Modulation of Rho and cytoskeletal protein attachment to membranes by a prenylcysteine analog.
The GTPases Rho regulate the assembly of polymerized actin structures. Their C-terminal sequences end with the CAAX motif that undergo a lipidation of the cysteine residue. Analogs to the C-terminal ends of Rho proteins, N-acetyl-S-all-trans, trans-farnesyl-L-cysteine and N-acetyl-S-all-trans-geranylgeranyl-L-cysteine, wereused to analyze the role of prenylation in their membrane association. Silver-stained gels indicated that N-acetyl-S-all-trans-geranylgeranyl-L-cysteine treatment released only a few proteins of 20, 46, and 60 kDa. Western blot analysis showed that N-acetyl-S-all-trans-geranylgeranyl-L-cysteine released RhoB (10%), RhoA (28%), and Cdc42 (95%) from membranes, whereas N-acetyl-S-all-trans and trans-farnesyl-L-cysteine did not. Rab1, which possesses two geranylgeranyl groups, was also strongly extracted by N-acetyl-S-all-trans-geranylgeranyl-L-cysteine, whereas Ras, which is farnesylated, was not. Furthermore, N-acetyl-S-all-trans-geranylgeranyl-L-cysteine was very efficient (95%) in dissociating actin and tubulin from membranes but not integral membrane protein P-glycoprotein and sodium/phosphate cotransporter NaP(i)-2. The extraction of Rho and cytoskeletal proteins occurred below the critical micellar concentration of N-acetyl-S-all-trans-geranylgeranyl-L-cysteine. Membrane treatments with 0.7 m KI totally extracted actin, whereas 70% of Cdc42 was released. Actin was, however, insoluble in Triton X-100-treated membranes, whereas this detergent extracted (80%) Cdc42. These data show that Rho proteins and actin are not physically bound together and suggest that their extraction from membranes by N-acetyl-S-all-trans-geranylgeranyl-L-cysteine likely occurs via different mechanisms.
Desrosiers
R R
RR
Laboratoire de médecine moléculaire, Hôpital Sainte-Justine, Université du Québec à Montréal, C.P. 8888, Succursale Centre-ville, Montréal, Québec H3C 3P8, Canada.
Gauthier
F
F
Lanthier
J
J
Béliveau
R
R
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism; D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000199:Actins / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology*; D003545:Cysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D004224:Diterpenes / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007672:Kidney Cortex / Q000502:physiology*; D007700:Kinetics; D008297:Male; D008871:Microvilli / Q000187:drug effects / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism; D020743:rhoB GTP-Binding Protein / Q000378:metabolism
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000199:Actins; D003598:Cytoskeletal Proteins; D004224:Diterpenes; D004791:Enzyme Inhibitors; C095716:N-acetyl-S-geranylgeranyl-cysteine; D020764:cdc42 GTP-Binding Protein; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein; D020743:rhoB GTP-Binding Protein; D003545:Cysteine; C068268:N-acetyl-S-farnesylcysteine; D000111:Acetylcysteine