Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1998 May 1;331(Pt 3):885–892. doi: 10.1042/bj3310885

LIM domains of cysteine-rich protein 1 (CRP1) are essential for its zyxin-binding function.

K L Schmeichel 1, M C Beckerle 1
PMCID: PMC1219431  PMID: 9560318

Abstract

Previous studies have demonstrated that the adhesion-plaque protein, zyxin, interacts specifically with a 23 kDa protein, called the cysteine-rich protein 1 (CRP1), which has been implicated in myogenesis. Primary sequence analyses have revealed that both zyxin and CRP1 exhibit multiple copies of a structural motif called the LIM domain. LIM domains, which are defined by the consensus CX2CX16-23HX2CX2CX2CX16-23CX2-3(C,H,D), are found in a variety of proteins that are involved in cell growth and differentiation. Recent studies have established that LIM domains are zinc-binding structures that mediate specific protein-protein interactions. For example, in the case of the zyxin-CRP1 interaction, one of zyxin's three LIM domains is necessary and sufficient for binding to CRP1. Because the CRP1 molecule is comprised primarily of two LIM domains, we were interested in the possibility that the binding site for zyxin on CRP1 might also be contained within a single LIM domain. Consistent with the hypothesis that the LIM domains of CRP1 are critical for the protein's zyxin-binding function, zinc-depleted CRP1 displays a reduced zyxin-binding activity. However, domain mapping analyses have revealed that neither of the two individual LIM domains of CRP1 can support a wild-type interaction with zyxin. Collectively, our results suggest that the binding site for zyxin on CRP1 is not contained within a single contiguous sequence of amino acids. Instead, the interaction appears to rely on the co-ordinate action of a number of residues that are displayed in both of CRP1's LIM domains.

Full Text

The Full Text of this article is available as a PDF (340.6 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abel T., Maniatis T. Gene regulation. Action of leucine zippers. Nature. 1989 Sep 7;341(6237):24–25. doi: 10.1038/341024a0. [DOI] [PubMed] [Google Scholar]
  2. Agulnick A. D., Taira M., Breen J. J., Tanaka T., Dawid I. B., Westphal H. Interactions of the LIM-domain-binding factor Ldb1 with LIM homeodomain proteins. Nature. 1996 Nov 21;384(6606):270–272. doi: 10.1038/384270a0. [DOI] [PubMed] [Google Scholar]
  3. Arber S., Halder G., Caroni P. Muscle LIM protein, a novel essential regulator of myogenesis, promotes myogenic differentiation. Cell. 1994 Oct 21;79(2):221–231. doi: 10.1016/0092-8674(94)90192-9. [DOI] [PubMed] [Google Scholar]
  4. Arber S., Hunter J. J., Ross J., Jr, Hongo M., Sansig G., Borg J., Perriard J. C., Chien K. R., Caroni P. MLP-deficient mice exhibit a disruption of cardiac cytoarchitectural organization, dilated cardiomyopathy, and heart failure. Cell. 1997 Feb 7;88(3):393–403. doi: 10.1016/s0092-8674(00)81878-4. [DOI] [PubMed] [Google Scholar]
  5. Archer V. E., Breton J., Sanchez-Garcia I., Osada H., Forster A., Thomson A. J., Rabbitts T. H. Cysteine-rich LIM domains of LIM-homeodomain and LIM-only proteins contain zinc but not iron. Proc Natl Acad Sci U S A. 1994 Jan 4;91(1):316–320. doi: 10.1073/pnas.91.1.316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bach I., Rhodes S. J., Pearse R. V., 2nd, Heinzel T., Gloss B., Scully K. M., Sawchenko P. E., Rosenfeld M. G. P-Lim, a LIM homeodomain factor, is expressed during pituitary organ and cell commitment and synergizes with Pit-1. Proc Natl Acad Sci U S A. 1995 Mar 28;92(7):2720–2724. doi: 10.1073/pnas.92.7.2720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Banecki B., Liberek K., Wall D., Wawrzynów A., Georgopoulos C., Bertoli E., Tanfani F., Zylicz M. Structure-function analysis of the zinc finger region of the DnaJ molecular chaperone. J Biol Chem. 1996 Jun 21;271(25):14840–14848. doi: 10.1074/jbc.271.25.14840. [DOI] [PubMed] [Google Scholar]
  8. Beckerle M. C. Zyxin: zinc fingers at sites of cell adhesion. Bioessays. 1997 Nov;19(11):949–957. doi: 10.1002/bies.950191104. [DOI] [PubMed] [Google Scholar]
  9. Blair S. S., Brower D. L., Thomas J. B., Zavortink M. The role of apterous in the control of dorsoventral compartmentalization and PS integrin gene expression in the developing wing of Drosophila. Development. 1994 Jul;120(7):1805–1815. doi: 10.1242/dev.120.7.1805. [DOI] [PubMed] [Google Scholar]
  10. Crawford A. W., Pino J. D., Beckerle M. C. Biochemical and molecular characterization of the chicken cysteine-rich protein, a developmentally regulated LIM-domain protein that is associated with the actin cytoskeleton. J Cell Biol. 1994 Jan;124(1-2):117–127. doi: 10.1083/jcb.124.1.117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dawid I. B., Toyama R., Taira M. LIM domain proteins. C R Acad Sci III. 1995 Mar;318(3):295–306. [PubMed] [Google Scholar]
  12. Durick K., Wu R. Y., Gill G. N., Taylor S. S. Mitogenic signaling by Ret/ptc2 requires association with enigma via a LIM domain. J Biol Chem. 1996 May 31;271(22):12691–12694. doi: 10.1074/jbc.271.22.12691. [DOI] [PubMed] [Google Scholar]
  13. Edmondson D. G., Olson E. N. Helix-loop-helix proteins as regulators of muscle-specific transcription. J Biol Chem. 1993 Jan 15;268(2):755–758. [PubMed] [Google Scholar]
  14. Gill G. N. The enigma of LIM domains. Structure. 1995 Dec 15;3(12):1285–1289. doi: 10.1016/s0969-2126(01)00265-9. [DOI] [PubMed] [Google Scholar]
  15. Johnson J. D., Zhang W., Rudnick A., Rutter W. J., German M. S. Transcriptional synergy between LIM-homeodomain proteins and basic helix-loop-helix proteins: the LIM2 domain determines specificity. Mol Cell Biol. 1997 Jul;17(7):3488–3496. doi: 10.1128/mcb.17.7.3488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jurata L. W., Kenny D. A., Gill G. N. Nuclear LIM interactor, a rhombotin and LIM homeodomain interacting protein, is expressed early in neuronal development. Proc Natl Acad Sci U S A. 1996 Oct 15;93(21):11693–11698. doi: 10.1073/pnas.93.21.11693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Konrat R., Weiskirchen R., Kräutler B., Bister K. Solution structure of the carboxyl-terminal LIM domain from quail cysteine-rich protein CRP2. J Biol Chem. 1997 May 2;272(18):12001–12007. doi: 10.1074/jbc.272.18.12001. [DOI] [PubMed] [Google Scholar]
  18. Kosa J. L., Michelsen J. W., Louis H. A., Olsen J. I., Davis D. R., Beckerle M. C., Winge D. R. Common metal ion coordination in LIM domain proteins. Biochemistry. 1994 Jan 18;33(2):468–477. doi: 10.1021/bi00168a011. [DOI] [PubMed] [Google Scholar]
  19. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  20. Liebhaber S. A., Emery J. G., Urbanek M., Wang X. K., Cooke N. E. Characterization of a human cDNA encoding a widely expressed and highly conserved cysteine-rich protein with an unusual zinc-finger motif. Nucleic Acids Res. 1990 Jul 11;18(13):3871–3879. doi: 10.1093/nar/18.13.3871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Louis H. A., Pino J. D., Schmeichel K. L., Pomiès P., Beckerle M. C. Comparison of three members of the cysteine-rich protein family reveals functional conservation and divergent patterns of gene expression. J Biol Chem. 1997 Oct 24;272(43):27484–27491. doi: 10.1074/jbc.272.43.27484. [DOI] [PubMed] [Google Scholar]
  22. Macalma T., Otte J., Hensler M. E., Bockholt S. M., Louis H. A., Kalff-Suske M., Grzeschik K. H., von der Ahe D., Beckerle M. C. Molecular characterization of human zyxin. J Biol Chem. 1996 Dec 6;271(49):31470–31478. doi: 10.1074/jbc.271.49.31470. [DOI] [PubMed] [Google Scholar]
  23. Michelsen J. W., Schmeichel K. L., Beckerle M. C., Winge D. R. The LIM motif defines a specific zinc-binding protein domain. Proc Natl Acad Sci U S A. 1993 May 15;90(10):4404–4408. doi: 10.1073/pnas.90.10.4404. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Michelsen J. W., Sewell A. K., Louis H. A., Olsen J. I., Davis D. R., Winge D. R., Beckerle M. C. Mutational analysis of the metal sites in an LIM domain. J Biol Chem. 1994 Apr 15;269(15):11108–11113. [PubMed] [Google Scholar]
  25. Osada H., Grutz G., Axelson H., Forster A., Rabbitts T. H. Association of erythroid transcription factors: complexes involving the LIM protein RBTN2 and the zinc-finger protein GATA1. Proc Natl Acad Sci U S A. 1995 Oct 10;92(21):9585–9589. doi: 10.1073/pnas.92.21.9585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pfaff S. L., Mendelsohn M., Stewart C. L., Edlund T., Jessell T. M. Requirement for LIM homeobox gene Isl1 in motor neuron generation reveals a motor neuron-dependent step in interneuron differentiation. Cell. 1996 Jan 26;84(2):309–320. doi: 10.1016/s0092-8674(00)80985-x. [DOI] [PubMed] [Google Scholar]
  27. Pérez-Alvarado G. C., Kosa J. L., Louis H. A., Beckerle M. C., Winge D. R., Summers M. F. Structure of the cysteine-rich intestinal protein, CRIP. J Mol Biol. 1996 Mar 22;257(1):153–174. doi: 10.1006/jmbi.1996.0153. [DOI] [PubMed] [Google Scholar]
  28. Pérez-Alvarado G. C., Miles C., Michelsen J. W., Louis H. A., Winge D. R., Beckerle M. C., Summers M. F. Structure of the carboxy-terminal LIM domain from the cysteine rich protein CRP. Nat Struct Biol. 1994 Jun;1(6):388–398. doi: 10.1038/nsb0694-388. [DOI] [PubMed] [Google Scholar]
  29. Sadler I., Crawford A. W., Michelsen J. W., Beckerle M. C. Zyxin and cCRP: two interactive LIM domain proteins associated with the cytoskeleton. J Cell Biol. 1992 Dec;119(6):1573–1587. doi: 10.1083/jcb.119.6.1573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Schmeichel K. L., Beckerle M. C. Molecular dissection of a LIM domain. Mol Biol Cell. 1997 Feb;8(2):219–230. doi: 10.1091/mbc.8.2.219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Schmeichel K. L., Beckerle M. C. The LIM domain is a modular protein-binding interface. Cell. 1994 Oct 21;79(2):211–219. doi: 10.1016/0092-8674(94)90191-0. [DOI] [PubMed] [Google Scholar]
  32. Shawlot W., Behringer R. R. Requirement for Lim1 in head-organizer function. Nature. 1995 Mar 30;374(6521):425–430. doi: 10.1038/374425a0. [DOI] [PubMed] [Google Scholar]
  33. Stronach B. E., Siegrist S. E., Beckerle M. C. Two muscle-specific LIM proteins in Drosophila. J Cell Biol. 1996 Sep;134(5):1179–1195. doi: 10.1083/jcb.134.5.1179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sánchez-García I., Rabbitts T. H. The LIM domain: a new structural motif found in zinc-finger-like proteins. Trends Genet. 1994 Sep;10(9):315–320. doi: 10.1016/0168-9525(94)90034-5. [DOI] [PubMed] [Google Scholar]
  35. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Valge-Archer V. E., Osada H., Warren A. J., Forster A., Li J., Baer R., Rabbitts T. H. The LIM protein RBTN2 and the basic helix-loop-helix protein TAL1 are present in a complex in erythroid cells. Proc Natl Acad Sci U S A. 1994 Aug 30;91(18):8617–8621. doi: 10.1073/pnas.91.18.8617. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wadman I., Li J., Bash R. O., Forster A., Osada H., Rabbitts T. H., Baer R. Specific in vivo association between the bHLH and LIM proteins implicated in human T cell leukemia. EMBO J. 1994 Oct 17;13(20):4831–4839. doi: 10.1002/j.1460-2075.1994.tb06809.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Warren A. J., Colledge W. H., Carlton M. B., Evans M. J., Smith A. J., Rabbitts T. H. The oncogenic cysteine-rich LIM domain protein rbtn2 is essential for erythroid development. Cell. 1994 Jul 15;78(1):45–57. doi: 10.1016/0092-8674(94)90571-1. [DOI] [PubMed] [Google Scholar]
  39. Weiskirchen R., Bister K. Suppression in transformed avian fibroblasts of a gene (crp) encoding a cysteine-rich protein containing LIM domains. Oncogene. 1993 Sep;8(9):2317–2324. [PubMed] [Google Scholar]
  40. Weiskirchen R., Pino J. D., Macalma T., Bister K., Beckerle M. C. The cysteine-rich protein family of highly related LIM domain proteins. J Biol Chem. 1995 Dec 1;270(48):28946–28954. doi: 10.1074/jbc.270.48.28946. [DOI] [PubMed] [Google Scholar]
  41. Wu R. Y., Gill G. N. LIM domain recognition of a tyrosine-containing tight turn. J Biol Chem. 1994 Oct 7;269(40):25085–25090. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES