Original Article | Published:

The activation loop tyrosine 823 is essential for the transforming capacity of the c-Kit oncogenic mutant D816V

Oncogene volume 34, pages 45814590 (27 August 2015) | Download Citation

Abstract

Oncogenic c-Kit mutations have been shown to display ligand-independent receptor activation and cell proliferation. A substitution of aspartate to valine at amino acid 816 (D816V) is one of the most commonly found oncogenic c-Kit mutations and is found in >90% of cases of mastocytosis and less commonly in germ-cell tumors, core-binding factor acute myeloid leukemia and mucosal melanomas. The mechanisms by which this mutation leads to constitutive activation and transformation are not fully understood. Previous studies have shown that the D816V mutation causes a structural change in the activation loop (A-loop), resulting in weaker binding of the A-loop to the juxtamembrane domain. In this paper, we have investigated the role of Y823, the only tyrosine residue in the A-loop, and its role in oncogenic transformation by c-Kit/D816V by introducing the Y823F mutation. Although dispensable for the kinase activity of c-Kit/D816V, the presence of Y823 was crucial for cell proliferation and survival. Furthermore, mutation of Y823 selectively downregulates the Ras/Erk and Akt pathways as well as the phosphorylation of STAT5 and reduces the transforming capacity of the D816V/c-Kit in vitro. We further show that mice injected with cells expressing c-Kit/D816V/Y823F display significantly reduced tumor size as well as tumor weight compared with controls. Finally, microarray analysis, comparing Y823F/D816V cells with cells expressing c-Kit/D816V, demonstrate that mutation of Y823 causes upregulation of proapoptotic genes, whereas genes of survival pathways are downregulated. Thus, phosphorylation of Y823 is not necessary for kinase activation, but essential for the transforming ability of the c-Kit/D816V mutant.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    . The biology of stem cell factor and its receptor C-kit. Int J Biochem Cell Biol 1999; 31: 1037–1051.

  2. 2.

    , , , . Biology and genetic aspects of gastrointestinal stromal tumors: KIT activation and cytogenetic alterations. Hum Pathol 2002; 33: 484–495.

  3. 3.

    , , . Allosteric communication across the native and mutated KIT receptor tyrosine kinase. PLoS Comput Biol 2012; 8: e1002661.

  4. 4.

    , , , , , et al. KIT mutations are common in testicular seminomas. Am J Pathol 2004; 164: 305–313.

  5. 5.

    , , , , , et al. KIT activating mutations: incidence in adult and pediatric acute myeloid leukemia, and identification of an internal tandem duplication. Haematologica 2004; 89: 920–925.

  6. 6.

    , , , , , et al. Prognostic impact of c-KIT mutations in core binding factor leukemias: an Italian retrospective study. Blood 2006; 107: 3463–3468.

  7. 7.

    , , , . Activating c-kit gene mutations in human germ cell tumors. Am J Pathol 1999; 154: 1643–1647.

  8. 8.

    , , , , , et al. Stem cell factor receptor (c-KIT) codon 816 mutations predict development of bilateral testicular germ-cell tumors. Cancer Res 2003; 63: 7674–7678.

  9. 9.

    , , , , , et al. Activating and dominant inactivating c-KIT catalytic domain mutations in distinct clinical forms of human mastocytosis. Proc Natl Acad Sci USA 1999; 96: 1609–1614.

  10. 10.

    , . A point mutation in the catalytic domain of c-kit induces growth factor independence, tumorigenicity, and differentiation of mast cells. Blood 1996; 87: 3117–3123.

  11. 11.

    , , , , , et al. Phosphatidylinositol 3 kinase contributes to the transformation of hematopoietic cells by the D816V c-Kit mutant. Blood 2001; 98: 1365–1373.

  12. 12.

    , , . Activating mutations of c-kit at codon 816 confer drug resistance in human leukemia cells. Leuk Lymphoma 2001; 41: 513–522.

  13. 13.

    , , , , , et al. Dasatinib (BMS-354825), a dual SRC/ABL kinase inhibitor, inhibits the kinase activity of wild-type, juxtamembrane, and activation loop mutant KIT isoforms associated with human malignancies. Cancer Res 2006; 66: 473–481.

  14. 14.

    , , , , , . Dasatinib (BMS-354825) inhibits KITD816V, an imatinib-resistant activating mutation that triggers neoplastic growth in most patients with systemic mastocytosis. Blood 2006; 108: 286–291.

  15. 15.

