Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2003 Mar 15;370(Pt 3):1033–1038. doi: 10.1042/BJ20020716

The adapter protein APS associates with the multifunctional docking sites Tyr-568 and Tyr-936 in c-Kit.

Patrik Wollberg 1, Johan Lennartsson 1, Eva Gottfridsson 1, Akihiko Yoshimura 1, Lars Rönnstrand 1
PMCID: PMC1223215  PMID: 12444928

Abstract

The adapter protein APS has previously been shown to be involved in recruiting the ubiquitin E3 ligase c-Cbl to the insulin receptor, the platelet-derived growth factor beta-receptor and the erythropoietin receptor, leading to increased degradation of the receptors and inhibition of mitogenesis. Here we demonstrate, by use of immobilized synthetic phosphopeptides corresponding to various autophosphorylated tyrosine residues in the receptor for stem-cell factor (c-Kit), that APS preferentially associates with phosphorylated Tyr-568 and Tyr-936. Tyr-568 has previously been identified as the binding site of the Src family of tyrosine kinases, the Csk-homologous kinase CHK, and the protein tyrosine phosphatase SHP-2. We have recently demonstrated that Tyr-936 is an autophosphorylation site involved in binding the adapter proteins Grb2 and Grb7. We could further demonstrate that the critical determinant for binding of APS is the presence of either a leucine or an isoleucine residue in the position +3 to the phosphorylated tyrosine. This allowed us to design mutants that selectively failed to associate with APS, while still associating with Src family members, SHP-2 and Grb2, respectively.

Full Text

The Full Text of this article is available as a PDF (213.1 KB).

Selected References

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

  1. Ahmed Z., Pillay T. S. Functional effects of APS and SH2-B on insulin receptor signalling. Biochem Soc Trans. 2001 Aug;29(Pt 4):529–534. doi: 10.1042/bst0290529. [DOI] [PubMed] [Google Scholar]
  2. Ahmed Z., Smith B. J., Kotani K., Wilden P., Pillay T. S. APS, an adapter protein with a PH and SH2 domain, is a substrate for the insulin receptor kinase. Biochem J. 1999 Aug 1;341(Pt 3):665–668. [PMC free article] [PubMed] [Google Scholar]
  3. Andrews D. M., Kitchin J., Seale P. W. Solid-phase synthesis of a range of O-phosphorylated peptides by post-assembly phosphitylation and oxidation. Int J Pept Protein Res. 1991 Nov;38(5):469–475. doi: 10.1111/j.1399-3011.1991.tb01528.x. [DOI] [PubMed] [Google Scholar]
  4. Arteaga C. L., Johnson M. D., Todderud G., Coffey R. J., Carpenter G., Page D. L. Elevated content of the tyrosine kinase substrate phospholipase C-gamma 1 in primary human breast carcinomas. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10435–10439. doi: 10.1073/pnas.88.23.10435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Blume-Jensen P., Siegbahn A., Stabel S., Heldin C. H., Rönnstrand L. Increased Kit/SCF receptor induced mitogenicity but abolished cell motility after inhibition of protein kinase C. EMBO J. 1993 Nov;12(11):4199–4209. doi: 10.1002/j.1460-2075.1993.tb06104.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Galli S. J., Zsebo K. M., Geissler E. N. The kit ligand, stem cell factor. Adv Immunol. 1994;55:1–96. doi: 10.1016/s0065-2776(08)60508-8. [DOI] [PubMed] [Google Scholar]
  7. Herbst R., Munemitsu S., Ullrich A. Oncogenic activation of v-kit involves deletion of a putative tyrosine-substrate interaction site. Oncogene. 1995 Jan 19;10(2):369–379. [PubMed] [Google Scholar]
  8. Joazeiro C. A., Wing S. S., Huang H., Leverson J. D., Hunter T., Liu Y. C. The tyrosine kinase negative regulator c-Cbl as a RING-type, E2-dependent ubiquitin-protein ligase. Science. 1999 Oct 8;286(5438):309–312. doi: 10.1126/science.286.5438.309. [DOI] [PubMed] [Google Scholar]
  9. Kozlowski M., Larose L., Lee F., Le D. M., Rottapel R., Siminovitch K. A. SHP-1 binds and negatively modulates the c-Kit receptor by interaction with tyrosine 569 in the c-Kit juxtamembrane domain. Mol Cell Biol. 1998 Apr;18(4):2089–2099. doi: 10.1128/mcb.18.4.2089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Lennartsson J., Blume-Jensen P., Hermanson M., Pontén E., Carlberg M., Rönnstrand L. Phosphorylation of Shc by Src family kinases is necessary for stem cell factor receptor/c-kit mediated activation of the Ras/MAP kinase pathway and c-fos induction. Oncogene. 1999 Sep 30;18(40):5546–5553. doi: 10.1038/sj.onc.1202929. [DOI] [PubMed] [Google Scholar]
  11. Lev S., Blechman J. M., Givol D., Yarden Y. Steel factor and c-kit protooncogene: genetic lessons in signal transduction. Crit Rev Oncog. 1994;5(2-3):141–168. doi: 10.1615/critrevoncog.v5.i2-3.30. [DOI] [PubMed] [Google Scholar]
  12. Levkowitz G., Waterman H., Ettenberg S. A., Katz M., Tsygankov A. Y., Alroy I., Lavi S., Iwai K., Reiss Y., Ciechanover A. Ubiquitin ligase activity and tyrosine phosphorylation underlie suppression of growth factor signaling by c-Cbl/Sli-1. Mol Cell. 1999 Dec;4(6):1029–1040. doi: 10.1016/s1097-2765(00)80231-2. [DOI] [PubMed] [Google Scholar]
  13. Linnekin D., DeBerry C. S., Mou S. Lyn associates with the juxtamembrane region of c-Kit and is activated by stem cell factor in hematopoietic cell lines and normal progenitor cells. J Biol Chem. 1997 Oct 24;272(43):27450–27455. doi: 10.1074/jbc.272.43.27450. [DOI] [PubMed] [Google Scholar]
  14. Liu Jun, Kimura Akiko, Baumann Christian A., Saltiel Alan R. APS facilitates c-Cbl tyrosine phosphorylation and GLUT4 translocation in response to insulin in 3T3-L1 adipocytes. Mol Cell Biol. 2002 Jun;22(11):3599–3609. doi: 10.1128/MCB.22.11.3599-3609.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Miyake S., Lupher M. L., Jr, Druker B., Band H. The tyrosine kinase regulator Cbl enhances the ubiquitination and degradation of the platelet-derived growth factor receptor alpha. Proc Natl Acad Sci U S A. 1998 Jul 7;95(14):7927–7932. doi: 10.1073/pnas.95.14.7927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Mori S., Rönnstrand L., Yokote K., Engström A., Courtneidge S. A., Claesson-Welsh L., Heldin C. H. Identification of two juxtamembrane autophosphorylation sites in the PDGF beta-receptor; involvement in the interaction with Src family tyrosine kinases. EMBO J. 1993 Jun;12(6):2257–2264. doi: 10.1002/j.1460-2075.1993.tb05879.