Analysis of K-Ras phosphorylation, translocation, and induction of apoptosis

Methods Enzymol. 2008;439:87-102. doi: 10.1016/S0076-6879(07)00407-7.


K-Ras is a member of a family of proteins that associate with the plasma membrane by virtue of a lipid modification that inserts into the membrane and a polybasic region that associates with the anionic head groups of inner leaflet phospholipids. In the case of K-Ras, the lipid is a C-terminal farnesyl isoprenoid adjacent to a polylysine sequence. The affinity of K-Ras for the plasma membrane can be modulated by diminishing the net charge of the polybasic region. Among the ways this can be accomplished is phosphorylation by protein kinase C (PKC) of serine 181 within the polybasic region. Phosphorylation at this site regulates a farnesyl-electrostatic switch that controls association of K-Ras with the plasma membrane. Surprisingly, engagement of the farnesyl-electrostatic switch promotes apoptosis. This chapter describes methods for directly analyzing the phosphorylation status of K-Ras using metabolic labeling with (32)P, for indirectly assessing the farnesyl-electrostatic switch by following GFP-tagged K-Ras in live cells, for artificially activating the farnesyl-electrostatic switch by directing the kinase domain of a PKC to activated K-Ras using a Ras-binding domain, and for assessing apoptosis of individual cells using a YFP-tagged caspase 3 biosensor.

MeSH terms

  • Animals
  • Apoptosis / drug effects*
  • COS Cells
  • Caspases / metabolism
  • Chlorocebus aethiops
  • Humans
  • Jurkat Cells
  • Phosphorylation
  • Protein Kinase C / metabolism
  • Protein Transport
  • Proto-Oncogene Proteins / metabolism*
  • Proto-Oncogene Proteins / physiology
  • Transfection / methods
  • ras Proteins / metabolism*
  • ras Proteins / physiology


  • Proto-Oncogene Proteins
  • Protein Kinase C
  • Caspases
  • ras Proteins