The ErbB2/Neu/HER2 receptor is a new calmodulin-binding protein.
Journal: 2004/November - Biochemical Journal
ISSN: 1470-8728
Abstract:
We have demonstrated previously that the EGFR (epidermal growth factor receptor) is a calmodulin (CaM)-binding protein. To establish whether or not the related receptor ErbB2/Neu/HER2 also binds CaM, we used human breast adenocarcinoma SK-BR-3 cells, because these cells overexpress this receptor thus facilitating the detection of this interaction. In the present paper, we show that ErbB2 could be pulled-down using CaM-agarose beads in a Ca2+-dependent manner, as detected by Western blot analysis using an anti-ErbB2 antibody. ErbB2 was also isolated by Ca2+-dependent CaM-affinity chromatography. We also demonstrate using an overlay technique with biotinylated CaM that CaM binds directly to the immunoprecipitated ErbB2. The binding of biotinylated CaM to ErbB2 depends strictly on the presence of Ca2+, since it was prevented by the presence of EGTA. Moreover, the addition of an excess of free CaM prevents the binding of its biotinylated form, demonstrating that this was a specific process. We excluded any interference with the EGFR, as SK-BR-3 cells express considerably lower levels of this receptor, and no detectable EGFR signal was observed by Western blot analysis in the immunoprecipitated ErbB2 preparations used to perform the overlay assays with biotinylated CaM. We also demonstrate that treating living cells with W7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide], a cell-permeant CaM antagonist, down-regulates ErbB2 phosphorylation, and show that W7 does not interfere non-specifically with the activity of ErbB tyrosine kinases. We also show that W7 inhibits the phosphorylation (activation) of both ERK1/2 (extracellular-signal-regulated kinases 1 and 2) and Akt/PKB (protein kinase B), in accordance with the inhibition observed in ErbB2 phosphorylation. In contrast, W7 treatment increased the phosphorylation (activation) of CREB (cAMP-response-element-binding protein) and ATF1 (activating transcription factor-1), two Ca2+-sensitive transcription factors that operate downstream of these ErbB2 signalling pathways, most likely because of the absence of calcineurin activity. We conclude that ErbB2 is a new CaM-binding protein, and that CaM plays a role in the regulation of this receptor and its downstream signalling pathways in vivo.
Relations:
Content
Citations
(12)
References
(43)
Diseases
(3)
Drugs
(1)
Chemicals
(9)
Genes
(2)
Organisms
(1)
Processes
(5)
Anatomy
(1)
Affiliates
(2)
Similar articles
Articles by the same authors
Discussion board
Biochem J 381(Pt 1): 257-266

The ErbB2/Neu/HER2 receptor is a new calmodulin-binding protein

Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, Arturo Duperier 4, E-28029 Madrid, Spain
Permanent address: Science and Research Center, Shijiazhuang Medical School, 450 Zhongshan Xi Road, Shijiazhuang, Hebei 05008, People's Republic of China.
Permanent address: Instituto de Biología Experimental, Facultad de Ciencias, Universidad Central de Venezuela, Caracas, Venezuela.
To whom correspondence should be addressed (e-mail se.mau.bii@obolalliv.oinotna).
Received 2004 Mar 30; Accepted 2004 Apr 14.

Abstract

We have demonstrated previously that the EGFR (epidermal growth factor receptor) is a calmodulin (CaM)-binding protein. To establish whether or not the related receptor ErbB2/Neu/HER2 also binds CaM, we used human breast adenocarcinoma SK-BR-3 cells, because these cells overexpress this receptor thus facilitating the detection of this interaction. In the present paper, we show that ErbB2 could be pulled-down using CaM–agarose beads in a Ca-dependent manner, as detected by Western blot analysis using an anti-ErbB2 antibody. ErbB2 was also isolated by Ca-dependent CaM-affinity chromatography. We also demonstrate using an overlay technique with biotinylated CaM that CaM binds directly to the immunoprecipitated ErbB2. The binding of biotinylated CaM to ErbB2 depends strictly on the presence of Ca, since it was prevented by the presence of EGTA. Moreover, the addition of an excess of free CaM prevents the binding of its biotinylated form, demonstrating that this was a specific process. We excluded any interference with the EGFR, as SK-BR-3 cells express considerably lower levels of this receptor, and no detectable EGFR signal was observed by Western blot analysis in the immunoprecipitated ErbB2 preparations used to perform the overlay assays with biotinylated CaM. We also demonstrate that treating living cells with W7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide], a cell-permeant CaM antagonist, down-regulates ErbB2 phosphorylation, and show that W7 does not interfere non-specifically with the activity of ErbB tyrosine kinases. We also show that W7 inhibits the phosphorylation (activation) of both ERK1/2 (extracellular-signal-regulated kinases 1 and 2) and Akt/PKB (protein kinase B), in accordance with the inhibition observed in ErbB2 phosphorylation. In contrast, W7 treatment increased the phosphorylation (activation) of CREB (cAMP-response-element-binding protein) and ATF1 (activating transcription factor-1), two Ca-sensitive transcription factors that operate downstream of these ErbB2 signalling pathways, most likely because of the absence of calcineurin activity. We conclude that ErbB2 is a new CaM-binding protein, and that CaM plays a role in the regulation of this receptor and its downstream signalling pathways in vivo.

Keywords: calmodulin, calmodulin-binding protein, epidermal growth factor receptor, ErbB2/Neu/HER2 receptor
Abbreviations: ATF1, activating transcription factor-1; [Ca]cyt, cytosolic concentration of free calcium; CaM, calmodulin; CaM-BD, CaM-binding domain; CaMK-II, CaM-dependent protein kinase II; CaMK-IV, CaM-dependent protein kinase IV; CREB, cAMP-response-element-binding protein; DMEM, Dulbecco's modified Eagle's medium; ECL®, enhanced chemiluminescence; EGF, epidermal growth factor; EGFR, EGF receptor; ERK, extracellular-signal-regulated kinase; FBS, foetal bovine serum; HRGβ1, heregulin-β1; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; NFAT, nuclear factor of activated T-cells; PI3K, phosphoinositide 3-kinase; PKB, protein kinase B; poly(L-Glu/L-Tyr), co-polymer of L-glutamic acid and L-tyrosine; W7, N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide; W12, N-(4-aminobutyl)-1-naphthalenesulphonamide; W13, N-(4-aminobutyl)-5-chloro-1-naphthalenesulphonamide
Abstract

Acknowledgments

We thank Amparo Jiménez for expert technical assistance, Dr Margarita Fernández and Dr Jorge Martín for gifts of anti-Akt/PKB antibodies, and Dr Miguel Quintanilla for the anti-α-tubulin antibody. This work was financed by grants (to A.V.) from the Comisión Interministerial de Ciencia y Tecnología (SAF2002-03258) and the Consejería de Educación de la Comunidad de Madrid (08.1/0027/2001-1). The generous support from the Instituto Carlos III, Fondo de Investigaciones Sanitarias (RTICCC C03/10) is also acknowledged. H.L. was supported by the Agencia Española de Cooperación Internacional (2002CN0013), A.dC. by a predoctoral fellowship from the Fundación Carolina and V.S. by a predoctoral fellowship from the Consejo de Desarrollo Científico y Humanístico de la Universidad Central de Venezuela.

Acknowledgments

References

  • 1. Schlessinger JCell signaling by receptor tyrosine kinases. Cell. 2000;103:211–225.[PubMed][Google Scholar]
  • 2. Carraway K. L., III, Sweeney C. Localization and modulation of erbB receptor tyrosine kinases. Curr. Opin. Cell Biol. 2001;13:125–130.[PubMed]
  • 3. Tzahar E., Yarden YThe ErbB-2/HER2 oncogenic receptor of adenocarcinomas: from orphanhood to multiple stromal ligands. Biochim. Biophys. Acta. 1998;1377:M25–M37.[PubMed][Google Scholar]
  • 4. Rubin I., Yarden YThe basic biology of HER2. Anal. Oncol. 2001;12:S3–S8.[PubMed][Google Scholar]
  • 5. Alroy I., Yarden YThe ErbB signaling network in embryogenesis and oncogenesis: signal diversification through combinatorial ligand–receptor interactions. FEBS Lett. 1997;410:83–86.[PubMed][Google Scholar]
  • 6. Olayioye M. A., Neve R. M., Lane H. A., Hynes N. E. The ErbB signaling network: receptor heterodimerization in development and cancer. EMBO J. 2000;19:3159–3167.
  • 7. Villalobo A., Ruano M. J., Palomo-Jiménez P. I., Li H., Martín-Nieto J. The epidermal growth factor receptor and the calcium signal. In: Pochet R., Donato R., Haiech J., Heizmann C., Gerke V., editors. Calcium: The Molecular Basis of Calcium Action in Biology and Medicine. Boston: Kluwer Academic Publishers; 2000. pp. 287–303. [PubMed]
  • 8. Feldner J. C., Brandt B. H. Cancer cell motility – on the road from c-erbB-2 receptor steered signaling to actin reorganization. Exp. Cell Res. 2002;272:93–108.[PubMed]
  • 9. Dittmar T., Husemann A., Schewe Y., Nofer J.-R., Niggemann B., Zänker K. S., Brandt B. H. Induction of cancer cell migration by epidermal growth factor is initiated by specific phosphorylation of tyrosine 1248 of c-erbB-2 receptor via EGFR. FASEB J. 2002;16:1823–1825.[PubMed]
  • 10. Persechini A., Cronk BThe relationship between the free concentrations of Ca and Ca–calmodulin in intact cells. J. Biol. Chem. 1999;274:6827–6830.[PubMed][Google Scholar]
  • 11. Martín-Nieto J., Cusidó-Hita D. M., Li H., Benguría A., Villalobo A. Regulation of ErbB receptors by calmodulin. In: Pandalai S. G., editor. Recent Research Developments in Biochemistry. Part I. Vol. 3. Trivandrum: Research Signpost; 2002. pp. 41–58. [PubMed]
  • 12. San José E., Benguría A., Geller P., Villalobo ACalmodulin inhibits the epidermal growth factor receptor tyrosine kinase. J. Biol. Chem. 1992;267:15237–15245.[PubMed][Google Scholar]
  • 13. Benguría A., Martín-Nieto J., Benaim G., Villalobo ARegulatory interaction between calmodulin and the epidermal growth factor receptor. Ann. N.Y. Acad. Sci. 1995;766:472–476.[PubMed][Google Scholar]
  • 14. Martín-Nieto J., Villalobo AThe human epidermal growth factor receptor contains a juxtamembrane calmodulin-binding site. Biochemistry. 1998;37:227–236.[PubMed][Google Scholar]
  • 15. Li H., Villalobo AEvidence for the direct interaction between calmodulin and the human epidermal growth factor receptor. Biochem. J. 2002;362:499–505.[Google Scholar]
  • 16. Li H., Ruano M. J., Villalobo A. Endogenous calmodulin interacts with the epidermal growth factor receptor in living cells. FEBS Lett. 2004;559:175–180.[PubMed]
  • 17. Countaway J. L., Nairn A. C., Davis R. J. Mechanism of desensitization of the epidermal growth factor receptor protein-tyrosine kinase. J. Biol. Chem. 1992;267:1129–1140.[PubMed]
  • 18. Theroux S. J., Latour D. A., Stanley K., Raden D. L., Davis R. J. Signal transduction by the epidermal growth factor receptor is attenuated by a COOH-terminal domain serine phosphorylation site. J. Biol. Chem. 1992;267:16620–16626.[PubMed]
  • 19. Feinmesser R. L., Wicks S. J., Taverner C. J., Chantry A. Ca/calmodulindependent kinase II phosphorylates the epidermal growth factor receptor on multiple sites in the cytoplasmic tail and serine 744 within the kinase domain to regulate signal generation. J. Biol. Chem. 1999;274:16168–16173.[PubMed]
  • 20. Aifa S., Johansen K., Nilsson U. K., Liedberg B., Lundström I., Svensson S. P. S. Interactions between the juxtamembrane domain of the EGFR and calmodulin measured by surface plasmon resonance. Cell. Signal. 2002;14:1005–1013.[PubMed]
  • 21. Hayashi N., Matsubara M., Takasaki A., Titani K., Taniguchi HAn expression system of rat calmodulin using T17 phage promoter in Escherichia coli. Protein Expression Purif. 1998;12:25–28.[PubMed][Google Scholar]
  • 22. Dicker P., Rozengurt EPhorbol esters and vasopressin stimulate DNA synthesis by a common mechanism. Nature (London) 1980;287:607–612.[PubMed][Google Scholar]
  • 23. Wolff D. J., Poirier P. G., Brostrom C. A., Brostrom M. A. Divalent cation binding properties of bovine brain Ca-dependent regulator protein. J. Biol. Chem. 1977;252:4108–4117.[PubMed]
  • 24. Billingsley M. L., Pennypacker K. R., Hoover C. G., Brigati D. J., Kincaid R. L. A rapid and sensitive method for detection and quantification of calcineurin and calmodulin-binding proteins using biotinylated calmodulin. Proc. Natl. Acad. Sci. U.S.A. 1985;82:7585–7589.
  • 25. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 1970;227:680–685.[PubMed]
  • 26. Kroll T., Odyvanova L., Clement J. H., Platzer C., Naumann A., Marr N., Höffken K., Wölfl S. Molecular characterization of breast cancer cell lines by expression profiling. J. Cancer Res. Clin. Oncol. 2002;128:125–134.[PubMed]
  • 27. Ullrich A., Coussens L., Hayflick J. S., Dull T. J., Gray A., Tam A. W., Lee J., Yarden Y., Libermann T. A., Schlessinger J., et al. Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells. Nature (London) 1984;309:418–425.[PubMed]
  • 28. O'Brian C. A., Ward N. E. Binding of protein kinase C to N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide through its ATP binding site. Biochem. Pharmacol. 1989;38:1737–1742.[PubMed]
  • 29. Verbeek B. S., Adriaansen-Slot S. S., Vroom T. M., Beckers T., Rijksen G. Overexpression of EGFR and c-erbB2 causes enhanced cell migration in human breast cancer cells and NIH3T3 fibroblasts. FEBS Lett. 1998;425:145–150.[PubMed]
  • 30. Nishikawa M., Tanaka T., Hidaka HCa-calmodulin-dependent phosphorylation and platelet secretion. Nature (London) 1980;287:863–865.[PubMed][Google Scholar]
  • 31. Wu B.-W., Wu Y., Wang J.-L., Lin J.-S., Yuan S.-Y., Li A., Cui W.-RStudy on the mechanism of epidermal growth factor-induced proliferation of hepatoma cells. World J. Gastroenterol. 2003;9:271–275.[Google Scholar]
  • 32. Feinmesser R. L., Gray K., Means A. R., Chantry A. HER-2/c-erbB2 is phosphorylated by calmodulin-dependent protein kinase II on a single site in the cytoplasmic tail at threonine-1172. Oncogene. 1996;12:2725–2730.[PubMed]
  • 33. Hernández-Sotomayor S. M., Arteaga C. L., Soler C., Carpenter G. Epidermal growth factor stimulates substrate-selective protein-tyrosine-phosphatase activity. Proc. Natl. Acad. Sci. U.S.A. 1993;90:7691–7695.
  • 34. Zhai Y., Wirth J., Kang S., Welsch C. W., Esselman W. J. LAR-PTPase cDNA transfection suppression of tumor growth of neu oncogene-transformed human breast carcinoma cells. Mol. Carcinog. 1995;14:103–110.[PubMed]
  • 35. Zhang X. B., Lee M. S., Zelivianski S., Lin M. F. Characterization of a prostate-specific tyrosine phosphatase by mutagenesis and expression in human prostate cancer cells. J. Biol. Chem. 2001;276:2544–2550.[PubMed]
  • 36. Tebar F., Villalonga P., Sorkina T., Agell N., Sorkin A., Enrich CCalmodulin regulates intracellular trafficking of epidermal growth factor receptor and the MAPK signaling pathway. Mol. Biol. Cell. 2002;13:2057–2068.[Google Scholar]
  • 37. Agell N., Bachs O., Rocamora N., Villalonga PModulation of the Ras/Raf/MEK/ERK pathway by Ca and calmodulin. Cell. Signalling. 2002;14:649–654.[PubMed][Google Scholar]
  • 38. Bosch M., Gil J., Nachs O., Agell NCalmodulin inhibitor W13 induces sustained activation of ERK2 and expression of p21. J. Biol. Chem. 1998;273:22145–22150.[PubMed][Google Scholar]
  • 39. Enslen H., Sun P., Brickey D., Soderling S. H., Klamo E., Soderling T. R. Characterization of Ca/calmodulin-dependent protein kinase IV: role in transcriptional regulation. J. Biol. Chem. 1994;269:15520–15527.[PubMed]
  • 40. Schwaninger M., Blume R., Krüger M., Lux G., Oetjen E., Knepel WInvolvement of the Ca-dependent phosphatase calcineurin in gene transcription that is stimulated by cAMP through cAMP response elements. J. Biol. Chem. 1995;270:8860–8866.[PubMed][Google Scholar]
  • 41. Wu G.-Y., Deisseroth K., Tsien R. W. Activity-dependent CREB phosphorylation: convergence of a fast, sensitive calmodulin kinase pathway and a slow, less sensitive mitogen-activated protein kinase pathway. Proc. Natl. Acad. Sci. U.S.A. 2001;98:2808–2813.
  • 42. Mellström B., Naranjo J. R. Mechanisms of Ca-dependent transcription. Curr. Opin. Neurobiol. 2001;11:312–319.[PubMed]
  • 43. Liu F., Thompson M. A., Wagner S., Greenberg M. E., Green M. R. Activating transcription factor-1 can mediate Ca- and cAMP-inducible transcriptional activation. J. Biol. Chem. 1993;268:6714–6720.[PubMed]
  • 44. Dolmetsch R. E., Lewis R. S., Goodnow C. C., Healy J. I. Differential activation of transcription factors induced by Ca response amplitude and duration. Nature (London) 1997;386:855–858.[PubMed]
  • 45. Holmberg C. I., Tran S. E. F., Eriksson J. E., Sistonen L. Multisite phosphorylation provides sophisticated regulation of transcription factors. Trends Biochem. Sci. 2002;27:619–627.[PubMed]
Collaboration tool especially designed for Life Science professionals.Drag-and-drop any entity to your messages.