Analysis of K-Ras phosphorylation, translocation, and induction of apoptosis.
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.
Quatela
Steven E
SE
Department of Medicine, Cancer Institute, New York University School of Medicine, New York, NY, USA.
Sung
Pamela J
PJ
Ahearn
Ian M
IM
Bivona
Trever G
TG
Philips
Mark R
MR
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D019556:COS Cells; D020169:Caspases / Q000378:metabolism; D002522:Chlorocebus aethiops; D006801:Humans; D019169:Jurkat Cells; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D021381:Protein Transport; D011518:Proto-Oncogene Proteins / Q000378:metabolism* / Q000502:physiology; D014162:Transfection / Q000379:methods; D018631:ras Proteins / Q000378:metabolism* / Q000502:physiology
D011518:Proto-Oncogene Proteins; D011493:Protein Kinase C; D020169:Caspases; D018631:ras Proteins