Tumor Necrosis Factor α Stimulation of Rac1 Activity

A number of proteins that play key roles in biological regulatory events undergo a process of post-translational modifications termed prenylation. The prenylation pathway consists of three enzymatic steps; the final processed protein is isoprenoidmodified and methylated on the C-terminal cysteine. This protein modification pathway plays a significant role in cancer biology because many oncogenic proteins undergo prenylation. Methylation of the C terminus by isoprenylcysteine carboxylmethyltransferase (Icmt) is the final step in the prenylation pathway. Cysmethynil, a specific Icmt inhibitor discovered in our laboratory, is able to inhibit Ras-mediated signaling, cell growth, and oncogenesis. We sought to examine the role of Icmt-mediated methylation on the behaviors of cancer cells associated with metastatic potential. Our results indicate that inhibition of methylation reduces migration of the highly metastatic MDA-MB-231 breast cancer cell line. In addition, cell adhesion and cell spreading are also significantly impacted by cysmethynil. To examine the mechanism of Icmt-dependent migration we focused on RhoA and Rac1, prenylated proteins that are important mediators of cell migration through their control of the actin cytoskeleton. Inhibition of Icmt significantly decreases the activation of both RhoA and Rac1; an increase in Rho GDP-dissociation inhibitor (RhoGDI) binding in the absence of methylation appears to contribute to this effect. Furthermore, in the absence of Icmt activity the addition of exogenous RhoA or Rac1 is able to partially rescue directed and random migration, respectively. These findings establish a role for Icmt-mediated methylation in cell migration and advance our understanding of the biological consequences of Rho methylation.Post-translational modifications of proteins play vital roles in many aspects of cell biology. Hence, identifying and understanding the biological impact of these processes is crucial to furthering our basic understanding of how cells function. Numerous proteins that control important biological regulatory events undergo a complex series of post-translational modifications that are directed by the presence of a so-called CaaX motif at their C terminus. This post-translational pathway, termed protein prenylation, is initiated by the attachment of an isoprenoid lipid to an invariant cysteine residue, the C of the CaaX motif (1, 2). Either a 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenoid is covalently attached to this cysteine by protein farnesyltransferase (FTase) 2 or protein geranylgeranyltransferase-I (GGTase-I), respectively (3). The prenylation step is followed by cleavage of the three C-terminal amino acids (the -AAX) by an endoplasmic reticulum (ER)-bound protease termed Rce1. Finally, the prenylated cysteine, which is now located at the C terminus, is methylated by isoprenylcysteine carboxylmethyltransferase (Icmt), another integral ER membrane protein (4, 5). The final result of these modifications is a protein that contains a prenylated and meth...

Section: Discussionsupporting

confidence: 81%

Section: Discussionmentioning

confidence: 99%

Section: Icmt Inhibition Impairs Cell Migration In a Dose-dependentmentioning

confidence: 99%

See 1 more Smart Citation

Role of Isoprenylcysteine Carboxylmethyltransferase-catalyzed Methylation in Rho Function and Migration

Cushman

Casey

2009

“…Additional post-prenylation modifications of Rac1, such as carboxyl methylation and proteolysis, are not required for complex formation with RhoGDI (77). On the contrary, lack of carboxyl methylation enhanced binding of Rac1 to RhoGDI in endothelial cells (78). Thus, to be able to conduct quantitative biochemical studies on Rac-RhoGDI interactions, we created stable Rac1(GDP)⅐RhoGDI and Rac2(GDP)⅐RhoGDI complexes in vitro by mixing recombinant Rac(1 or 2)(GDP), expressed in E. coli and prenylated in vitro, with a 2-or 4-fold excess of recombinant RhoGDI, also expressed in E. coli.…”

Section: Generation Of Rac1(gdp)⅐rhogdi and Rac2(gdp)⅐rhogdi Complexementioning

confidence: 97%

Liposomes Comprising Anionic but Not Neutral Phospholipids Cause Dissociation of Rac(1 or 2)·RhoGDI Complexes and Support Amphiphile-independent NADPH Oxidase Activation by Such Complexes

Ugolev

Molshanski-Mor

Weinbaum

et al. 2006

Activation of the phagocyte NADPH oxidase involves the assembly of a membrane-localized cytochrome b 559 with the cytosolic components p47 phox , p67 phox , p40 phox , and the GTPase Rac (1 or 2). In resting phagocytes, Rac is found in the cytosol as a prenylated protein in the GDP-bound form, associated with the Rho GDP dissociation inhibitor (RhoGDI). In the process of NADPH oxidase activation, Rac is dissociated from RhoGDI and translocates to the membrane, in concert with the other cytosolic components. The mechanism responsible for dissociation of Rac from RhoGDI is poorly understood. We generated Rac(1 or 2)⅐RhoGDI complexes in vitro from recombinant Rac(1 or 2), prenylated enzymatically, and recombinant RhoGDI, and purified these by anion exchange chromatography. Exposing Rac(1 or 2)(GDP)⅐RhoGDI complexes to liposomes containing four different anionic phospholipids caused the dissociation of Rac(1 or 2)(GDP) from RhoGDI and its binding to the anionic liposomes. Rac2(GDP)⅐RhoGDI complexes were more resistant to dissociation, reflecting the lesser positive charge of Rac2. Liposomes consisting of neutral phospholipid did not cause dissociation of Rac(1 or 2)⅐RhoGDI complexes. Rac1 exchanged to the hydrolysis-resistant GTP analogue, GMPPNP, associated with RhoGDI with lower affinity than Rac1(GDP) and Rac1(GMPPNP)⅐RhoGDI complexes were more readily dissociated by anionic liposomes. Rac1(GMPPNP)⅐RhoGDI complexes elicited NADPH oxidase activation in native phagocyte membrane liposomes in the presence of p67 phox , without the need for an anionic amphiphile, as activator. Both Rac1(GDP)⅐RhoGDI and Rac1(GMPPNP)⅐RhoGDI complexes elicited amphiphileindependent, p67 phox -dependent NADPH oxidase activation in phagocyte membrane liposomes enriched in anionic phospholipids but not in membrane liposomes enriched in neutral phospholipids.

“…A recent study found that the membrane association and biological function of farnesylated Ras proteins, but not of geranylgeranylated Rho GTPases, were impaired in the absence of Rce1 or Icmt expression (22). However, other studies using pharmacologic inhibitors suggested that geranylgeranylated protein function also depends on these modifications (51,52). Therefore, to elucidate whether the specific isoprenoid modification of a Rho GTPase dictates a dependence on Rce1-catalyzed -AAX cleavage and Icmt-mediated carboxyl methylation, we evaluated the subcellular localization of Rho GTPases in MEF cell lines deficient in Rce1 (Rce1 Ϫ/Ϫ ) or Icmt (Icmt Ϫ/Ϫ ).…”

Section: The Membrane Association And/or Function Of Rho Gtpases Is Dmentioning

confidence: 99%

Rho Family GTPase Modification and Dependence on CAAX Motif-signaled Posttranslational Modification

Roberts

Mitin

Keller

et al. 2008

291

300

Rho GTPases (20 human members) comprise a major branch of the Ras superfamily of small GTPases, and aberrant Rho GTPase function has been implicated in oncogenesis and other human diseases. Although many of our current concepts of Rho GTPases are based on the three classical members (RhoA, Rac1, and Cdc42), recent studies have revealed the diversity of biological functions mediated by other family members. A key basis for the functional diversity of Rho GTPases is their association with distinct subcellular compartments, which is dictated in part by three posttranslational modifications signaled by their carboxyl-terminal CAAX (where C represents cysteine, A is an aliphatic amino acid, and X is a terminal amino acid) tetrapeptide motifs. CAAX motifs are substrates for the prenyltransferase-catalyzed addition of either farnesyl or geranylgeranyl isoprenoid lipids, Rce1-catalyzed endoproteolytic cleavage of the AAX amino acids, and Icmt-catalyzed carboxyl methylation of the isoprenylcysteine. We utilized pharmacologic, biochemical, and genetic approaches to determine the sequence requirements and roles of CAAX signal modifications in dictating the subcellular locations and functions of the Rho GTPase family. Although the classical Rho GTPases are modified by geranylgeranylation, we found that a majority of the other Rho GTPases are substrates for farnesyltransferase. We found that the membrane association and/or function of Rho GTPases are differentially dependent on Rce1-and Icmt-mediated modifications. Our results further delineate the sequence requirements for prenyltransferase specificity and functional roles for protein prenylation in Rho GTPase function. We conclude that a majority of Rho GTPases are targets for pharmacologic inhibitors of farnesyltransferase, Rce1, and Icmt.Rho proteins are members of the Ras superfamily of small GTPases and function as GDP/GTP-regulated switches (1, 2). Much of our current understanding of the biochemistry and biology of the Rho family has come from the extensive evaluation of three classical members, RhoA, Rac1, and Cdc42 (3).Similar to Ras, Rho GDP/GTP cycling is regulated by guanine nucleotide exchange factors that promote the formation of the active GTP-bound form (4) and GTPase-activating proteins that catalyze the intrinsic GTPase activity and promote the formation of inactive GDP-bound Rho (5). Active, GTP-bound Rho GTPases bind preferentially to downstream effectors, stimulating diverse cytoplasmic signaling cascades that control actin reorganization and regulate cell shape, polarity, motility, adhesion, and membrane trafficking (6). As such, it is thought that activated Rho proteins contribute to cancer progression by influencing the ability of cells to migrate and thus to invade and metastasize. In addition to these alterations in cellular function, aberrant activation of Rho proteins has also been shown to contribute to other cancer phenotypes by promoting cell growth, proliferation, survival, and angiogenesis (7). Therefore, defining pharmacologic approaches fo...