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Odai H., Sasaki K., Iwamatsu A., Hanazono Y., Tanaka T., Mitani K., Yazaki Y., Hirai H. The proto-oncogene product c-Cbl becomes tyrosine phosphorylated by stimulation with GM-CSF or Epo and constitutively binds to the SH3 domain of Grb2/Ash in human hematopoietic cells. J Biol Chem. 1995 May 5;270(18):10800–10805. doi: 10.1074/jbc.270.18.10800. [DOI] [PubMed] [Google Scholar]
  18. Ohtsuka Satoshi, Takaki Satoshi, Iseki Masanori, Miyoshi Kanta, Nakagata Naomi, Kataoka Yuki, Yoshida Nobuaki, Takatsu Kiyoshi, Yoshimura Akihiko. SH2-B is required for both male and female reproduction. Mol Cell Biol. 2002 May;22(9):3066–3077. doi: 10.1128/MCB.22.9.3066-3077.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Price D. J., Rivnay B., Fu Y., Jiang S., Avraham S., Avraham H. Direct association of Csk homologous kinase (CHK) with the diphosphorylated site Tyr568/570 of the activated c-KIT in megakaryocytes. J Biol Chem. 1997 Feb 28;272(9):5915–5920. doi: 10.1074/jbc.272.9.5915. [DOI] [PubMed] [Google Scholar]
  20. Qian X., Ginty D. D. SH2-B and APS are multimeric adapters that augment TrkA signaling. Mol Cell Biol. 2001 Mar;21(5):1613–1620. doi: 10.1128/MCB.21.5.1613-1620.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Qian X., Riccio A., Zhang Y., Ginty D. D. Identification and characterization of novel substrates of Trk receptors in developing neurons. Neuron. 1998 Nov;21(5):1017–1029. doi: 10.1016/s0896-6273(00)80620-0. [DOI] [PubMed] [Google Scholar]
  22. Rönnstrand L., Arvidsson A. K., Kallin A., Rorsman C., Hellman U., Engström U., Wernstedt C., Heldin C. H. SHP-2 binds to Tyr763 and Tyr1009 in the PDGF beta-receptor and mediates PDGF-induced activation of the Ras/MAP kinase pathway and chemotaxis. Oncogene. 1999 Jun 24;18(25):3696–3702. doi: 10.1038/sj.onc.1202705. [DOI] [PubMed] [Google Scholar]
  23. Rönnstrand L., Mori S., Arridsson A. K., Eriksson A., Wernstedt C., Hellman U., Claesson-Welsh L., Heldin C. H. Identification of two C-terminal autophosphorylation sites in the PDGF beta-receptor: involvement in the interaction with phospholipase C-gamma. EMBO J. 1992 Nov;11(11):3911–3919. doi: 10.1002/j.1460-2075.1992.tb05484.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Serve H., Hsu Y. C., Besmer P. Tyrosine residue 719 of the c-kit receptor is essential for binding of the P85 subunit of phosphatidylinositol (PI) 3-kinase and for c-kit-associated PI 3-kinase activity in COS-1 cells. J Biol Chem. 1994 Feb 25;269(8):6026–6030. [PubMed] [Google Scholar]
  25. Takaki Satoshi, Morita Hatsue, Tezuka Yoshinari, Takatsu Kiyoshi. Enhanced hematopoiesis by hematopoietic progenitor cells lacking intracellular adaptor protein, Lnk. J Exp Med. 2002 Jan 21;195(2):151–160. doi: 10.1084/jem.20011170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Thömmes K., Lennartsson J., Carlberg M., Rönnstrand L. Identification of Tyr-703 and Tyr-936 as the primary association sites for Grb2 and Grb7 in the c-Kit/stem cell factor receptor. Biochem J. 1999 Jul 1;341(Pt 1):211–216. [PMC free article] [PubMed] [Google Scholar]
  27. Wakioka T., Sasaki A., Mitsui K., Yokouchi M., Inoue A., Komiya S., Yoshimura A. APS, an adaptor protein containing Pleckstrin homology (PH) and Src homology-2 (SH2) domains inhibits the JAK-STAT pathway in collaboration with c-Cbl. Leukemia. 1999 May;13(5):760–767. doi: 10.1038/sj.leu.2401397. [DOI] [PubMed] [Google Scholar]
  28. Yokouchi M., Suzuki R., Masuhara M., Komiya S., Inoue A., Yoshimura A. Cloning and characterization of APS, an adaptor molecule containing PH and SH2 domains that is tyrosine phosphorylated upon B-cell receptor stimulation. Oncogene. 1997 Jul 3;15(1):7–15. doi: 10.1038/sj.onc.1201163. [DOI] [PubMed] [Google Scholar]
  29. Yokouchi M., Wakioka T., Sakamoto H., Yasukawa H., Ohtsuka S., Sasaki A., Ohtsubo M., Valius M., Inoue A., Komiya S. APS, an adaptor protein containing PH and SH2 domains, is associated with the PDGF receptor and c-Cbl and inhibits PDGF-induced mitogenesis. Oncogene. 1999 Jan 21;18(3):759–767. doi: 10.1038/sj.onc.1202326. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES