Both the C-terminal polylysine region and the farnesylation of K-RasB are important for its specific interaction with calmodulin

PLoS One. 2011;6(7):e21929. doi: 10.1371/journal.pone.0021929. Epub 2011 Jul 5.


Background: Ras protein, as one of intracellular signal switches, plays various roles in several cell activities such as differentiation and proliferation. There is considerable evidence showing that calmodulin (CaM) binds to K-RasB and dissociates K-RasB from membrane and that the inactivation of CaM is able to induce K-RasB activation. However, the mechanism for the interaction of CaM with K-RasB is not well understood.

Methodology/principal findings: Here, by applying fluorescence spectroscopy and isothermal titration calorimetry, we have obtained thermodynamic parameters for the interaction between these two proteins and identified the important elements of K-RasB for its interaction with Ca(2+)/CaM. One K-RasB molecule interacts with one CaM molecule in a GTP dependent manner with moderate, micromolar affinity at physiological pH and physiologic ionic strength. Mutation in the polybasic domain of K-Ras decreases the binding affinity. By using a chimera in which the C-terminal polylysine region of K-RasB has been replaced with that of H-Ras and vice versa, we find that at physiological pH, H-Ras-(KKKKKK) and Ca(2+)/CaM formed a 1:1 complex with an equilibrium association constant around 10(5) M(-1), whereas no binding reaction of K-RasB-(DESGPC) with Ca(2+)/CaM is detected. Furthermore, the interaction of K-RasB with Ca(2+)/CaM is found to be enhanced by the farnesylation of K-RasB.

Conclusions/significance: We demonstrate that the polylysine region of K-RasB not only contributes importantly to the interaction of K-RasB with Ca(2+)/CaM, but also defines its isoform specific interaction with Ca(2+)/CaM. The farnesylation of K-RasB is also important for its specific interaction with Ca(2+)/CaM. Information obtained here can enhance our understanding of how CaM interacts with K-RasB in physiological environments.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Algorithms
  • Amino Acid Sequence
  • Animals
  • Blotting, Western
  • Calcium / chemistry
  • Calcium / metabolism*
  • Calmodulin / chemistry
  • Calmodulin / genetics
  • Calmodulin / metabolism*
  • Calorimetry
  • Cattle
  • Escherichia coli / genetics
  • Kinetics
  • Light
  • Models, Chemical
  • Polylysine / chemistry
  • Polylysine / genetics
  • Polylysine / metabolism*
  • Prenylation
  • Protein Binding / radiation effects
  • Proto-Oncogene Proteins p21(ras) / chemistry
  • Proto-Oncogene Proteins p21(ras) / genetics
  • Proto-Oncogene Proteins p21(ras) / metabolism*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Spectrometry, Fluorescence
  • Thermodynamics


  • Calmodulin
  • Recombinant Proteins
  • Polylysine
  • Proto-Oncogene Proteins p21(ras)
  • Calcium