Logo of molcellbPermissionsJournals.ASM.orgJournalMCB ArticleJournal InfoAuthorsReviewers
Mol Cell Biol. 1995 Jan; 15(1): 433–444.
PMCID: PMC231986

The serum response factor nuclear localization signal: general implications for cyclic AMP-dependent protein kinase activity in control of nuclear translocation.


We have identified a basic sequence in the N-terminal region of the 67-kDa serum response factor (p67SRF or SRF) responsible for its nuclear localization. A peptide containing this nuclear localization signal (NLS) translocates rabbit immunoglobulin G (IgG) into the nucleus as efficiently as a peptide encoding the simian virus 40 NLS. This effect is abolished by substituting any two of the four basic residues in this NLS. Overexpression of a modified form of SRF in which these basic residues have been mutated confirms the absolute requirement for this sequence, and not the other basic amino acid sequences adjacent to it, in the nuclear localization of SRF. Since this NLS is in close proximity to potential phosphorylation sites for the cAMP-dependent protein kinase (A-kinase), we further investigated if A-kinase plays a role in the nuclear location of SRF. The nuclear transport of SRF proteins requires basal A-kinase activity, since inhibition of A-kinase by using either the specific inhibitory peptide PKIm or type II regulatory subunits (RII) completely prevents the nuclear localization of plasmid-expressed tagged SRF or an SRF-NLS-IgG conjugate. Direct phosphorylation of SRF by A-kinase can be discounted in this effect, since mutation of the putative phosphorylation sites in either the NLS peptide or the encoded full-length SRF protein had no effect on nuclear transport of the mutants. Finally, in support of an implication of A-kinase-dependent phosphorylation in a more general mechanism affecting nuclear import, we show that the nuclear transport of a simian virus 40-NLS-conjugated IgG or purified cyclin A protein is also blocked by inhibition of A-kinase, even though neither contains any potential sites for phosphorylation by A-kinase or can be phosphorylated by A-kinase in vitro.

Full Text

The Full Text of this article is available as a PDF (980K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Adam SA, Lobl TJ, Mitchell MA, Gerace L. Identification of specific binding proteins for a nuclear location sequence. Nature. 1989 Jan 19;337(6204):276–279. [PubMed]
  • Bornslaeger EA, Mattei P, Schultz RM. Involvement of cAMP-dependent protein kinase and protein phosphorylation in regulation of mouse oocyte maturation. Dev Biol. 1986 Apr;114(2):453–462. [PubMed]
  • Chambers AE, Kotecha S, Towers N, Mohun TJ. Muscle-specific expression of SRF-related genes in the early embryo of Xenopus laevis. EMBO J. 1992 Dec;11(13):4981–4991. [PMC free article] [PubMed]
  • Davis LI, Fink GR. The NUP1 gene encodes an essential component of the yeast nuclear pore complex. Cell. 1990 Jun 15;61(6):965–978. [PubMed]
  • Dingwall C, Laskey RA. Protein import into the cell nucleus. Annu Rev Cell Biol. 1986;2:367–390. [PubMed]
  • Dingwall C, Laskey RA. Nuclear targeting sequences--a consensus? Trends Biochem Sci. 1991 Dec;16(12):478–481. [PubMed]
  • Dingwall C, Robbins J, Dilworth SM, Roberts B, Richardson WD. The nucleoplasmin nuclear location sequence is larger and more complex than that of SV-40 large T antigen. J Cell Biol. 1988 Sep;107(3):841–849. [PMC free article] [PubMed]
  • Fernandez A, Mery J, Vandromme M, Basset M, Cavadore JC, Lamb NJ. Effective intracellular inhibition of the cAMP-dependent protein kinase by microinjection of a modified form of the specific inhibitor peptide PKi in living fibroblasts. Exp Cell Res. 1991 Aug;195(2):468–477. [PubMed]
  • Garcia-Bustos J, Heitman J, Hall MN. Nuclear protein localization. Biochim Biophys Acta. 1991 Mar 7;1071(1):83–101. [PubMed]
  • Gashler AL, Swaminathan S, Sukhatme VP. A novel repression module, an extensive activation domain, and a bipartite nuclear localization signal defined in the immediate-early transcription factor Egr-1. Mol Cell Biol. 1993 Aug;13(8):4556–4571. [PMC free article] [PubMed]
  • Gauthier-Rouvière C, Basset M, Blanchard JM, Cavadore JC, Fernandez A, Lamb NJ. Casein kinase II induces c-fos expression via the serum response element pathway and p67SRF phosphorylation in living fibroblasts. EMBO J. 1991 Oct;10(10):2921–2930. [PMC free article] [PubMed]
  • Gauthier-Rouvière C, Caï QQ, Lautredou N, Fernandez A, Blanchard JM, Lamb NJ. Expression and purification of the DNA-binding domain of SRF: SRF-DB, a part of a DNA-binding protein which can act as a dominant negative mutant in vivo. Exp Cell Res. 1993 Dec;209(2):208–215. [PubMed]
  • Gauthier-Rouvière C, Cavadore JC, Blanchard JM, Lamb NJ, Fernandez A. p67SRF is a constitutive nuclear protein implicated in the modulation of genes required throughout the G1 period. Cell Regul. 1991 Jul;2(7):575–588. [PMC free article] [PubMed]
  • Gilman MZ, Wilson RN, Weinberg RA. Multiple protein-binding sites in the 5'-flanking region regulate c-fos expression. Mol Cell Biol. 1986 Dec;6(12):4305–4316. [PMC free article] [PubMed]
  • Gómez-Márquez J, Segade F. Prothymosin alpha is a nuclear protein. FEBS Lett. 1988 Jan 4;226(2):217–219. [PubMed]
  • Girard F, Strausfeld U, Fernandez A, Lamb NJ. Cyclin A is required for the onset of DNA replication in mammalian fibroblasts. Cell. 1991 Dec 20;67(6):1169–1179. [PubMed]
  • House C, Kemp BE. Protein kinase C contains a pseudosubstrate prototope in its regulatory domain. Science. 1987 Dec 18;238(4834):1726–1728. [PubMed]
  • Janknecht R, Hipskind RA, Houthaeve T, Nordheim A, Stunnenberg HG. Identification of multiple SRF N-terminal phosphorylation sites affecting DNA binding properties. EMBO J. 1992 Mar;11(3):1045–1054. [PMC free article] [PubMed]
  • Kalderon D, Richardson WD, Markham AF, Smith AE. Sequence requirements for nuclear location of simian virus 40 large-T antigen. Nature. 1984 Sep 6;311(5981):33–38. [PubMed]
  • Klamut HJ, Gangopadhyay SB, Worton RG, Ray PN. Molecular and functional analysis of the muscle-specific promoter region of the Duchenne muscular dystrophy gene. Mol Cell Biol. 1990 Jan;10(1):193–205. [PMC free article] [PubMed]
  • Lamb NJ, Cavadore JC, Labbe JC, Maurer RA, Fernandez A. Inhibition of cAMP-dependent protein kinase plays a key role in the induction of mitosis and nuclear envelope breakdown in mammalian cells. EMBO J. 1991 Jun;10(6):1523–1533. [PMC free article] [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]
  • Lanford RE, Kanda P, Kennedy RC. Induction of nuclear transport with a synthetic peptide homologous to the SV40 T antigen transport signal. Cell. 1986 Aug 15;46(4):575–582. [PubMed]
  • Li R, Shi Y, Thomas JO. Intracellular distribution of a nuclear localization signal binding protein. Exp Cell Res. 1992 Oct;202(2):355–365. [PubMed]
  • Maller JL, Krebs EG. Progesterone-stimulated meiotic cell division in Xenopus oocytes. Induction by regulatory subunit and inhibition by catalytic subunit of adenosine 3':5'-monophosphate-dependent protein kinase. J Biol Chem. 1977 Mar 10;252(5):1712–1718. [PubMed]
  • McVey D, Brizuela L, Mohr I, Marshak DR, Gluzman Y, Beach D. Phosphorylation of large tumour antigen by cdc2 stimulates SV40 DNA replication. Nature. 1989 Oct 12;341(6242):503–507. [PubMed]
  • Meier UT, Blobel G. A nuclear localization signal binding protein in the nucleolus. J Cell Biol. 1990 Dec;111(6 Pt 1):2235–2245. [PMC free article] [PubMed]
  • Meier UT, Blobel G. Nopp140 shuttles on tracks between nucleolus and cytoplasm. Cell. 1992 Jul 10;70(1):127–138. [PubMed]
  • Meijer L, Dostmann W, Genieser HG, Butt E, Jastorff B. Starfish oocyte maturation: evidence for a cyclic AMP-dependent inhibitory pathway. Dev Biol. 1989 May;133(1):58–66. [PubMed]
  • Minty A, Kedes L. Upstream regions of the human cardiac actin gene that modulate its transcription in muscle cells: presence of an evolutionarily conserved repeated motif. Mol Cell Biol. 1986 Jun;6(6):2125–2136. [PMC free article] [PubMed]
  • Mosialos G, Hamer P, Capobianco AJ, Laursen RA, Gilmore TD. A protein kinase-A recognition sequence is structurally linked to transformation by p59v-rel and cytoplasmic retention of p68c-rel. Mol Cell Biol. 1991 Dec;11(12):5867–5877. [PMC free article] [PubMed]
  • Nakabeppu Y, Nathans D. The basic region of Fos mediates specific DNA binding. EMBO J. 1989 Dec 1;8(12):3833–3841. [PMC free article] [PubMed]
  • Nasmyth K, Adolf G, Lydall D, Seddon A. The identification of a second cell cycle control on the HO promoter in yeast: cell cycle regulation of SW15 nuclear entry. Cell. 1990 Aug 24;62(4):631–647. [PubMed]
  • Norman C, Runswick M, Pollock R, Treisman R. Isolation and properties of cDNA clones encoding SRF, a transcription factor that binds to the c-fos serum response element. Cell. 1988 Dec 23;55(6):989–1003. [PubMed]
  • Norris JL, Manley JL. Selective nuclear transport of the Drosophila morphogen dorsal can be established by a signaling pathway involving the transmembrane protein Toll and protein kinase A. Genes Dev. 1992 Sep;6(9):1654–1667. [PubMed]
  • Pollock R, Treisman R. Human SRF-related proteins: DNA-binding properties and potential regulatory targets. Genes Dev. 1991 Dec;5(12A):2327–2341. [PubMed]
  • Prywes R, Roeder RG. Purification of the c-fos enhancer-binding protein. Mol Cell Biol. 1987 Oct;7(10):3482–3489. [PMC free article] [PubMed]
  • Rihs HP, Jans DA, Fan H, Peters R. The rate of nuclear cytoplasmic protein transport is determined by the casein kinase II site flanking the nuclear localization sequence of the SV40 T-antigen. EMBO J. 1991 Mar;10(3):633–639. [PMC free article] [PubMed]
  • Rivera VM, Miranti CK, Misra RP, Ginty DD, Chen RH, Blenis J, Greenberg ME. A growth factor-induced kinase phosphorylates the serum response factor at a site that regulates its DNA-binding activity. Mol Cell Biol. 1993 Oct;13(10):6260–6273. [PMC free article] [PubMed]
  • Roberts BL, Richardson WD, Smith AE. The effect of protein context on nuclear location signal function. Cell. 1987 Jul 31;50(3):465–475. [PubMed]
  • Roux P, Blanchard JM, Fernandez A, Lamb N, Jeanteur P, Piechaczyk M. Nuclear localization of c-Fos, but not v-Fos proteins, is controlled by extracellular signals. Cell. 1990 Oct 19;63(2):341–351. [PubMed]
  • Shi Y, Thomas JO. The transport of proteins into the nucleus requires the 70-kilodalton heat shock protein or its cytosolic cognate. Mol Cell Biol. 1992 May;12(5):2186–2192. [PMC free article] [PubMed]
  • Silver P, Sadler I, Osborne MA. Yeast proteins that recognize nuclear localization sequences. J Cell Biol. 1989 Sep;109(3):983–989. [PMC free article] [PubMed]
  • Silver PA. How proteins enter the nucleus. Cell. 1991 Feb 8;64(3):489–497. [PubMed]
  • Stochaj U, Bossie MA, van Zee K, Whalen AM, Silver PA. Analysis of conserved binding proteins for nuclear localization sequences. J Cell Sci. 1993 Jan;104(Pt 1):89–95. [PubMed]
  • Stochaj U, Osborne M, Kurihara T, Silver P. A yeast protein that binds nuclear localization signals: purification localization, and antibody inhibition of binding activity. J Cell Biol. 1991 Jun;113(6):1243–1254. [PMC free article] [PubMed]
  • Stochaj U, Silver PA. A conserved phosphoprotein that specifically binds nuclear localization sequences is involved in nuclear import. J Cell Biol. 1992 May;117(3):473–482. [PMC free article] [PubMed]
  • Treisman R. Transient accumulation of c-fos RNA following serum stimulation requires a conserved 5' element and c-fos 3' sequences. Cell. 1985 Oct;42(3):889–902. [PubMed]
  • Treisman R. Identification and purification of a polypeptide that binds to the c-fos serum response element. EMBO J. 1987 Sep;6(9):2711–2717. [PMC free article] [PubMed]
  • Treisman R, Ammerer G. The SRF and MCM1 transcription factors. Curr Opin Genet Dev. 1992 Apr;2(2):221–226. [PubMed]
  • Vandromme M, Carnac G, Gauthier-Rouvière C, Fesquet D, Lamb N, Fernandez A. Nuclear import of the myogenic factor MyoD requires cAMP-dependent protein kinase activity but not the direct phosphorylation of MyoD. J Cell Sci. 1994 Feb;107(Pt 2):613–620. [PubMed]
  • Vandromme M, Gauthier-Rouvière C, Carnac G, Lamb N, Fernandez A. Serum response factor p67SRF is expressed and required during myogenic differentiation of both mouse C2 and rat L6 muscle cell lines. J Cell Biol. 1992 Sep;118(6):1489–1500. [PMC free article] [PubMed]
  • Vriz S, Lemaitre JM, Leibovici M, Thierry N, Méchali M. Comparative analysis of the intracellular localization of c-Myc, c-Fos, and replicative proteins during cell cycle progression. Mol Cell Biol. 1992 Aug;12(8):3548–3555. [PMC free article] [PubMed]
  • Wilson IA, Niman HL, Houghten RA, Cherenson AR, Connolly ML, Lerner RA. The structure of an antigenic determinant in a protein. Cell. 1984 Jul;37(3):767–778. [PubMed]
  • Yamasaki L, Kanda P, Lanford RE. Identification of four nuclear transport signal-binding proteins that interact with diverse transport signals. Mol Cell Biol. 1989 Jul;9(7):3028–3036. [PMC free article] [PubMed]
  • Zeilig CE, Goldberg ND. Cell-cycle-related changes of 3':5'-cyclic GMP levels in Novikoff hepatoma cells. Proc Natl Acad Sci U S A. 1977 Mar;74(3):1052–1056. [PMC free article] [PubMed]

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


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


  • MedGen
    Related information in MedGen
  • PubMed
    PubMed citations for these articles
  • Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...