Modulation of Rho and Cytoskeletal Protein Attachment to Membranes by a Prenylcysteine Analog

Abstract

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, were used to analyze the role of prenylation in their membrane association. Silver-stained gels indicated that Nacetyl-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-Sall-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-Lcysteine. 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.

Annexin 2 Regulates Intestinal Epithelial Cell Spreading and Wound Closure through Rho-Related Signaling

“…Following release from its GDP dissociation inhibitor, Rho can be targeted to membranes where it is subsequently activated. 54,55 Consistent with this notion, we observed not only a decrease in the amount of membrane-associated Rho but also reduced active Rho following annexin 2 knockdown. This finding supports a role of annexin 2 in mediating Rho membrane association that is required for its activation at these sites.…”

In vitro and clinical evaluation of SIG1273: a cosmetic functional ingredient with a broad spectrum of anti‐aging and antioxidant activities

“…Isoprenylcysteine (IPC) small molecules have been identified as a novel class of topical anti‐inflammatory and antimicrobial cosmetic functional ingredients (CFI) . IPCs have been shown to inhibit signaling activation in the membrane, by competing with isoprenoid groups for prenyl‐binding sites on membrane‐associated proteins . In addition, IPCs modulate signal transduction by inhibiting heterotrimeric G‐protein formation and/or presumably by blocking downstream G‐protein–effector interactions and more recently by binding and activating PPARγ .…”

Synthesis of high specific activity 35S‐labelled N‐methanesulfonyl farnesylcysteine and a photoactive analog

“…e eliveau and co-workers have estimated the critical micellular concentration of AGGC to be 340 mM, substantially above the amounts used in Figure 5. 31 Interestingly, AGGCE was a significantly better competitor than AGGC: the latter offered no protection while AGGCE reduced GDI cross-linking to 44% of that of GDI irradiated with [ 35 S]3 alone. Competition experiments previously performed using GDI and [ 35 S]4 with these geranylgeranylcysteines also indicated a similar pattern of cross-linking reduction, with AGGCE being the more effective competitor.…”