    , , , . Semaxinib (SU5416) as a therapeutic agent targeting oncogenic Kit mutants resistant to imatinib mesylate. Oncogene 2007; 26: 3904–3908.

  16. 16.

    , , , , , et al. PKC412 inhibits in vitro growth of neoplastic human mast cells expressing the D816V-mutated variant of KIT: comparison with AMN107, imatinib, and cladribine (2CdA) and evaluation of cooperative drug effects. Blood 2006; 107: 752–759.

  17. 17.

    , , , , , et al. Identification of proapoptotic Bim as a tumor suppressor in neoplastic mast cells: role of KIT D816V and effects of various targeted drugs. Blood 2009; 114: 5342–5351.

  18. 18.

    , , , , REMA. Recent advances in the understanding of mastocytosis: the role of KIT mutations. Br J Haematol 2007; 138: 12–30.

  19. 19.

    , , , . Molecular basis of the constitutive activity and STI571 resistance of Asp816Val mutant KIT receptor tyrosine kinase. J Mol Graph Model 2004; 23: 139–152.

  20. 20.

    , , , , , et al. Function of activation loop tyrosine phosphorylation in the mechanism of c-Kit auto-activation and its implication in sunitinib resistance. J Biochem 2010; 147: 601–609.

  21. 21.

    , , . Phosphorylation of the activation loop tyrosine 823 in c-Kit is crucial for cell survival and proliferation. J Biol Chem 2013; 288: 22460–22468.

  22. 22.

    , , , , , et al. Constitutively activating mutations of c-kit receptor tyrosine kinase confer factor-independent growth and tumorigenicity of factor-dependent hematopoietic cell lines. Blood 1995; 85: 790–798.

  23. 23.

    , , , , , et al. Relocalization of KIT D816V to cell surface after dasatinib treatment: potential clinical implications. Clin Lymphoma Myeloma Leuk 2013; 13: 62–69.

  24. 24.

    , . Oncogenic signaling from the hematopoietic growth factor receptors c-Kit and Flt3. Cell Signal 2009; 21: 1717–1726.

  25. 25.

    , , . The D816V mutation of c-Kit circumvents a requirement for Src family kinases in c-Kit signal transduction. J Biol Chem 2009; 284: 11039–11047.

  26. 26.

    , , . JAK-STAT signaling: from interferons to cytokines. J Biol Chem 2007; 282: 20059–20063.

  27. 27.

    , , , , , . Mechanisms of STAT protein activation by oncogenic KIT mutants in neoplastic mast cells. J Biol Chem 2011; 286: 5956–5966.

  28. 28.

    , . Oncogenic activation of tyrosine kinases. Curr Opin Genet Dev 1994; 4: 15–24.

  29. 29.

    , , , , , et al. Identification of mutations in the coding sequence of the proto-oncogene c-kit in a human mast cell leukemia cell line causing ligand-independent activation of c-kit product. J Clin Invest 1993; 92: 1736–1744.

  30. 30.

    , , , , , et al. KIT-D816V oncogenic activity is controlled by the juxtamembrane docking site Y568-Y570. Oncogene 2013.

  31. 31.

    , , , , , . Differential roles of PI3-kinase and Kit tyrosine 821 in Kit receptor-mediated proliferation, survival and cell adhesion in mast cells. EMBO J 1995; 14: 473–483.

  32. 32.

    , , , . Phosphorylation of Y845 on the epidermal growth factor receptor mediates binding to the mitochondrial protein cytochrome c oxidase subunit II. Mol Cell Biol 2004; 24: 7059–7071.

  33. 33.

    , , , . Transactivating agonists of the EGF receptor require Tyr 845 phosphorylation for induction of DNA synthesis. Mol Carcinog 2005; 44: 262–273.

  34. 34.

    , , . Mutation of tyrosine residue 857 in the PDGF beta-receptor affects cell proliferation but not migration. Cell Signal 2010; 22: 1363–1368.

  35. 35.

    , , , , , . The tyrosine 343 residue of nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK) is important for its interaction with SHP1, a cytoplasmic tyrosine phosphatase with tumor suppressor functions. J Biol Chem 2010; 285: 19813–19820.

  36. 36.

    , , , . Site-directed mutagenesis and yeast two-hybrid studies of the insulin and insulin-like growth factor-1 receptors: the Src homology-2 domain-containing protein hGrb10 binds to the autophosphorylated tyrosine residues in the kinase domain of the insulin receptor. Mol Endocrinol 1997; 11: 1757–1765.

  37. 37.

