Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

K-Ras4B phosphorylation at Ser181 is inhibited by calmodulin and modulates K-Ras activity and function

Oncogene volume 29, pages 5911–5922 (2010)Cite this article

Subjects

Abstract

Fine tuning of Ras activity is widely known as a mechanism to induce different cellular responses. Recently, we have shown that calmodulin (CaM) binds to K-Ras and that K-Ras phosphorylation inhibits its interaction with CaM. In this study we report that CaM inhibits K-Ras phosphorylation at Ser181 by protein kinase C (PKC) in vivo, and this is a mechanism to modulate K-Ras activity and signaling. Although CaM inhibition increased the activation of endogenous K-Ras, PKC inhibition decreased its activation status. We demonstrate that K-Ras phosphorylation decreased susceptibility to p120GAP activity. Accordingly, we also observed that non-phosphorylable K-Ras mutant exhibits a less sustained activation profile and do not efficiently activate AKT at low growth factor doses compared with wild-type K-Ras. It is interesting that the physiological responses induced by K-Ras are affected by this phosphorylation; when K-Ras cannot be phosphorylated it exhibits a remarkably decreased ability to stimulate proliferation in non-saturated serum conditions. Finally, we demonstrate that phosphorylation also regulates oncogenic K-Ras functions, as focus formation capacity, mobility and apoptosis resistance upon adriamycin treatment of cells expressing oncogenic K-Ras that cannot be phosphorylated are highly compromised. Moreover, at low serum concentration proliferation and survival is practically inhibited when cells cannot phosphorylate oncogenic K-Ras. In this condition, K-Ras phosphorylation is essential to ensure a proper activation of mitogen-activated protein kinase and PI3K/AKT pathways. In summary, our findings suggest that the interplay between CaM interaction and PKC phosphorylation is essential to regulate non-oncogenic and oncogenic K-Ras activity and functionality.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9

Similar content being viewed by others

References

  • Agell N, Bachs O, Rocamora N, Villalonga P . (2002). Modulation of the Ras/Raf/MEK/ERK pathway by Ca(2+), and calmodulin. Cell Signal 14: 649–654.

    Article  CAS  Google Scholar 

  • Agell N, Jaumot M, Rodriguez-Vilarrupla A, Brun S, Abella N, Canela N et al. (2006). The diverging roles of calmodulin and PKC in the regulation of p21 intracellular localization. Cell Cycle 5: 3–6.

    Article  CAS  Google Scholar 

  • Ashery U, Yizhar O, Rotblat B, Elad-Sfadia G, Barkan B, Haklai R et al. (2006). Spatiotemporal organization of Ras signaling: rasosomes and the galectin switch. Cell Mol Neurobiol 26: 471–495.

    Article  CAS  Google Scholar 

  • Ballester R, Furth ME, Rosen OM . (1987). Phorbol ester- and protein kinase C-mediated phosphorylation of the cellular Kirsten ras gene product. J Biol Chem 262: 2688–2695.

    CAS  PubMed  Google Scholar 

  • Bivona TG, Quatela SE, Bodemann BO, Ahearn IM, Soskis MJ, Mor A et al. (2006). PKC regulates a farnesyl-electrostatic switch on K-Ras that promotes its association with Bcl-XL on mitochondria and induces apoptosis. Mol Cell 21: 481–493.

    Article  CAS  Google Scholar 

  • Bos JL . (1989). Ras oncogenes in human cancer. Cancer Res 49: 4682–4689.

    CAS  Google Scholar 

  • Bosch M, Gil J, Bachs O, Agell N . (1998). Calmodulin inhibitor W13 induces sustained activation of ERK2 and expression of p21(cip1). J Biol Chem 273: 22145–22150.

    Article  CAS  Google Scholar 

  • Chakravarthy B, Morley P, Whitfield J . (1999). Ca2+-calmodulin and protein kinase Cs: a hypothetical synthesis of their conflicting convergences on shared substrate domains. Trends in Neurosci 22: 12–16.

    Article  CAS  Google Scholar 

  • Downward J . (1996). Control of ras activation. Cancer Surv 27: 87–100.

    CAS  PubMed  Google Scholar 

  • Downward J, Graves JD, Warne PH, Rayter S, Cantrell DA . (1990). Stimulation of p21ras upon T-cell activation. Nature 346: 719–723.

    Article  CAS  Google Scholar 

  • Drosten M, Dhawahir A, Sum EY, Urosevic J, Lechuga CG, Esteban LM et al. (2010). Genetic analysis of Ras signalling pathways in cell proliferation, migration and survival. EMBO J 29: 1091–1104.

    Article  CAS  Google Scholar 

  • Elad-Sfadia G, Haklai R, Balan E, Kloog Y . (2004). Galectin-3 augments K-Ras activation and triggers a Ras signal that attenuates ERK but not phosphoinositide 3-kinase activity. J Biol Chem 279: 34922–34930.

    Article  CAS  Google Scholar 

  • Elad-Sfadia G, Haklai R, Ballan E, Gabius HJ, Kloog Y . (2002). Galectin-1 augments Ras activation and diverts Ras signals to Raf-1 at the expense of phosphoinositide 3-kinase. J Biol Chem 277: 37169–37175.

    Article  CAS  Google Scholar 

  • Hamilton M, Liao J, Cathcart MK, Wolfman A . (2001). Constitutive association of c-N-Ras with c-Raf-1 and protein kinase C epsilon in latent signaling modules. J Biol Chem 276: 29079–29090.

    Article  CAS  Google Scholar 

  • Hancock JF . (2003). Ras proteins: different signals from different locations. Nat Rev Mol Cell Biol 4: 373–384.

    Article  CAS  Google Scholar 

  • Johnson L, Greenbaum D, Cichowski K, Mercer K, Murphy E, Schmitt E et al. (1997). K-ras is an essential gene in the mouse with partial functional overlap with N-ras. Genes Dev 11: 2468–2481.

    Article  CAS  Google Scholar 

  • Kahan C, Seuwen K, Meloche S, Pouyssegur J . (1992). Coordinate, biphasic activation of p44 mitogen-activated protein kinase and S6 kinase by growth factors in hamster fibroblasts. Evidence for thrombin-induced signals different from phosphoinositide turnover and adenylylcyclase inhibition. J Biol Chem 267: 13369–13375.

    CAS  PubMed  Google Scholar 

  • Klee C, Vanaman T . (1982). Calmodulin. Adv Protein Chem 35: 213–321.

    Article  CAS  Google Scholar 

  • Liao J, Planchon SM, Wolfman JC, Wolfman A . (2006). Growth Factor-dependent AKT Activation and Cell Migration Requires the Function of c-K(B)-Ras versus other cellular Ras isoforms. J Biol Chem 281: 29730–29738.

    Article  CAS  Google Scholar 

  • Liou JS, Chen JS, Faller DV . (2004). Characterization of p21Ras-mediated apoptosis induced by protein kinase C inhibition and application to human tumor cell lines. J Cell Physiol 198: 277–294.

    Article  CAS  Google Scholar 

  • Lopez-Alcala C, Alvarez-Moya B, Villalonga P, Calvo M, Bachs O, Agell N . (2008). Identification of essential interacting elements in K-Ras/calmodulin binding and its role in K-Ras localization. J Biol Chem 283: 10621–10631.

    Article  CAS  Google Scholar 

  • Lu KP, Means AR . (1993). Regulation of the cell cycle by calcium and calmodulin. Endocrine Rev 14: 40–58.

    Article  CAS  Google Scholar 

  • Malumbres M, Barbacid M . (2003). RAS oncogenes: the first 30 years (vol 3, pg 459, 2003). Nat Rev Cancer 3: 708.

    Article  CAS  Google Scholar 

  • Marshall C . (1999). How do small GTPase signal transduction pathways regulate cell cycle entry? Curr Opin Cell Biol 11: 732–736.

    Article  CAS  Google Scholar 

  • Marshall CJ . (1995). Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell 80: 179–185.

    Article  CAS  Google Scholar 

  • Matallanas D, Sanz-Moreno V, Arozarena I, Calvo F, Agudo-Ibanez L, Santos E et al. (2006). Distinct utilization of effectors and biological outcomes resulting from site-specific Ras activation: Ras functions in lipid rafts and Golgi complex are dispensable for proliferation and transformation. Mol Cell Biol 26: 100–116.

    Article  CAS  Google Scholar 

  • Moreto J, Llado A, Vidal-Quadras M, Calvo M, Pol A, Enrich C et al. (2008). Calmodulin modulates H-Ras mediated Raf-1 activation. Cell Signal 20: 1092–1103.

    Article  CAS  Google Scholar 

  • Omerovic J, Laude AJ, Prior IA . (2007). Ras proteins: paradigms for compartmentalised and isoform-specific signalling. Cell Mol Life Sci 64: 2575–2589.

    Article  CAS  Google Scholar 

  • Plowman SJ, Ariotti N, Goodall A, Parton RG, Hancock JF . (2008). Electrostatic interactions positively regulate K-Ras nanocluster formation and function. Mol Cell Biol 28: 4377–4385.

    Article  CAS  Google Scholar 

  • Prior IA, Muncke C, Parton RG, Hancock JF . (2003). Direct visualization of Ras proteins in spatially distinct cell surface microdomains. J Cell Biol 160: 165–170.

    Article  CAS  Google Scholar 

  • Pumiglia KM, Decker SJ . (1997). Cell cycle arrest mediated by the MEK/mitogen-activated protein kinase pathway. Proc Natl Acad Sci USA 94: 448–452.

    Article  CAS  Google Scholar 

  • Qui MS, Green SH . (1992). PC12 cell neuronal differentiation is associated with prolonged p21ras activity and consequent prolonged ERK activity. Neuron 9: 705–717.

    Article  CAS  Google Scholar 

  • Rodriguez-Vilarrupla A, Jaumot M, Abella N, Canela N, Brun S, Diaz C et al. (2005). Binding of calmodulin to the carboxy-terminal region of p21 induces nuclear accumulation via inhibition of protein kinase C-mediated phosphorylation of Ser153. Mol Cell Biol 25: 7364–7374.

    Article  CAS  Google Scholar 

  • Roovers K, Assoian RK . (2000). Integrating the MAP kinase signal into the G1 phase cell cycle machinery. Bioessays 22: 818–826.

    Article  CAS  Google Scholar 

  • Schulman H . (1993). The multifunctional Ca2+/calmodulin-dependent protein kinases. Curr Opin Cell Biol 5: 247–253.

    Article  CAS  Google Scholar 

  • Shalom-Feuerstein R, Plowman SJ, Rotblat B, Ariotti N, Tian T, Hancock JF et al. (2008). K-ras nanoclustering is subverted by overexpression of the scaffold protein galectin-3. Cancer Res 68: 6608–6616.

    Article  CAS  Google Scholar 

  • Umanoff H, Edelmann W, Pellicer A, Kucherlapati R . (1995). The murine N-ras gene is not essential for growth and development. Proc Natl Acad Sci USA 92: 1709–1713.

    Article  CAS  Google Scholar 

  • Veigl ML, Sedwick WD, Vanaman TC . (1982). Calmodulin and Ca2+ in normal and transformed cells. Fed Proc 41: 2283–2288.

    CAS  PubMed  Google Scholar 

  • Villalonga P, López-Alcalá C, Bosch M, Chiloeches A, Rocamora N, Gil J et al. (2001). Calmodulin binds to K-RAs but H- or N-Ras, and modulates its downstream signaling. Mol Cell Biol 21: 7345–7354.

    Article  CAS  Google Scholar 

  • Villalonga P, Lopez-Alcala C, Chiloeches A, Gil J, Marais R, Bachs O et al. (2002). Calmodulin prevents activation of Ras by PKC in 3T3 fibroblasts. J Biol Chem 277: 37929–37935.

    Article  CAS  Google Scholar 

  • Xia S, Chen Z, Forman LW, Faller DV . (2009). PKCdelta survival signaling in cells containing an activated p21Ras protein requires PDK1. Cell Signal 21: 502–508.

    Article  CAS  Google Scholar 

  • Xia S, Forman LW, Faller DV . (2007). Protein kinase C delta is required for survival of cells expressing activated p21RAS. J Biol Chem 282: 13199–13210.

    Article  CAS  Google Scholar 

  • Yan J, Roy S, Apolloni A, Lane A, Hancock JF . (1998). Ras isoforms vary in their ability to activate Raf-1 and phosphoinositide 3-kinase. J Biol Chem 273: 24052–24056.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Dr M Barbacid (CNIO, Spain) for the generous gift of Rasless and K-Ras−/− MEFs. We are also indebt with Anna Bosch and Maria Calvo (Confocal microscopy facility, SCT-University of Barcelona) for their help of performing the cell mobility assays. This study was supported by grant SAF2007-60491 from the Ministerio de Educación y Ciencia (Spain). Blanca Alvarez-Moya was recipient of a pre-doctoral fellowship from the Generalitat de Catalunya (Spain).

Author information

Authors and Affiliations

  1. Departament de Biologia Cel.lular, Immunologia i Neurociències. Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Facultat de Medicina, Universitat de Barcelona, Barcelona, Spain

    B Alvarez-Moya, C López-Alcalá, O Bachs & N Agell

  2. Molecular Oncology Programme, Centro Nacional de Investigaciones Oncológicas (CNIO), Melchor Fernández Almagro 3, Madrid, Spain

    M Drosten

Authors
  1. B Alvarez-Moya
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. C López-Alcalá
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. M Drosten
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. O Bachs
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. N Agell
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to N Agell.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies the paper on the Oncogene website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Alvarez-Moya, B., López-Alcalá, C., Drosten, M. et al. K-Ras4B phosphorylation at Ser181 is inhibited by calmodulin and modulates K-Ras activity and function. Oncogene 29, 5911–5922 (2010). https://doi.org/10.1038/onc.2010.298

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/onc.2010.298

Keywords

This article is cited by

Search

Advanced search

Quick links