Differential Interference of Chlorpromazine with the Membrane Interactions of Oncogenic K-Ras and Its Effects on Cell Growth

Abstract

Membrane anchorage of Ras proteins is important for their signaling and oncogenic potential. K-Ras4B (K-Ras), the Ras isoform most often mutated in human cancers, is the only Ras isoform where a polybasic motif contributes essential electrostatic interactions with the negatively charged cytoplasmic leaflet. Here we studied the effects of the cationic amphiphilic drug chlorpromazine (CPZ) on the membrane association of oncogenic K-Ras(G12V), cell proliferation, and apoptosis. Combining live cell microscopy, FRAP beam size analysis, and cell fractionation studies, we show that CPZ reduces the association of GFP-K-Ras(G12V) with the plasma membrane and increases its exchange between plasma membrane and cytoplasmic pools. These effects appear to depend on electrostatic interactions because the membrane association of another related protein that has a membrane-interacting polybasic cluster (Rac1(G12V)) was also affected, whereas that of H-Ras was not. The weakened association with the plasma membrane led to a higher fraction of GFP-K-Ras(G12V) in the cytoplasm and in internal membranes, accompanied by either cell cycle arrest (PANC-1 cells) or apoptosis (Rat-1 fibroblasts), the latter being in correlation with the targeting of K-Ras(G12V) to mitochondria. In accord with these results, CPZ compromised the transformed phenotype of PANC-1 cells, as indicated by inhibition of cell migration and growth in soft agar.

Protein expression of targets of the FMRP regulon is altered in brains of subjects with schizophrenia and mood disorders

“…Treatment of a rat pancreas cell line (PANC-1) with chlorpromazine resulted in increased GFP-RAC1 expression (Table 5; Eisenberg et al, 2008). Hill et al (2006) reported on a significant decrease in RAC1 mRNA expression in white matter of prefrontal cortex in subjects with schizophrenia (Hill et al, 2006).…”

Fendiline Inhibits K-Ras Plasma Membrane Localization and Blocks K-Ras Signal Transmission

“…Several drugs that are weak amphiphilic bases, including chlorpromazine, scored weakly positive in the assay. The effect of this class of drug on K-Ras localization has been described previously (47), and the mechanism of action is believed to involve direct impairment of the electrostatic interaction of the K-Ras polybasic domain with the negatively charged inner leaflet of the plasma membrane. These compounds were not pursued further.…”

Differential Effects of Prenylation andS-Acylation on Type I and II ROPS Membrane Interaction and Function

“…FRAP studies (Axelrod et al, 1976;Koppel et al, 1976) were carried out essentially as described previously (Illenberger et al, 2003;Henis et al, 2006;Eisenberg et al, 2008). FRAP measurements were carried out at 22°C on the abaxial side of cauline leaves taken from 2-week-old plants.…”