    , , , . STATs in oncogenesis. Oncogene 2000; 19: 2474–2488.

  38. 38.

    , , , . Signal transducer and activator of transcription proteins in leukemias. Blood 2003; 101: 2940–2954.

  39. 39.

    , , , , . Stat3 activation is required for cellular transformation by v-src. Mol Cell Biol 1998; 18: 2553–2558.

  40. 40.

    , , . Tyrosyl phosphorylation and DNA binding activity of signal transducers and activators of transcription (STAT) proteins in hematopoietic cell lines transformed by Bcr/Abl. J Exp Med 1996; 183: 811–820.

  41. 41.

    , , , , . Cloning and characterization of SCHIP-1, a novel protein interacting specifically with spliced isoforms and naturally occurring mutant NF2 proteins. Mol Cell Biol 2000; 20: 1699–1712.

  42. 42.

    , , , , . The role of annexin A3 playing in cancers. Clin Transl Oncol 2013; 15: 106–110.

  43. 43.

    , , , , , et al. Interleukin-6 receptor enhances early colonization of the murine omentum by upregulation of a mannose family receptor, LY75, in ovarian tumor cells. Clin Exp Metastasis 2011; 28: 887–897.

  44. 44.

    , , , , , . Inpp5f is a polyphosphoinositide phosphatase that regulates cardiac hypertrophic responsiveness. Circ Res 2009; 105: 1240–1247.

  45. 45.

    , , . Abnormal hematopoietic phenotypes in Pim kinase triple knockout mice. J Hematol Oncol 2013; 6: 12.

  46. 46.

    , . Pim kinases in cancer: diagnostic, prognostic and treatment opportunities. Biochem Pharmacol 2013; 85: 629–643.

  47. 47.

    , , , , , . Transcription and translation are primary targets of Pim kinase inhibitor SGI-1776 in mantle cell lymphoma. Blood 2012; 120: 3491–3500.

  48. 48.

    , , , , , et al. Dissection of PIM serine/threonine kinases in FLT3-ITD-induced leukemogenesis reveals PIM1 as regulator of CXCL12-CXCR4-mediated homing and migration. J Exp Med 2009; 206: 1957–1970.

  49. 49.

    , , . Ubiquitin specific protease 18 (Usp18) is a WT1 transcriptional target. Exp Cell Res 2013; 319: 612–622.

  50. 50.

    , , , . Epigenetic silencing of Bcl-2, CEBPA and p14ARF by the AML1-ETO oncoprotein contributing to growth arrest and differentiation block in the U937 cell line. Oncol Rep 2013; 30: 185–192.

  51. 51.

    , . MYC, BCL2, BCL6 in DLBCL: impact for clinics in the future? Blood 2013; 121: 2165–2166.

  52. 52.

    , , , , . Increased Kit/SCF receptor induced mitogenicity but abolished cell motility after inhibition of protein kinase C. EMBO J 1993; 12: 4199–4209.

  53. 53.

    , , . Gab2 is involved in differential phosphoinositide 3-kinase signaling by two splice forms of c-Kit. J Biol Chem 2008; 283: 27444–27451.

Download references

Acknowledgements

This work was supported by grants from the Swedish Research Council, the Swedish Cancer Foundation, Gunnar Nilsson Cancer foundation, the Stiftelsen Olle Engkvist Byggmästare, Royal Physiographic Society in Lund, Ollie och Elof Ericssons Stiftelse and Stiftelsen Lars Hiertas Minne.

Author information

Author notes

    • S Agarwal

    Current address: Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund University, Lund, Sweden.

Affiliations

  1. Translational Cancer Research, Lund University, Lund, Sweden

    • S Agarwal
    • , J U Kazi
    • , S Mohlin
    • , S Påhlman
    •  & L Rönnstrand
  2. Lund Stem Cell Center, Lund University, Lund, Sweden

    • S Agarwal
    • , J U Kazi
    •  & L Rönnstrand
  3. CREATE Health, Lund University, Lund, Sweden

    • S Mohlin
    •  & S Påhlman

Authors

  1. Search for S Agarwal in:

  2. Search for J U Kazi in:

  3. Search for S Mohlin in:

  4. Search for S Påhlman in:

  5. Search for L Rönnstrand in:

Competing interests

The authors declare no conflict of interest.

Corresponding author

Correspondence to L Rönnstrand.

Supplementary information

About this article

Publication history

Received

Revised

Accepted

Published

DOI

https://doi.org/10.1038/onc.2014.383

Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)

Further reading