A, Bollen M, Stalmans W. Inactivation of nuclear inhibitory polypeptides of protein phosphatase-1 (NIPP-1) by protein kinase A. J Biol Chem. 1993 Jun 25;268(18):13172–13177. [PubMed]
  • Boshart M, Weber F, Jahn G, Dorsch-Häsler K, Fleckenstein B, Schaffner W. A very strong enhancer is located upstream of an immediate early gene of human cytomegalovirus. Cell. 1985 Jun;41(2):521–530. [PubMed]
  • Brautigan DL, Sunwoo J, Labbé JC, Fernandez A, Lamb NJ. Cell cycle oscillation of phosphatase inhibitor-2 in rat fibroblasts coincident with p34cdc2 restriction. Nature. 1990 Mar 1;344(6261):74–78. [PubMed]
  • Brindle PK, Montminy MR. The CREB family of transcription activators. Curr Opin Genet Dev. 1992 Apr;2(2):199–204. [PubMed]
  • Capecchi MR. High efficiency transformation by direct microinjection of DNA into cultured mammalian cells. Cell. 1980 Nov;22(2 Pt 2):479–488. [PubMed]
  • Chang LS, Pater MM, Hutchinson NI, di Mayorca G. Transformation by purified early genes of simian virus 40. Virology. 1984 Mar;133(2):341–353. [PubMed]
  • Chrivia JC, Kwok RP, Lamb N, Hagiwara M, Montminy MR, Goodman RH. Phosphorylated CREB binds specifically to the nuclear protein CBP. Nature. 1993 Oct 28;365(6449):855–859. [PubMed]
  • Cobb MH, Boulton TG, Robbins DJ. Extracellular signal-regulated kinases: ERKs in progress. Cell Regul. 1991 Dec;2(12):965–978. [PMC free article] [PubMed]
  • Cohen P. The structure and regulation of protein phosphatases. Annu Rev Biochem. 1989;58:453–508. [PubMed]
  • Fernandez A, Brautigan DL, Mumby M, Lamb NJ. Protein phosphatase type-1, not type-2A, modulates actin microfilament integrity and myosin light chain phosphorylation in living nonmuscle cells. J Cell Biol. 1990 Jul;111(1):103–112. [PMC free article] [PubMed]
  • Friedmann T, Doolittle RF, Walter G. Amino acid sequence homology between polyoma and SV40 tumour antigens deduced from nucleotide sequences. Nature. 1978 Jul 20;274(5668):291–293. [PubMed]
  • Gonzalez GA, Menzel P, Leonard J, Fischer WH, Montminy MR. Characterization of motifs which are critical for activity of the cyclic AMP-responsive transcription factor CREB. Mol Cell Biol. 1991 Mar;11(3):1306–1312. [PMC free article] [PubMed]
  • Gonzalez GA, Montminy MR. Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133. Cell. 1989 Nov 17;59(4):675–680. [PubMed]
  • Gonzalez GA, Yamamoto KK, Fischer WH, Karr D, Menzel P, Biggs W, 3rd, Vale WW, Montminy MR. A cluster of phosphorylation sites on the cyclic AMP-regulated nuclear factor CREB predicted by its sequence. Nature. 1989 Feb 23;337(6209):749–752. [PubMed]
  • Gotoh Y, Nishida E, Sakai H. Okadaic acid activates microtubule-associated protein kinase in quiescent fibroblastic cells. Eur J Biochem. 1990 Nov 13;193(3):671–674. [PubMed]
  • Guan K, Hakes DJ, Wang Y, Park HD, Cooper TG, Dixon JE. A yeast protein phosphatase related to the vaccinia virus VH1 phosphatase is induced by nitrogen starvation. Proc Natl Acad Sci U S A. 1992 Dec 15;89(24):12175–12179. [PubMed]
  • Habener JF. Cyclic AMP response element binding proteins: a cornucopia of transcription factors. Mol Endocrinol. 1990 Aug;4(8):1087–1094. [PubMed]
  • Hagiwara M, Alberts A, Brindle P, Meinkoth J, Feramisco J, Deng T, Karin M, Shenolikar S, Montminy M. Transcriptional attenuation following cAMP induction requires PP-1-mediated dephosphorylation of CREB. Cell. 1992 Jul 10;70(1):105–113. [PubMed]
  • Hagiwara M, Brindle P, Harootunian A, Armstrong R, Rivier J, Vale W, Tsien R, Montminy MR. Coupling of hormonal stimulation and transcription via the cyclic AMP-responsive factor CREB is rate limited by nuclear entry of protein kinase A. Mol Cell Biol. 1993 Aug;13(8):4852–4859. [PMC free article] [PubMed]
  • Haystead TA, Weiel JE, Litchfield DW, Tsukitani Y, Fischer EH, Krebs EG. Okadaic acid mimics the action of insulin in stimulating protein kinase activity in isolated adipocytes. The role of protein phosphatase 2a in attenuation of the signal. J Biol Chem. 1990 Sep 25;265(27):16571–16580. [PubMed]
  • Hemmings HC, Jr, Girault JA, Nairn AC, Bertuzzi G, Greengard P. Distribution of protein phosphatase inhibitor-1 in brain and peripheral tissues of various species: comparison with DARPP-32. J Neurochem. 1992 Sep;59(3):1053–1061. [PubMed]
  • Hemmings HC, Jr, Nairn AC, Elliott JI, Greengard P. Synthetic peptide analogs of DARPP-32 (Mr 32,000 dopamine- and cAMP-regulated phosphoprotein), an inhibitor of protein phosphatase-1. Phosphorylation, dephosphorylation, and inhibitory activity. J Biol Chem. 1990 Nov 25;265(33):20369–20376. [PubMed]
  • Herberg FW, Bell SM, Taylor SS. Expression of the catalytic subunit of cAMP-dependent protein kinase in Escherichia coli: multiple isozymes reflect different phosphorylation states. Protein Eng. 1993 Sep;6(7):771–777. [PubMed]
  • Joshi B, Rundell K. Association of simian virus 40 small-t antigen with the 61-kilodalton component of a cellular protein complex. J Virol. 1990 Nov;64(11):5649–5651. [PMC free article] [PubMed]
  • Kalderon D, Roberts BL, Richardson WD, Smith AE. A short amino acid sequence able to specify nuclear location. Cell. 1984 Dec;39(3 Pt 2):499–509. [PubMed]
  • Kim SJ, Lafyatis R, Kim KY, Angel P, Fujiki H, Karin M, Sporn MB, Roberts AB. Regulation of collagenase gene expression by okadaic acid, an inhibitor of protein phosphatases. Cell Regul. 1990 Feb;1(3):269–278. [PMC free article] [PubMed]
  • Kuret J, Bell H, Cohen P. Identification of high levels of protein phosphatase-1 in rat liver nuclei. FEBS Lett. 1986 Jul 28;203(2):197–202. [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Lamb NJ, Fernandez A, Conti MA, Adelstein R, Glass DB, Welch WJ, Feramisco JR. Regulation of actin microfilament integrity in living nonmuscle cells by the cAMP-dependent protein kinase and the myosin light chain kinase. J Cell Biol. 1988 Jun;106(6):1955–1971. [PMC free article] [PubMed]
  • Lee CQ, Yun YD, Hoeffler JP, Habener JF. Cyclic-AMP-responsive transcriptional activation of CREB-327 involves interdependent phosphorylated subdomains. EMBO J. 1990 Dec;9(13):4455–4465. [PubMed]
  • Liu JS, Park EA, Gurney AL, Roesler WJ, Hanson RW. Cyclic AMP induction of phosphoenolpyruvate carboxykinase (GTP) gene transcription is mediated by multiple promoter elements. J Biol Chem. 1991 Oct 5;266(28):19095–19102. [PubMed]
  • Meinkoth J, Alberts AS, Feramisco JR. Construction of mammalian cell lines with indicator genes driven by regulated promoters. Ciba Found Symp. 1990;150:47–56. [PubMed]
  • Meinkoth JL, Ji Y, Taylor SS, Feramisco JR. Dynamics of the distribution of cyclic AMP-dependent protein kinase in living cells. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9595–9599. [PubMed]
  • Meinkoth JL, Montminy MR, Fink JS, Feramisco JR. Induction of a cyclic AMP-responsive gene in living cells requires the nuclear factor CREB. Mol Cell Biol. 1991 Mar;11(3):1759–1764. [PMC free article] [PubMed]
  • Montminy MR, Bilezikjian LM. Binding of a nuclear protein to the cyclic-AMP response element of the somatostatin gene. Nature. 1987 Jul 9;328(6126):175–178. [PubMed]
  • Mumby MC, Russell KL, Garrard LJ, Green DD. Cardiac contractile protein phosphatases. Purification of two enzyme forms and their characterization with subunit-specific antibodies. J Biol Chem. 1987 May 5;262(13):6257–6265. [PubMed]
  • Mumby MC, Walter G. Protein phosphatases and DNA tumor viruses: transformation through the back door? Cell Regul. 1991 Aug;2(8):589–598. [PMC free article] [PubMed]
  • Mumby MC, Walter G. Protein serine/threonine phosphatases: structure, regulation, and functions in cell growth. Physiol Rev. 1993 Oct;73(4):673–699. [PubMed]
  • Patwardhan S, Gashler A, Siegel MG, Chang LC, Joseph LJ, Shows TB, Le Beau MM, Sukhatme VP. EGR3, a novel member of the Egr family of genes encoding immediate-early transcription factors. Oncogene. 1991 Jun;6(6):917–928. [PubMed]
  • Riabowol KT, Fink JS, Gilman MZ, Walsh DA, Goodman RH, Feramisco JR. The catalytic subunit of cAMP-dependent protein kinase induces expression of genes containing cAMP-responsive enhancer elements. Nature. 1988 Nov 3;336(6194):83–86. [PubMed]
  • Rose DW, McCabe G, Feramisco JR, Adler M. Expression of c-fos and AP-1 activity in senescent human fibroblasts is not sufficient for DNA synthesis. J Cell Biol. 1992 Dec;119(6):1405–1411. [PMC free article] [PubMed]
  • Rundell K. Complete interaction of cellular 56,000- and 32,000-Mr proteins with simian virus 40 small-t antigen in productively infected cells. J Virol. 1987 Apr;61(4):1240–1243. [PMC free article] [PubMed]
  • Scheidtmann KH, Mumby MC, Rundell K, Walter G. Dephosphorylation of simian virus 40 large-T antigen and p53 protein by protein phosphatase 2A: inhibition by small-t antigen. Mol Cell Biol. 1991 Apr;11(4):1996–2003. [PMC free article] [PubMed]
  • Schönthal A. Okadaic acid--a valuable new tool for the study of signal transduction and cell cycle regulation? New Biol. 1992 Jan;4(1):16–21. [PubMed]
  • Schönthal A, Tsukitani Y, Feramisco JR. Transcriptional and post-transcriptional regulation of c-fos expression by the tumor promoter okadaic acid. Oncogene. 1991 Mar;6(3):423–430. [PubMed]
  • Shenolikar S, Nairn AC. Protein phosphatases: recent progress. Adv Second Messenger Phosphoprotein Res. 1991;23:1–121. [PubMed]
  • Sola MM, Langan T, Cohen P. p34cdc2 phosphorylation sites in histone H1 are dephosphorylated by protein phosphatase 2A1. Biochim Biophys Acta. 1991 Sep 3;1094(2):211–216. [PubMed]
  • Sontag E, Fedorov S, Kamibayashi C, Robbins D, Cobb M, Mumby M. The interaction of SV40 small tumor antigen with protein phosphatase 2A stimulates the map kinase pathway and induces cell proliferation. Cell. 1993 Dec 3;75(5):887–897. [PubMed]
  • Sun H, Charles CH, Lau LF, Tonks NK. MKP-1 (3CH134), an immediate early gene product, is a dual specificity phosphatase that dephosphorylates MAP kinase in vivo. Cell. 1993 Nov 5;75(3):487–493. [PubMed]
  • Thévenin C, Kim SJ, Kehrl JH. Inhibition of protein phosphatases by okadaic acid induces AP1 in human T cells. J Biol Chem. 1991 May 25;266(15):9363–9366. [PubMed]
  • Thorburn AM, Alberts AS. Efficient expression of miniprep plasmid DNA after needle micro-injection into somatic cells. Biotechniques. 1993 Mar;14(3):356–358. [PubMed]
  • Tung HY, Alemany S, Cohen P. The protein phosphatases involved in cellular regulation. 2. Purification, subunit structure and properties of protein phosphatases-2A0, 2A1, and 2A2 from rabbit skeletal muscle. Eur J Biochem. 1985 Apr 15;148(2):253–263. [PubMed]
  • Usui H, Imazu M, Maeta K, Tsukamoto H, Azuma K, Takeda M. Three distinct forms of type 2A protein phosphatase in human erythrocyte cytosol. J Biol Chem. 1988 Mar 15;263(8):3752–3761. [PubMed]
  • Wadzinski BE, Wheat WH, Jaspers S, Peruski LF, Jr, Lickteig RL, Johnson GL, Klemm DJ. Nuclear protein phosphatase 2A dephosphorylates protein kinase A-phosphorylated CREB and regulates CREB transcriptional stimulation. Mol Cell Biol. 1993 May;13(5):2822–2834. [PMC free article] [PubMed]
  • Walter G, Carbone-Wiley A, Joshi B, Rundell K. Homologous cellular proteins associated with simian virus 40 small T antigen and polyomavirus medium T antigen. J Virol. 1988 Dec;62(12):4760–4762. [PMC free article] [PubMed]
  • Walter G, Ruediger R, Slaughter C, Mumby M. Association of protein phosphatase 2A with polyoma virus medium tumor antigen. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2521–2525. [PubMed]
  • Yang SI, Lickteig RL, Estes R, Rundell K, Walter G, Mumby MC. Control of protein phosphatase 2A by simian virus 40 small-t antigen. Mol Cell Biol. 1991 Apr;11(4):1988–1995. [PMC free article] [PubMed]

Articles from Molecular and Cellular Biology are provided here courtesy of American Society for Microbiology (ASM)