Semisynthesis of hyperphosphorylated type I TGFβ receptor: Addressing the mechanism of kinase activation [20]

Journal of the American Chemical Society

Volumn 122, Issue 34, 2000, Pages 8337-8338

  Huse, Morgan ^a   Holford, Mande N ^a   Kuriyan, John ^a   Muir, Tom W ^a  

^a ROCKEFELLER UNIVERSITY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

CYTOKINE; CYTOKINE RECEPTOR; TRANSFORMING GROWTH FACTOR BETA; TRANSFORMING GROWTH FACTOR BETA RECEPTOR;

CRYSTAL STRUCTURE; ENZYME ACTIVATION; ENZYME PHOSPHORYLATION; LETTER; PEPTIDE ANALYSIS; PROTEIN EXPRESSION; SIGNAL TRANSDUCTION;

EID: 0034734354     PISSN: 00027863     EISSN: None     Source Type: Journal    
DOI: 10.1021/ja001763p     Document Type: Letter

References (20)

1. Massagué, J. Annu. Rev. Biochem. 1998, 67, 753-791.
2. Johnson, L. N.; Noble M. E. M.; Owen, D. J. Cell 1996, 85, 149-158.
3. Wieser, R.; Wrana, J. L.; Massagué, J. EMBO J. 1995, 14, 2199-2208.
4. Huse, M.; Chen Y.-G.; Massagué, J.; Kuriyan, J. Cell 1999, 96, 425-436.
5. Willis, S. A.; Zimmerman, C. M.; Li, L.; Mathews, L. S. Mol. Endocrinol. 1996, 10, 367-379.
6. We initially tried, unsuccessfully, to produce homogeneously phosphorylated TβR-I by coexpression with TβR-II and by in vitro phosphorylation.
7. Dawson, P. E.; Muir T. W.; Clark-Lewis, I.; Kent S. B. H. Science 1994, 266, 776-779.
8. 196, and further purified using γ-phosphate conjugated ATP sepharose.
9. The ligation site was chosen on the basis of analysis of the crystal structure of unphosphorylated TβR-I as well as inspection of the sequence conservation in that portion of the GS region.
10. Camarero, J. A.; Cotton, G. J.; Adeva, A.; Muir T. W. J. Pept. Res. 1998, 51, 303-316.
11. The biotin moiety did not effect the kinase activity of the ligation product (data not shown).
12. Perich, J. W.; Terzi, E.; Carnazzi, E.; Seyer, R.; Trifilieff, E. Int. J. Pept. Protein Res. 1994, 44, 305-312.
13. Ingenito, R.; Bianchi, E.; Fattori, D.; Pessi, A. J. Am. Chem. Soc. 1999, 121, 11369-11374.
14. Shin, Y.; Winans, K. A.; Backes, B. J.; Kent, S. B. H.; Ellman, J. A.; Bertozzi, C. B. J. Am. Chem. Soc. 1999, 121, 11684-11689.
15. GS-4 was initially biotinylated at the N-terminus. Cleavage revealed a contaminating degradation product 144 Da less than the expected mass. Peptide mapping studies localized the modification to the N-terminus of the peptide. The contaminant did not bind avidin beads, and we concluded that the biotin had fragmented at some point during the activation/cleavage process.
16. Mass analysis of side products and amino acid analysis of the resin after cleavage revealed incomplete deprotection of the phosphate groups and incomplete thiolysis of the peptide from the resin, respectively. This accounts for the modest yield of the GS-4 peptide.
17. In a typical ligation reaction, 100 nmol of peptide were reacted with 10 nmol GSΔTβR-I in 100 μL of 100 mM Hepes pH 8.0, 200 mM NaCl, 50 mM MESNA at 4 °C for 12-24 h.
18. 32P]ATP. Samples were subjected to gel electrophoresis and visualized by autoradiography.
19. Muir T. W.; Sondhi, D.; Cole, P. A. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 6705-6710.
20. Cotton, G. J.; Ayers, B.; Xu, R.; Muir T. W. J. Am. Chem. Soc. 1999, 121, 1100-1101.