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. 1995 Dec 5;92(25):11761–11765. doi: 10.1073/pnas.92.25.11761

The specificity of the transforming growth factor beta receptor kinases determined by a spatially addressable peptide library.

K Luo 1, P Zhou 1, H F Lodish 1
PMCID: PMC40482  PMID: 8524844

Abstract

Type I and II receptors for the transforming growth factor beta (TGF-beta) are transmembrane serine/threonine kinases that are essential for TGF-beta signaling. However, little is known about their in vivo substrates or signal transduction pathways. To determine the substrate specificity of these kinases, we developed combinatorial peptide libraries synthesized on a hydrophilic matrix that is easily accessible to proteins in aqueous solutions. When we subjected these libraries to phosphorylation by the cAMP-dependent protein kinase, we obtained the optimal peptide sequence RRXS (I/L/V), in perfect agreement with the substrate sequence deduced from mutagenesis and crystal structure analyses. By using the same libraries, we showed that the optimal substrate peptide for both the type I and II TGF-beta receptors was KKKKKK(S/T)XXX. Since the two kinases are thought to play different roles in intracellular signal transduction, it was a surprise to find that they have almost identical substrate specificity. Our method is direct, sensitive, and simple and provides information about the kinase specificity for all the amino acid residues at each position.

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Selected References

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  1. Attisano L., Cárcamo J., Ventura F., Weis F. M., Massagué J., Wrana J. L. Identification of human activin and TGF beta type I receptors that form heteromeric kinase complexes with type II receptors. Cell. 1993 Nov 19;75(4):671–680. doi: 10.1016/0092-8674(93)90488-c. [DOI] [PubMed] [Google Scholar]
  2. Attisano L., Wrana J. L., Cheifetz S., Massagué J. Novel activin receptors: distinct genes and alternative mRNA splicing generate a repertoire of serine/threonine kinase receptors. Cell. 1992 Jan 10;68(1):97–108. doi: 10.1016/0092-8674(92)90209-u. [DOI] [PubMed] [Google Scholar]
  3. Bassing C. H., Yingling J. M., Howe D. J., Wang T., He W. W., Gustafson M. L., Shah P., Donahoe P. K., Wang X. F. A transforming growth factor beta type I receptor that signals to activate gene expression. Science. 1994 Jan 7;263(5143):87–89. doi: 10.1126/science.8272871. [DOI] [PubMed] [Google Scholar]
  4. Darnell J. E., Jr, Kerr I. M., Stark G. R. Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science. 1994 Jun 3;264(5164):1415–1421. doi: 10.1126/science.8197455. [DOI] [PubMed] [Google Scholar]
  5. Estevez M., Attisano L., Wrana J. L., Albert P. S., Massagué J., Riddle D. L. The daf-4 gene encodes a bone morphogenetic protein receptor controlling C. elegans dauer larva development. Nature. 1993 Oct 14;365(6447):644–649. doi: 10.1038/365644a0. [DOI] [PubMed] [Google Scholar]
  6. Frangioni J. V., Neel B. G. Solubilization and purification of enzymatically active glutathione S-transferase (pGEX) fusion proteins. Anal Biochem. 1993 Apr;210(1):179–187. doi: 10.1006/abio.1993.1170. [DOI] [PubMed] [Google Scholar]
  7. Franzén P., ten Dijke P., Ichijo H., Yamashita H., Schulz P., Heldin C. H., Miyazono K. Cloning of a TGF beta type I receptor that forms a heteromeric complex with the TGF beta type II receptor. Cell. 1993 Nov 19;75(4):681–692. doi: 10.1016/0092-8674(93)90489-d. [DOI] [PubMed] [Google Scholar]
  8. Kemp B. E., Graves D. J., Benjamini E., Krebs E. G. Role of multiple basic residues in determining the substrate specificity of cyclic AMP-dependent protein kinase. J Biol Chem. 1977 Jul 25;252(14):4888–4894. [PubMed] [Google Scholar]
  9. Knighton D. R., Zheng J. H., Ten Eyck L. F., Xuong N. H., Taylor S. S., Sowadski J. M. Structure of a peptide inhibitor bound to the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. Science. 1991 Jul 26;253(5018):414–420. doi: 10.1126/science.1862343. [DOI] [PubMed] [Google Scholar]
  10. Koenig B. B., Cook J. S., Wolsing D. H., Ting J., Tiesman J. P., Correa P. E., Olson C. A., Pecquet A. L., Ventura F., Grant R. A. Characterization and cloning of a receptor for BMP-2 and BMP-4 from NIH 3T3 cells. Mol Cell Biol. 1994 Sep;14(9):5961–5974. doi: 10.1128/mcb.14.9.5961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Lemmon M. A., Schlessinger J. Regulation of signal transduction and signal diversity by receptor oligomerization. Trends Biochem Sci. 1994 Nov;19(11):459–463. doi: 10.1016/0968-0004(94)90130-9. [DOI] [PubMed] [Google Scholar]
  12. Lin H. Y., Moustakas A., Knaus P., Wells R. G., Henis Y. I., Lodish H. F. The soluble exoplasmic domain of the type II transforming growth factor (TGF)-beta receptor. A heterogeneously glycosylated protein with high affinity and selectivity for TGF-beta ligands. J Biol Chem. 1995 Feb 10;270(6):2747–2754. doi: 10.1074/jbc.270.6.2747. [DOI] [PubMed] [Google Scholar]
  13. Lin H. Y., Wang X. F. Expression cloning of TGF-beta receptors. Mol Reprod Dev. 1992 Jun;32(2):105–110. doi: 10.1002/mrd.1080320205. [DOI] [PubMed] [Google Scholar]
  14. Mathews L. S., Vale W. W. Expression cloning of an activin receptor, a predicted transmembrane serine kinase. Cell. 1991 Jun 14;65(6):973–982. doi: 10.1016/0092-8674(91)90549-e. [DOI] [PubMed] [Google Scholar]
  15. Mathews L. S., Vale W. W., Kintner C. R. Cloning of a second type of activin receptor and functional characterization in Xenopus embryos. Science. 1992 Mar 27;255(5052):1702–1705. doi: 10.1126/science.1313188. [DOI] [PubMed] [Google Scholar]
  16. Pinilla C., Appel J. R., Blanc P., Houghten R. A. Rapid identification of high affinity peptide ligands using positional scanning synthetic peptide combinatorial libraries. Biotechniques. 1992 Dec;13(6):901–905. [PubMed] [Google Scholar]
  17. Sadowski H. B., Shuai K., Darnell J. E., Jr, Gilman M. Z. A common nuclear signal transduction pathway activated by growth factor and cytokine receptors. Science. 1993 Sep 24;261(5129):1739–1744. doi: 10.1126/science.8397445. [DOI] [PubMed] [Google Scholar]
  18. Shuai K., Stark G. R., Kerr I. M., Darnell J. E., Jr A single phosphotyrosine residue of Stat91 required for gene activation by interferon-gamma. Science. 1993 Sep 24;261(5129):1744–1746. doi: 10.1126/science.7690989. [DOI] [PubMed] [Google Scholar]
  19. Songyang Z., Blechner S., Hoagland N., Hoekstra M. F., Piwnica-Worms H., Cantley L. C. Use of an oriented peptide library to determine the optimal substrates of protein kinases. Curr Biol. 1994 Nov 1;4(11):973–982. doi: 10.1016/s0960-9822(00)00221-9. [DOI] [PubMed] [Google Scholar]
  20. Songyang Z., Carraway K. L., 3rd, Eck M. J., Harrison S. C., Feldman R. A., Mohammadi M., Schlessinger J., Hubbard S. R., Smith D. P., Eng C. Catalytic specificity of protein-tyrosine kinases is critical for selective signalling. Nature. 1995 Feb 9;373(6514):536–539. doi: 10.1038/373536a0. [DOI] [PubMed] [Google Scholar]
  21. Till J. H., Annan R. S., Carr S. A., Miller W. T. Use of synthetic peptide libraries and phosphopeptide-selective mass spectrometry to probe protein kinase substrate specificity. J Biol Chem. 1994 Mar 11;269(10):7423–7428. [PubMed] [Google Scholar]
  22. Wrana J. L., Attisano L., Cárcamo J., Zentella A., Doody J., Laiho M., Wang X. F., Massagué J. TGF beta signals through a heteromeric protein kinase receptor complex. Cell. 1992 Dec 11;71(6):1003–1014. doi: 10.1016/0092-8674(92)90395-s. [DOI] [PubMed] [Google Scholar]
  23. Wrana J. L., Attisano L., Wieser R., Ventura F., Massagué J. Mechanism of activation of the TGF-beta receptor. Nature. 1994 Aug 4;370(6488):341–347. doi: 10.1038/370341a0. [DOI] [PubMed] [Google Scholar]
  24. Wu J., Ma Q. N., Lam K. S. Identifying substrate motifs of protein kinases by a random library approach. Biochemistry. 1994 Dec 13;33(49):14825–14833. doi: 10.1021/bi00253a022. [DOI] [PubMed] [Google Scholar]
  25. ten Dijke P., Yamashita H., Ichijo H., Franzén P., Laiho M., Miyazono K., Heldin C. H. Characterization of type I receptors for transforming growth factor-beta and activin. Science. 1994 Apr 1;264(5155):101–104. doi: 10.1126/science.8140412. [DOI] [PubMed] [Google Scholar]
  26. ten Dijke P., Yamashita H., Sampath T. K., Reddi A. H., Estevez M., Riddle D. L., Ichijo H., Heldin C. H., Miyazono K. Identification of type I receptors for osteogenic protein-1 and bone morphogenetic protein-4. J Biol Chem. 1994 Jun 24;269(25):16985–16988. [PubMed] [Google Scholar]

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