Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1997 Oct;17(10):5707–5718. doi: 10.1128/mcb.17.10.5707

Self-association of the single-KH-domain family members Sam68, GRP33, GLD-1, and Qk1: role of the KH domain.

T Chen 1, B B Damaj 1, C Herrera 1, P Lasko 1, S Richard 1
PMCID: PMC232419  PMID: 9315629

Abstract

Sam68 is a member of a growing family of proteins that contain a single KH domain embedded in a larger conserved domain of approximately 170 amino acids. Loops 1 and 4 of this KH domain family are longer than the corresponding loops in other KH domains and contain conserved residues. KH domains are protein motifs that are involved in RNA binding and are often present in multiple copies. Here we demonstrate by coimmunoprecipitation studies that Sam68 self-associated and that cellular RNA was required for the association. Deletion studies demonstrated that the Sam68 KH domain loops 1 and 4 were required for self-association. The Sam68 interaction was also observed in Saccharomyces cerevisiae by the two-hybrid system. In situ chemical cross-linking studies in mammalian cells demonstrated that Sam68 oligomerized in vivo. These Sam68 complexes bound homopolymeric RNA and the SH3 domains of p59fyn and phospholipase Cgamma1 in vitro, demonstrating that Sam68 associates with RNA and signaling molecules as a multimer. The formation of the Sam68 complex was inhibited by p59fyn, suggesting that tyrosine phosphorylation regulates Sam68 oligomerization. Other Sam68 family members including Artemia salina GRP33, Caenorhabditis elegans GLD-1, and mouse Qk1 also oligomerized. In addition, Sam68, GRP33, GLD-1, and Qk1 associated with other KH domain proteins such as Bicaudal C. These observations indicate that the single KH domain found in the Sam68 family, in addition to mediating protein-RNA interactions, mediates protein-protein interactions.

Full Text

The Full Text of this article is available as a PDF (1.9 MB).

Selected References

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

  1. Arning S., Grüter P., Bilbe G., Krämer A. Mammalian splicing factor SF1 is encoded by variant cDNAs and binds to RNA. RNA. 1996 Aug;2(8):794–810. [PMC free article] [PubMed] [Google Scholar]
  2. Ashley C. T., Jr, Wilkinson K. D., Reines D., Warren S. T. FMR1 protein: conserved RNP family domains and selective RNA binding. Science. 1993 Oct 22;262(5133):563–566. doi: 10.1126/science.7692601. [DOI] [PubMed] [Google Scholar]
  3. Barlat I., Maurier F., Duchesne M., Guitard E., Tocque B., Schweighoffer F. A role for Sam68 in cell cycle progression antagonized by a spliced variant within the KH domain. J Biol Chem. 1997 Feb 7;272(6):3129–3132. doi: 10.1074/jbc.272.6.3129. [DOI] [PubMed] [Google Scholar]
  4. Bunnell S. C., Henry P. A., Kolluri R., Kirchhausen T., Rickles R. J., Berg L. J. Identification of Itk/Tsk Src homology 3 domain ligands. J Biol Chem. 1996 Oct 11;271(41):25646–25656. doi: 10.1074/jbc.271.41.25646. [DOI] [PubMed] [Google Scholar]
  5. Burd C. G., Dreyfuss G. Conserved structures and diversity of functions of RNA-binding proteins. Science. 1994 Jul 29;265(5172):615–621. doi: 10.1126/science.8036511. [DOI] [PubMed] [Google Scholar]
  6. Cruz-Alvarez M., Pellicer A. Cloning of a full-length complementary DNA for an Artemia salina glycine-rich protein. Structural relationship with RNA binding proteins. J Biol Chem. 1987 Oct 5;262(28):13377–13380. [PubMed] [Google Scholar]
  7. De Boulle K., Verkerk A. J., Reyniers E., Vits L., Hendrickx J., Van Roy B., Van den Bos F., de Graaff E., Oostra B. A., Willems P. J. A point mutation in the FMR-1 gene associated with fragile X mental retardation. Nat Genet. 1993 Jan;3(1):31–35. doi: 10.1038/ng0193-31. [DOI] [PubMed] [Google Scholar]
  8. Durfee T., Becherer K., Chen P. L., Yeh S. H., Yang Y., Kilburn A. E., Lee W. H., Elledge S. J. The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit. Genes Dev. 1993 Apr;7(4):555–569. doi: 10.1101/gad.7.4.555. [DOI] [PubMed] [Google Scholar]
  9. Ebersole T. A., Chen Q., Justice M. J., Artzt K. The quaking gene product necessary in embryogenesis and myelination combines features of RNA binding and signal transduction proteins. Nat Genet. 1996 Mar;12(3):260–265. doi: 10.1038/ng0396-260. [DOI] [PubMed] [Google Scholar]
  10. Evan G. I., Lewis G. K., Bishop J. M. Isolation of monoclonal antibodies specific for products of avian oncogene myb. Mol Cell Biol. 1984 Dec;4(12):2843–2850. doi: 10.1128/mcb.4.12.2843. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fields S., Song O. A novel genetic system to detect protein-protein interactions. Nature. 1989 Jul 20;340(6230):245–246. doi: 10.1038/340245a0. [DOI] [PubMed] [Google Scholar]
  12. Francis R., Barton M. K., Kimble J., Schedl T. gld-1, a tumor suppressor gene required for oocyte development in Caenorhabditis elegans. Genetics. 1995 Feb;139(2):579–606. doi: 10.1093/genetics/139.2.579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Francis R., Maine E., Schedl T. Analysis of the multiple roles of gld-1 in germline development: interactions with the sex determination cascade and the glp-1 signaling pathway. Genetics. 1995 Feb;139(2):607–630. doi: 10.1093/genetics/139.2.607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fumagalli S., Totty N. F., Hsuan J. J., Courtneidge S. A. A target for Src in mitosis. Nature. 1994 Apr 28;368(6474):871–874. doi: 10.1038/368871a0. [DOI] [PubMed] [Google Scholar]
  15. Fusaki N., Iwamatsu A., Iwashima M., Fujisawa J. i. Interaction between Sam68 and Src family tyrosine kinases, Fyn and Lck, in T cell receptor signaling. J Biol Chem. 1997 Mar 7;272(10):6214–6219. doi: 10.1074/jbc.272.10.6214. [DOI] [PubMed] [Google Scholar]
  16. Gibson T. J., Thompson J. D., Heringa J. The KH domain occurs in a diverse set of RNA-binding proteins that include the antiterminator NusA and is probably involved in binding to nucleic acid. FEBS Lett. 1993 Jun 21;324(3):361–366. doi: 10.1016/0014-5793(93)80152-k. [DOI] [PubMed] [Google Scholar]
  17. Hardy R. J., Loushin C. L., Friedrich V. L., Jr, Chen Q., Ebersole T. A., Lazzarini R. A., Artzt K. Neural cell type-specific expression of QKI proteins is altered in quakingviable mutant mice. J Neurosci. 1996 Dec 15;16(24):7941–7949. doi: 10.1523/JNEUROSCI.16-24-07941.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jones A. R., Francis R., Schedl T. GLD-1, a cytoplasmic protein essential for oocyte differentiation, shows stage- and sex-specific expression during Caenorhabditis elegans germline development. Dev Biol. 1996 Nov 25;180(1):165–183. doi: 10.1006/dbio.1996.0293. [DOI] [PubMed] [Google Scholar]
  19. Jones A. R., Schedl T. Mutations in gld-1, a female germ cell-specific tumor suppressor gene in Caenorhabditis elegans, affect a conserved domain also found in Src-associated protein Sam68. Genes Dev. 1995 Jun 15;9(12):1491–1504. doi: 10.1101/gad.9.12.1491. [DOI] [PubMed] [Google Scholar]
  20. Lawe D. C., Hahn C., Wong A. J. The Nck SH2/SH3 adaptor protein is present in the nucleus and associates with the nuclear protein SAM68. Oncogene. 1997 Jan 16;14(2):223–231. doi: 10.1038/sj.onc.1200821. [DOI] [PubMed] [Google Scholar]
  21. Lock P., Fumagalli S., Polakis P., McCormick F., Courtneidge S. A. The human p62 cDNA encodes Sam68 and not the RasGAP-associated p62 protein. Cell. 1996 Jan 12;84(1):23–24. doi: 10.1016/s0092-8674(00)80989-7. [DOI] [PubMed] [Google Scholar]
  22. Maa M. C., Leu T. H., Trandel B. J., Chang J. H., Parsons S. J. A protein that is highly related to GTPase-activating protein-associated p62 complexes with phospholipase C gamma. Mol Cell Biol. 1994 Aug;14(8):5466–5473. doi: 10.1128/mcb.14.8.5466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Mahone M., Saffman E. E., Lasko P. F. Localized Bicaudal-C RNA encodes a protein containing a KH domain, the RNA binding motif of FMR1. EMBO J. 1995 May 1;14(9):2043–2055. doi: 10.1002/j.1460-2075.1995.tb07196.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. McBride A. E., Schlegel A., Kirkegaard K. Human protein Sam68 relocalization and interaction with poliovirus RNA polymerase in infected cells. Proc Natl Acad Sci U S A. 1996 Mar 19;93(6):2296–2301. doi: 10.1073/pnas.93.6.2296. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Musco G., Stier G., Joseph C., Castiglione Morelli M. A., Nilges M., Gibson T. J., Pastore A. Three-dimensional structure and stability of the KH domain: molecular insights into the fragile X syndrome. Cell. 1996 Apr 19;85(2):237–245. doi: 10.1016/s0092-8674(00)81100-9. [DOI] [PubMed] [Google Scholar]
  26. Pawson T. Protein modules and signalling networks. Nature. 1995 Feb 16;373(6515):573–580. doi: 10.1038/373573a0. [DOI] [PubMed] [Google Scholar]
  27. Pieretti M., Zhang F. P., Fu Y. H., Warren S. T., Oostra B. A., Caskey C. T., Nelson D. L. Absence of expression of the FMR-1 gene in fragile X syndrome. Cell. 1991 Aug 23;66(4):817–822. doi: 10.1016/0092-8674(91)90125-i. [DOI] [PubMed] [Google Scholar]
  28. Richard S., Yu D., Blumer K. J., Hausladen D., Olszowy M. W., Connelly P. A., Shaw A. S. Association of p62, a multifunctional SH2- and SH3-domain-binding protein, with src family tyrosine kinases, Grb2, and phospholipase C gamma-1. Mol Cell Biol. 1995 Jan;15(1):186–197. doi: 10.1128/mcb.15.1.186. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Richard S., Zingg H. H. Identification of a retinoic acid response element in the human oxytocin promoter. J Biol Chem. 1991 Nov 15;266(32):21428–21433. [PubMed] [Google Scholar]
  30. SIDMAN R. L., DICKIE M. M., APPEL S. H. MUTANT MICE (QUAKING AND JIMPY) WITH DEFICIENT MYELINATION IN THE CENTRAL NERVOUS SYSTEM. Science. 1964 Apr 17;144(3616):309–311. doi: 10.1126/science.144.3616.309. [DOI] [PubMed] [Google Scholar]
  31. Siomi H., Choi M., Siomi M. C., Nussbaum R. L., Dreyfuss G. Essential role for KH domains in RNA binding: impaired RNA binding by a mutation in the KH domain of FMR1 that causes fragile X syndrome. Cell. 1994 Apr 8;77(1):33–39. doi: 10.1016/0092-8674(94)90232-1. [DOI] [PubMed] [Google Scholar]
  32. Siomi H., Matunis M. J., Michael W. M., Dreyfuss G. The pre-mRNA binding K protein contains a novel evolutionarily conserved motif. Nucleic Acids Res. 1993 Mar 11;21(5):1193–1198. doi: 10.1093/nar/21.5.1193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Siomi M. C., Zhang Y., Siomi H., Dreyfuss G. Specific sequences in the fragile X syndrome protein FMR1 and the FXR proteins mediate their binding to 60S ribosomal subunits and the interactions among them. Mol Cell Biol. 1996 Jul;16(7):3825–3832. doi: 10.1128/mcb.16.7.3825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Taggart A. K., Pugh B. F. Dimerization of TFIID when not bound to DNA. Science. 1996 May 31;272(5266):1331–1333. doi: 10.1126/science.272.5266.1331. [DOI] [PubMed] [Google Scholar]
  35. Taylor S. J., Anafi M., Pawson T., Shalloway D. Functional interaction between c-Src and its mitotic target, Sam 68. J Biol Chem. 1995 Apr 28;270(17):10120–10124. doi: 10.1074/jbc.270.17.10120. [DOI] [PubMed] [Google Scholar]
  36. Taylor S. J., Shalloway D. An RNA-binding protein associated with Src through its SH2 and SH3 domains in mitosis. Nature. 1994 Apr 28;368(6474):867–871. doi: 10.1038/368867a0. [DOI] [PubMed] [Google Scholar]
  37. Toda T., Iida A., Miwa T., Nakamura Y., Imai T. Isolation and characterization of a novel gene encoding nuclear protein at a locus (D11S636) tightly linked to multiple endocrine neoplasia type 1 (MEN1). Hum Mol Genet. 1994 Mar;3(3):465–470. doi: 10.1093/hmg/3.3.465. [DOI] [PubMed] [Google Scholar]
  38. Trüb T., Frantz J. D., Miyazaki M., Band H., Shoelson S. E. The role of a lymphoid-restricted, Grb2-like SH3-SH2-SH3 protein in T cell receptor signaling. J Biol Chem. 1997 Jan 10;272(2):894–902. doi: 10.1074/jbc.272.2.894. [DOI] [PubMed] [Google Scholar]
  39. Verkerk A. J., Pieretti M., Sutcliffe J. S., Fu Y. H., Kuhl D. P., Pizzuti A., Reiner O., Richards S., Victoria M. F., Zhang F. P. Identification of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome. Cell. 1991 May 31;65(5):905–914. doi: 10.1016/0092-8674(91)90397-h. [DOI] [PubMed] [Google Scholar]
  40. Vogel L. B., Fujita D. J. p70 phosphorylation and binding to p56lck is an early event in interleukin-2-induced onset of cell cycle progression in T-lymphocytes. J Biol Chem. 1995 Feb 10;270(6):2506–2511. doi: 10.1074/jbc.270.6.2506. [DOI] [PubMed] [Google Scholar]
  41. Wang L. L., Richard S., Shaw A. S. P62 association with RNA is regulated by tyrosine phosphorylation. J Biol Chem. 1995 Feb 3;270(5):2010–2013. doi: 10.1074/jbc.270.5.2010. [DOI] [PubMed] [Google Scholar]
  42. Weng Z., Thomas S. M., Rickles R. J., Taylor J. A., Brauer A. W., Seidel-Dugan C., Michael W. M., Dreyfuss G., Brugge J. S. Identification of Src, Fyn, and Lyn SH3-binding proteins: implications for a function of SH3 domains. Mol Cell Biol. 1994 Jul;14(7):4509–4521. doi: 10.1128/mcb.14.7.4509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wilson R., Ainscough R., Anderson K., Baynes C., Berks M., Bonfield J., Burton J., Connell M., Copsey T., Cooper J. 2.2 Mb of contiguous nucleotide sequence from chromosome III of C. elegans. Nature. 1994 Mar 3;368(6466):32–38. doi: 10.1038/368032a0. [DOI] [PubMed] [Google Scholar]
  44. Wong G., Müller O., Clark R., Conroy L., Moran M. F., Polakis P., McCormick F. Molecular cloning and nucleic acid binding properties of the GAP-associated tyrosine phosphoprotein p62. Cell. 1992 May 1;69(3):551–558. doi: 10.1016/0092-8674(92)90455-l. [DOI] [PubMed] [Google Scholar]
  45. Zhang Y., O'Connor J. P., Siomi M. C., Srinivasan S., Dutra A., Nussbaum R. L., Dreyfuss G. The fragile X mental retardation syndrome protein interacts with novel homologs FXR1 and FXR2. EMBO J. 1995 Nov 1;14(21):5358–5366. doi: 10.1002/j.1460-2075.1995.tb00220.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Zorn A. M., Grow M., Patterson K. D., Ebersole T. A., Chen Q., Artzt K., Krieg P. A. Remarkable sequence conservation of transcripts encoding amphibian and mammalian homologues of quaking, a KH domain RNA-binding protein. Gene. 1997 Apr 1;188(2):199–206. doi: 10.1016/s0378-1119(96)00795-0. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES