Differential interference of chlorpromazine with the membrane interactions of oncogenic K-Ras and its effects on cell growth.
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.
Eisenberg
Sharon
S
Department of Neurobiology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.
Giehl
Klaudia
K
Henis
Yoav I
YI
Ehrlich
Marcelo
M
D020816:Amino Acid Motifs / Q000502:physiology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002453:Cell Cycle / Q000187:drug effects*; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology*; D002465:Cell Movement / Q000187:drug effects; D002746:Chlorpromazine / Q000494:pharmacology*; D003593:Cytoplasm / Q000201:enzymology*; D018492:Dopamine Antagonists / Q000494:pharmacology*; D007527:Isoenzymes / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology; D021381:Protein Transport / Q000187:drug effects; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D051381:Rats; D055672:Static Electricity; D020830:rac1 GTP-Binding Protein / Q000378:metabolism
D018492:Dopamine Antagonists; D007527:Isoenzymes; C499134:Rac1 protein, rat; D016283:Proto-Oncogene Proteins p21(ras); D020830:rac1 GTP-Binding Protein; D002746:Chlorpromazine