Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1993 Sep;13(9):5560–5566. doi: 10.1128/mcb.13.9.5560

A region of the 85-kilodalton (kDa) subunit of phosphatidylinositol 3-kinase binds the 110-kDa catalytic subunit in vivo.

A Klippel 1, J A Escobedo 1, Q Hu 1, L T Williams 1
PMCID: PMC360276  PMID: 8395006

Abstract

Phosphatidylinositol (PI) 3-kinase is a heterodimer consisting of an 85-kDa subunit (p85) and 110-kDa subunit (p110). The 85-kDa noncatalytic subunit, which contains two Src homology 2 (SH2) domains, one SH3 domain, and a domain homologous to the carboxy terminus of the breakpoint cluster region gene product, is known to mediate the association of the PI 3-kinase complex with activated growth factor receptors. We previously demonstrated that the C-terminal SH2 domain of p85 is responsible for the interaction of PI 3-kinase with phosphorylated platelet-derived growth factor receptor. To define the region in p85 that directs the complex formation with the PI 3-kinase catalytic subunit, a series of truncated p85 mutants was analyzed for association with p110 in vivo. We found that a fragment of p85 containing the region between the two SH2 domains was sufficient to promote the interaction with p110 in vivo. The complex between the fragment of p85 and p110 had PI 3-kinase activity that was comparable in magnitude to the activity of p110 associated with full-length p85. The binding with p110 was abolished when this domain in p85 was disrupted. These results identify a novel structural and functional element that is responsible for localizing the catalytic subunit of PI 3-kinase.

Full text

PDF

Images in this article

5563Image
on p.5563
5564Image
on p.5564

Selected References

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

  1. Auger K. R., Serunian L. A., Soltoff S. P., Libby P., Cantley L. C. PDGF-dependent tyrosine phosphorylation stimulates production of novel polyphosphoinositides in intact cells. Cell. 1989 Apr 7;57(1):167–175. doi: 10.1016/0092-8674(89)90182-7. [DOI] [PubMed] [Google Scholar]
  2. Cantley L. C., Auger K. R., Carpenter C., Duckworth B., Graziani A., Kapeller R., Soltoff S. Oncogenes and signal transduction. Cell. 1991 Jan 25;64(2):281–302. doi: 10.1016/0092-8674(91)90639-g. [DOI] [PubMed] [Google Scholar]
  3. Carpenter C. L., Auger K. R., Duckworth B. C., Hou W. M., Schaffhausen B., Cantley L. C. A tightly associated serine/threonine protein kinase regulates phosphoinositide 3-kinase activity. Mol Cell Biol. 1993 Mar;13(3):1657–1665. doi: 10.1128/mcb.13.3.1657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Carpenter C. L., Duckworth B. C., Auger K. R., Cohen B., Schaffhausen B. S., Cantley L. C. Purification and characterization of phosphoinositide 3-kinase from rat liver. J Biol Chem. 1990 Nov 15;265(32):19704–19711. [PubMed] [Google Scholar]
  5. Cooper J. A., Kashishian A. In vivo binding properties of SH2 domains from GTPase-activating protein and phosphatidylinositol 3-kinase. Mol Cell Biol. 1993 Mar;13(3):1737–1745. doi: 10.1128/mcb.13.3.1737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Coughlin S. R., Escobedo J. A., Williams L. T. Role of phosphatidylinositol kinase in PDGF receptor signal transduction. Science. 1989 Mar 3;243(4895):1191–1194. doi: 10.1126/science.2466336. [DOI] [PubMed] [Google Scholar]
  7. Escobedo J. A., Kaplan D. R., Kavanaugh W. M., Turck C. W., Williams L. T. A phosphatidylinositol-3 kinase binds to platelet-derived growth factor receptors through a specific receptor sequence containing phosphotyrosine. Mol Cell Biol. 1991 Feb;11(2):1125–1132. doi: 10.1128/mcb.11.2.1125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Escobedo J. A., Navankasattusas S., Kavanaugh W. M., Milfay D., Fried V. A., Williams L. T. cDNA cloning of a novel 85 kd protein that has SH2 domains and regulates binding of PI3-kinase to the PDGF beta-receptor. Cell. 1991 Apr 5;65(1):75–82. doi: 10.1016/0092-8674(91)90409-r. [DOI] [PubMed] [Google Scholar]
  9. Escobedo J. A., Williams L. T. A PDGF receptor domain essential for mitogenesis but not for many other responses to PDGF. Nature. 1988 Sep 1;335(6185):85–87. doi: 10.1038/335085a0. [DOI] [PubMed] [Google Scholar]
  10. Evan G. I., Lewis G. K., Ramsay G., Bishop J. M. Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol Cell Biol. 1985 Dec;5(12):3610–3616. doi: 10.1128/mcb.5.12.3610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fantl W. J., Escobedo J. A., Martin G. A., Turck C. W., del Rosario M., McCormick F., Williams L. T. Distinct phosphotyrosines on a growth factor receptor bind to specific molecules that mediate different signaling pathways. Cell. 1992 May 1;69(3):413–423. doi: 10.1016/0092-8674(92)90444-h. [DOI] [PubMed] [Google Scholar]
  12. Groger R. K., Morrow D. M., Tykocinski M. L. Directional antisense and sense cDNA cloning using Epstein-Barr virus episomal expression vectors. Gene. 1989 Sep 30;81(2):285–294. doi: 10.1016/0378-1119(89)90189-3. [DOI] [PubMed] [Google Scholar]
  13. Hiles I. D., Otsu M., Volinia S., Fry M. J., Gout I., Dhand R., Panayotou G., Ruiz-Larrea F., Thompson A., Totty N. F. Phosphatidylinositol 3-kinase: structure and expression of the 110 kd catalytic subunit. Cell. 1992 Aug 7;70(3):419–429. doi: 10.1016/0092-8674(92)90166-a. [DOI] [PubMed] [Google Scholar]
  14. Kavanaugh W. M., Klippel A., Escobedo J. A., Williams L. T. Modification of the 85-kilodalton subunit of phosphatidylinositol-3 kinase in platelet-derived growth factor-stimulated cells. Mol Cell Biol. 1992 Aug;12(8):3415–3424. doi: 10.1128/mcb.12.8.3415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Klippel A., Escobedo J. A., Fantl W. J., Williams L. T. The C-terminal SH2 domain of p85 accounts for the high affinity and specificity of the binding of phosphatidylinositol 3-kinase to phosphorylated platelet-derived growth factor beta receptor. Mol Cell Biol. 1992 Apr;12(4):1451–1459. doi: 10.1128/mcb.12.4.1451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Koch C. A., Anderson D., Moran M. F., Ellis C., Pawson T. SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins. Science. 1991 May 3;252(5006):668–674. doi: 10.1126/science.1708916. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Matthias P., Müller M. M., Schreiber E., Rusconi S., Schaffner W. Eukaryotic expression vectors for the analysis of mutant proteins. Nucleic Acids Res. 1989 Aug 11;17(15):6418–6418. doi: 10.1093/nar/17.15.6418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. McGlade C. J., Ellis C., Reedijk M., Anderson D., Mbamalu G., Reith A. D., Panayotou G., End P., Bernstein A., Kazlauskas A. SH2 domains of the p85 alpha subunit of phosphatidylinositol 3-kinase regulate binding to growth factor receptors. Mol Cell Biol. 1992 Mar;12(3):991–997. doi: 10.1128/mcb.12.3.991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Morgan S. J., Smith A. D., Parker P. J. Purification and characterization of bovine brain type I phosphatidylinositol kinase. Eur J Biochem. 1990 Aug 17;191(3):761–767. doi: 10.1111/j.1432-1033.1990.tb19185.x. [DOI] [PubMed] [Google Scholar]
  21. Morrison D. K., Kaplan D. R., Escobedo J. A., Rapp U. R., Roberts T. M., Williams L. T. Direct activation of the serine/threonine kinase activity of Raf-1 through tyrosine phosphorylation by the PDGF beta-receptor. Cell. 1989 Aug 25;58(4):649–657. doi: 10.1016/0092-8674(89)90100-1. [DOI] [PubMed] [Google Scholar]
  22. Otsu M., Hiles I., Gout I., Fry M. J., Ruiz-Larrea F., Panayotou G., Thompson A., Dhand R., Hsuan J., Totty N. Characterization of two 85 kd proteins that associate with receptor tyrosine kinases, middle-T/pp60c-src complexes, and PI3-kinase. Cell. 1991 Apr 5;65(1):91–104. doi: 10.1016/0092-8674(91)90411-q. [DOI] [PubMed] [Google Scholar]
  23. Panayotou G., Bax B., Gout I., Federwisch M., Wroblowski B., Dhand R., Fry M. J., Blundell T. L., Wollmer A., Waterfield M. D. Interaction of the p85 subunit of PI 3-kinase and its N-terminal SH2 domain with a PDGF receptor phosphorylation site: structural features and analysis of conformational changes. EMBO J. 1992 Dec;11(12):4261–4272. doi: 10.1002/j.1460-2075.1992.tb05524.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Reedijk M., Liu X., van der Geer P., Letwin K., Waterfield M. D., Hunter T., Pawson T. Tyr721 regulates specific binding of the CSF-1 receptor kinase insert to PI 3'-kinase SH2 domains: a model for SH2-mediated receptor-target interactions. EMBO J. 1992 Apr;11(4):1365–1372. doi: 10.1002/j.1460-2075.1992.tb05181.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Shibasaki F., Homma Y., Takenawa T. Two types of phosphatidylinositol 3-kinase from bovine thymus. Monomer and heterodimer form. J Biol Chem. 1991 May 5;266(13):8108–8114. [PubMed] [Google Scholar]
  26. Skolnik E. Y., Margolis B., Mohammadi M., Lowenstein E., Fischer R., Drepps A., Ullrich A., Schlessinger J. Cloning of PI3 kinase-associated p85 utilizing a novel method for expression/cloning of target proteins for receptor tyrosine kinases. Cell. 1991 Apr 5;65(1):83–90. doi: 10.1016/0092-8674(91)90410-z. [DOI] [PubMed] [Google Scholar]
  27. Whitman M., Downes C. P., Keeler M., Keller T., Cantley L. Type I phosphatidylinositol kinase makes a novel inositol phospholipid, phosphatidylinositol-3-phosphate. Nature. 1988 Apr 14;332(6165):644–646. doi: 10.1038/332644a0. [DOI] [PubMed] [Google Scholar]
  28. Wilson I. A., Niman H. L., Houghten R. A., Cherenson A. R., Connolly M. L., Lerner R. A. The structure of an antigenic determinant in a protein. Cell. 1984 Jul;37(3):767–778. doi: 10.1016/0092-8674(84)90412-4. [DOI] [PubMed] [Google Scholar]
  29. Yoakim M., Hou W., Liu Y., Carpenter C. L., Kapeller R., Schaffhausen B. S. Interactions of polyomavirus middle T with the SH2 domains of the pp85 subunit of phosphatidylinositol-3-kinase. J Virol. 1992 Sep;66(9):5485–5491. doi: 10.1128/jvi.66.9.5485-5491.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Yonezawa K., Ueda H., Hara K., Nishida K., Ando A., Chavanieu A., Matsuba H., Shii K., Yokono K., Fukui Y. Insulin-dependent formation of a complex containing an 85-kDa subunit of phosphatidylinositol 3-kinase and tyrosine-phosphorylated insulin receptor substrate 1. J Biol Chem. 1992 Dec 25;267(36):25958–25965. [PubMed] [Google Scholar]

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

RESOURCES