Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1994 Aug 16;91(17):8132–8136. doi: 10.1073/pnas.91.17.8132

Insect cell-expressed p180erbB3 possesses an impaired tyrosine kinase activity.

P M Guy 1, J V Platko 1, L C Cantley 1, R A Cerione 1, K L Carraway 3rd 1
PMCID: PMC44559  PMID: 8058768

Abstract

Protein kinases share a number of highly conserved or invariant amino acid residues in their catalytic domains, suggesting that these residues are necessary for kinase activity. In p180erbB3, a receptor tyrosine kinase belonging to the epidermal growth factor (EGF) receptor subfamily, three of these residues are altered, suggesting that this protein might have an impaired protein tyrosine kinase activity. To test this hypothesis, we have expressed human EGF receptor and bovine p180erbB3 in insect cells via baculovirus infection and have compared their autophosphorylation and substrate phosphorylation activities. We have found that, while the EGF receptor readily undergoes EGF-stimulated autophosphorylation and catalyzes the incorporation of phosphate into the model substrates (E4Y1)n (random 4:1 copolymer of glutamic acid and tyrosine) and GST-p85 (glutathione S-transferase fusion protein with the 85-kDa subunit of phosphatidylinositol 3-kinase), p180erbB3 autophosphorylation and substrate phosphorylation are at least 2 orders of magnitude less efficient. However, p180erbB3 is capable of binding the ATP analog 5'-p-fluorosulfonylbenzoyladenosine, indicating that the lack of observed kinase activity is probably not due to nonfunctional or denatured receptors expressed by the insect cells. On the basis of these results, we propose that p180erbB3 possesses an impaired intrinsic tyrosine kinase activity.

Full text

PDF
8132

Images in this article

8133Image
on p.8133
8134Image
on p.8134
8135Image
on p.8135

Selected References

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

  1. Anostario M., Jr, Harrison M. L., Geahlen R. L. Immunochemical detection of adenine nucleotide-binding proteins with antibodies to 5'-p-fluorosulfonylbenzoyladenosine. Anal Biochem. 1990 Oct;190(1):60–65. doi: 10.1016/0003-2697(90)90133-t. [DOI] [PubMed] [Google Scholar]
  2. Buhrow S. A., Cohen S., Staros J. V. Affinity labeling of the protein kinase associated with the epidermal growth factor receptor in membrane vesicles from A431 cells. J Biol Chem. 1982 Apr 25;257(8):4019–4022. [PubMed] [Google Scholar]
  3. Carraway K. L., 3rd, Sliwkowski M. X., Akita R., Platko J. V., Guy P. M., Nuijens A., Diamonti A. J., Vandlen R. L., Cantley L. C., Cerione R. A. The erbB3 gene product is a receptor for heregulin. J Biol Chem. 1994 May 13;269(19):14303–14306. [PubMed] [Google Scholar]
  4. Gibbs C. S., Zoller M. J. Rational scanning mutagenesis of a protein kinase identifies functional regions involved in catalysis and substrate interactions. J Biol Chem. 1991 May 15;266(14):8923–8931. [PubMed] [Google Scholar]
  5. Greenfield C., Patel G., Clark S., Jones N., Waterfield M. D. Expression of the human EGF receptor with ligand-stimulatable kinase activity in insect cells using a baculovirus vector. EMBO J. 1988 Jan;7(1):139–146. doi: 10.1002/j.1460-2075.1988.tb02793.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Guy P. M., Carraway K. L., 3rd, Cerione R. A. Biochemical comparisons of the normal and oncogenic forms of insect cell-expressed neu tyrosine kinases. J Biol Chem. 1992 Jul 15;267(20):13851–13856. [PubMed] [Google Scholar]
  7. Hanks S. K., Quinn A. M., Hunter T. The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science. 1988 Jul 1;241(4861):42–52. doi: 10.1126/science.3291115. [DOI] [PubMed] [Google Scholar]
  8. Knighton D. R., Cadena D. L., Zheng J., Ten Eyck L. F., Taylor S. S., Sowadski J. M., Gill G. N. Structural features that specify tyrosine kinase activity deduced from homology modeling of the epidermal growth factor receptor. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):5001–5005. doi: 10.1073/pnas.90.11.5001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Knighton D. R., Zheng J. H., Ten Eyck L. F., Ashford V. A., Xuong N. H., Taylor S. S., Sowadski J. M. Crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. Science. 1991 Jul 26;253(5018):407–414. doi: 10.1126/science.1862342. [DOI] [PubMed] [Google Scholar]
  10. Koland J. G., Cerione R. A. Activation of the EGF receptor tyrosine kinase by divalent metal ions: comparison of holoreceptor and isolated kinase domain properties. Biochim Biophys Acta. 1990 May 22;1052(3):489–498. doi: 10.1016/0167-4889(90)90160-f. [DOI] [PubMed] [Google Scholar]
  11. Koland J. G., Cerione R. A. Growth factor control of epidermal growth factor receptor kinase activity via an intramolecular mechanism. J Biol Chem. 1988 Feb 15;263(5):2230–2237. [PubMed] [Google Scholar]
  12. Koland J. G., O'Brien K. M., Cerione R. A. Expression of epidermal growth factor receptor sequences as E. coli fusion proteins: applications in the study of tyrosine kinase function. Biochem Biophys Res Commun. 1990 Jan 15;166(1):90–100. doi: 10.1016/0006-291x(90)91915-f. [DOI] [PubMed] [Google Scholar]
  13. Kraus M. H., Fedi P., Starks V., Muraro R., Aaronson S. A. Demonstration of ligand-dependent signaling by the erbB-3 tyrosine kinase and its constitutive activation in human breast tumor cells. Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):2900–2904. doi: 10.1073/pnas.90.7.2900. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kraus M. H., Issing W., Miki T., Popescu N. C., Aaronson S. A. Isolation and characterization of ERBB3, a third member of the ERBB/epidermal growth factor receptor family: evidence for overexpression in a subset of human mammary tumors. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9193–9197. doi: 10.1073/pnas.86.23.9193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Moran M. F., Koch C. A., Sadowski I., Pawson T. Mutational analysis of a phosphotransfer motif essential for v-fps tyrosine kinase activity. Oncogene. 1988 Dec;3(6):665–672. [PubMed] [Google Scholar]
  17. Plowman G. D., Whitney G. S., Neubauer M. G., Green J. M., McDonald V. L., Todaro G. J., Shoyab M. Molecular cloning and expression of an additional epidermal growth factor receptor-related gene. Proc Natl Acad Sci U S A. 1990 Jul;87(13):4905–4909. doi: 10.1073/pnas.87.13.4905. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Slamon D. J., Clark G. M., Wong S. G., Levin W. J., Ullrich A., McGuire W. L. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science. 1987 Jan 9;235(4785):177–182. doi: 10.1126/science.3798106. [DOI] [PubMed] [Google Scholar]
  19. Slamon D. J., Godolphin W., Jones L. A., Holt J. A., Wong S. G., Keith D. E., Levin W. J., Stuart S. G., Udove J., Ullrich A. Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science. 1989 May 12;244(4905):707–712. doi: 10.1126/science.2470152. [DOI] [PubMed] [Google Scholar]
  20. Sliwkowski M. X., Schaefer G., Akita R. W., Lofgren J. A., Fitzpatrick V. D., Nuijens A., Fendly B. M., Cerione R. A., Vandlen R. L., Carraway K. L., 3rd Coexpression of erbB2 and erbB3 proteins reconstitutes a high affinity receptor for heregulin. J Biol Chem. 1994 May 20;269(20):14661–14665. [PubMed] [Google Scholar]
  21. Soltoff S. P., Carraway K. L., 3rd, Prigent S. A., Gullick W. G., Cantley L. C. ErbB3 is involved in activation of phosphatidylinositol 3-kinase by epidermal growth factor. Mol Cell Biol. 1994 Jun;14(6):3550–3558. doi: 10.1128/mcb.14.6.3550. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Songyang Z., Shoelson S. E., Chaudhuri M., Gish G., Pawson T., Haser W. G., King F., Roberts T., Ratnofsky S., Lechleider R. J. SH2 domains recognize specific phosphopeptide sequences. Cell. 1993 Mar 12;72(5):767–778. doi: 10.1016/0092-8674(93)90404-e. [DOI] [PubMed] [Google Scholar]
  23. Tan J. C., Nocka K., Ray P., Traktman P., Besmer P. The dominant W42 spotting phenotype results from a missense mutation in the c-kit receptor kinase. Science. 1990 Jan 12;247(4939):209–212. doi: 10.1126/science.1688471. [DOI] [PubMed] [Google Scholar]
  24. Ullrich A., Schlessinger J. Signal transduction by receptors with tyrosine kinase activity. Cell. 1990 Apr 20;61(2):203–212. doi: 10.1016/0092-8674(90)90801-k. [DOI] [PubMed] [Google Scholar]
  25. Yarden Y., Ullrich A. Growth factor receptor tyrosine kinases. Annu Rev Biochem. 1988;57:443–478. doi: 10.1146/annurev.bi.57.070188.002303. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES