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

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 isoprenoid-modified 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 methylated cysteine at its C terminus. Numerous studies have demonstrated that this post-translational processing not only facilitates protein association with cellular membranes, but also can play important roles in protein-protein interactions and protein stability (1, 6, 7). Thus, it is clear that CaaX processing is necessary for the biological activities of these proteins.The prenylation pathway has been targeted for potential anticancer therapy because most members of the Ras superfamily, which contains many known oncogenes, undergo CAAX processing. The Ras superfamily consists of five large subfamilies; the two most well-characterized are the Ras and Rho subfamilies (8). Both Ras and Rho proteins are processed by the CaaX pathway; Ras family members are farnesylated, while most Rho family members are geranylgeranylated. These monomeric GTPases cycle between a GDP-bound inactive state and a GTP-bound active state. In their active states, Ras and Rho subfamily members control numerous cell signaling pathways that are involved in cell proliferation, differentiation, migration, polarity, and morphology (9).Abnormally high activity of Ras and Rho signaling pathways contribute to initiation and progression of many types of cancer (10, 11). For example, many breast cancers that are highly metastatic express abnormally high levels of Rho proteins (12). Rho proteins control migration and invasion of cells by tightly coordinating changes in the actin and microtubule cytoskeletons. Acting through their effectors, Rho proteins rearrange the actin cytoskeleton to respond to chemo-attractant gradients, polarize cells, and control migration and invasion. While cell migration is necessary for development, leukocyte function, and other normal cell biologies, dysregulation of migration and invasion results in cancer metastasis (13). Metastasis is an important and deadly progression of cancer and understanding the biology of migrating cancer cells is crucial for therapeutic targeting of this aspect of cancer.Pharmacologic targeting of the enzymes involved in the CaaX-processing pathway has emerged as a promising anticancer strategy. In particular, there has been much effort in designing inhibitors against the protein prenyltransferases, most notably FTase (14, 15). There is also recent evidence that inhibition of geranygeranylation of Rho proteins also impacts oncogenesis and metastasis (16–18). However, the overall success of the FTase inhibitors (FTIs) in the clinical setting has been somewhat disappointing. One possible reason is a phenomenon termed “alternate prenylation” in which some FTase substrates, most notably K- and N- Ras, are modified by GGTase and escape inhibition by FTIs (19–21). Because the Rce1 protease and Icmt methyltransferase act on all CaaX proteins, problems such as alternate prenylation would not arise if these enzymes were targeted. Hence, while protein prenyltransferase inhibitors still show some promise as anticancer agents, the emerging view that global attenuation of CaaX protein function may be advantageous in blocking cancer cell growth has increased interest in studying the two downstream enzymes involved in CaaX processing.While the biological consequences of prenylation are fairly well understood, the precise roles of C-terminal methylation in CaaX protein function are still elusive. Depending on the CaaX protein, methylation has been ascribed to roles in localization, protein-protein interactions and protein stability (11). The development of an Icmt knock-out mouse model has furthered our understanding of Icmt function (22, 23). Localization studies conducted in cells with genetically deleted Icmt have shown that methylation is important for proper membrane association of Ras proteins. However, the localization of Rho proteins in the absence of Icmt activity appears to be more complicated and may vary depending on family member and activation status (24–26). Importantly, inhibition of CaaX protein methylation via either genetic or pharmacologic targeting has shown a clear impact on oncogenic transformation and tumor growth (23, 27, 28).Defining the role of Icmt-mediated methylation in complex cellular behaviors such as migration and invasion is crucial for furthering our understanding of the impact of CaaX protein methylation on the biology of normal and cancer cells. In the current study, we have assessed the impact of Icmt inhibition on cell biological processes associated with the function of Rho proteins, specifically cell adhesion, morphology, and migration. We found that inhibition of Icmt results in a disruption of the actin cytoskeleton and impairs ligand-mediated activation of RhoA and Rac1, a potential consequence of increased RhoGDI binding to both RhoA and Rac1 when their methylation is impaired. Further, we show that the impact of Icmt inhibition on cell migration is due at least in part to impairment of RhoA and Rac1function. These findings establish a role for Icmt-mediated methylation in cell migration and further elucidate the role that methylation plays in the function of Rho GTPases.     

Postprenylation CAAX processing is required for proper localization of Ras but not Rho GTPases

The CAAX motif at the C terminus of most monomeric GTPases is required for membrane targeting because it signals for a series of three posttranslational modifications that include isoprenylation, endoproteolytic release of the C-terminal- AAX amino acids, and carboxyl methylation of the newly exposed isoprenylcysteine. The individual contributions of these modifications to protein trafficking and function are unknown. To address this issue, we performed a series of experiments with mouse embryonic fibroblasts (MEFs) lacking Rce1 (responsible for removal of the -AAX sequence) or Icmt (responsible for carboxyl methylation of the isoprenylcysteine). In MEFs lacking Rce1 or Icmt, farnesylated Ras proteins were mislocalized. In contrast, the intracellular localizations of geranylgeranylated Rho GTPases were not perturbed. Consistent with the latter finding, RhoGDI binding and actin remodeling were normal in Rce1- and Icmt-deficient cells. Swapping geranylgeranylation for farnesylation on Ras proteins or vice versa on Rho proteins reversed the differential sensitivities to Rce1 and Icmt deficiency. These results suggest that postprenylation CAAX processing is required for proper localization of farnesylated Ras but not geranygeranylated Rho proteins.     

srGAP2 Arginine Methylation Regulates Cell Migration and Cell Spreading through Promoting Dimerization

The Slit-Robo GTPase-activating proteins (srGAPs) are critical for neuronal migration through inactivation of Rho GTPases Cdc42, Rac1, and RhoA. Here we report that srGAP2 physically interacts with protein arginine methyltransferase 5 (PRMT5). srGAP2 localizes to the cytoplasm and plasma membrane protrusion. srGAP2 knockdown reduces cell adhesion spreading and increases cell migration, but has no effect on cell proliferation. PRMT5 binds to the N terminus of srGAP2 (225–538 aa) and methylates its C-terminal arginine residue Arg-927. The methylation mutant srGAP2-R927A fails to rescue the cell spreading rate, is unable to localize to the plasma membrane leading edge, and perturbs srGAP2 homodimer formation mediated by the F-BAR domain. These results suggest that srGAP2 arginine methylation plays important roles in cell spreading and cell migration through influencing membrane protrusion.     

Why three Rho proteins? RhoA, RhoB, RhoC, and cell motility

Higher vertebrates have 3 Rho GTPases, RhoA, RhoB, and RhoC, which share 85% amino acid sequence identity. Here, we compare and contrast the roles of RhoA, B, and C in the regulation of the cytoskeleton and cell motility. Despite their similarity, some regulators and effectors show preferential interaction with RhoA, B, or C, and the three proteins show differences in function in cells. RhoA plays a key role in the regulation of actomyosin contractility. RhoB, which is localized primarily on endosomes, has been shown to regulate cytokine trafficking and cell survival, while RhoC may be more important in cell locomotion. In cancer cells, the expression and activity of RhoA, B, and C is altered in different ways. Together, this evidence suggests that although the 3 isoforms of Rho are structurally highly homologous, they have different cellular functions.     

Tech: a RhoA GEF selectively expressed in hippocampal and cortical neurons

Recent studies implicating the Rho family of small G proteins in the regulation of neuronal morphology have focused attention on identifying key components of Rho signaling pathways in neurons. To this end, we have conducted studies aimed at defining the localization and function of Tech, a Rho guanine nucleotide exchange factor (GEF) family member that is highly enriched in brain. We have found that Tech is selectively expressed in cortical and hippocampal neurons with prominent Tech immunostaining apparent in the cell bodies and dendrites of these cells. In vitro studies with prototypical members of the major Rho subfamilies, RhoA, Rac1 and Cdc42, indicate that Tech binds selectively to and activates RhoA. To assess whether Tech may be involved in the regulation of neuronal morphology, we examined the effects of Tech constructs on the morphology of cortical neurons grown in primary culture. We found that a constitutively active Tech construct, Tech 245DeltaC, decreases the number of dendritic processes present on these neurons. This reduction appears to be mediated by activation of RhoA as it is blocked by insertion of a point mutation into the DH domain of Tech which blocks its ability to activate RhoA or coexpression of a dominant negative RhoA construct. As Tech protein levels increase during post-natal development and remain at peak levels into adulthood, these results indicate that Tech regulates RhoA signaling pathways in developing and mature forebrain neurons.     

A novel strategy for specifically down-regulating individual Rho GTPase activity in tumor cells

The Rho family GTPases RhoA, RhoB, and RhoC regulate the actin cytoskeleton, cell movement, and cell growth. Unlike Ras, up-regulation or overexpression of these GDP/GTP binding molecular switches, but not activating point mutations, has been associated with human cancer. Although they share over 85% sequence identity, RhoA, RhoB, and RhoC appear to play distinct roles in cell transformation and metastasis. In NIH 3T3 cells, RhoA or RhoB overexpression causes transformation whereas RhoC increases the cell migration rate. To specifically target RhoA, RhoB, or RhoC function, we have generated a set of chimeric molecules by fusing the RhoGAP domain of p190, a GTPase-activating protein that accelerates the intrinsic GTPase activity of all three Rho GTPases, with the C-terminal hypervariable sequences of RhoA, RhoB, or RhoC. The p190-Rho chimeras were active as GTPase-activating proteins toward RhoA in vitro, co-localized with the respective active Rho proteins, and specifically down-regulated Rho protein activities in cells depending on which Rho GTPase sequences were included in the chimeras. In particular, the p190-RhoA-C chimera specifically inhibited RhoA-induced transformation whereas p190-RhoC-C specifically reversed the migration phenotype induced by the active RhoC. In human mammary epithelial-RhoC breast cancer cells, p190-RhoC-C, but not p190-RhoA-C or p190-RhoB-C, reversed the anchorage-independent growth and invasion phenotypes caused by RhoC overexpression. In the highly metastatic A375-M human melanoma cells, p190-RhoC-C specifically reversed migration, and invasion phenotypes attributed to RhoC up-regulation. Thus, we have developed a novel strategy utilizing RhoGAP-Rho chimeras to specifically down-regulate individual Rho activity and demonstrate that this approach may be applied to multiple human tumor cells to reverse the growth and/or invasion phenotypes associated with disregulation of a distinct subtype of Rho GTPase.     

Methylation of Ribosomal Protein L42 Regulates Ribosomal Function and Stress-adapted Cell Growth

Lysine methylation is one of the most common protein modifications. Although lysine methylation of histones has been extensively studied and linked to gene regulation, that of non-histone proteins remains incompletely understood. Here, we show a novel regulatory role of ribosomal protein methylation. Using an in vitro methyltransferase assay, we found that Schizosaccharomyces pombe Set13, a SET domain protein encoded by SPAC688.14, specifically methylates lysine 55 of ribosomal protein L42 (Rpl42). Mass spectrometric analysis revealed that endogenous Rpl42 is monomethylated at lysine 55 in wild-type S. pombe cells and that the methylation is lost in Δset13 mutant cells. Δset13 and Rpl42 methylation-deficient mutant S. pombe cells showed higher cycloheximide sensitivity and defects in stress-responsive growth control compared with wild type. Genetic analyses suggested that the abnormal growth phenotype was distinct from the conserved stress-responsive pathway that modulates translation initiation. Furthermore, the Rpl42 methylation-deficient mutant cells showed a reduced ability to survive after entering stationary phase. These results suggest that Rpl42 methylation plays direct roles in ribosomal function and cell proliferation control independently of the general stress-response pathway.     

Membrane trafficking at the ER/Golgi interface: functional implications of RhoA and Rac1

N-WASP and Arp2/3, the components of the actin nucleation/polymerization signaling pathway governed by Cdc42, are located in Golgi membranes and regulate ER/Golgi interface protein transport. In the present study, we examined whether RhoA and Rac1, like Cdc42, are also involved in this early secretory pathway. Unlike Cdc42, RhoA and Rac1 were not observed in the Golgi complex of different clonal cell lines nor were they present in isolated Golgi membranes. Expression of constitutively active or inactive mutants of RhoA or Rac1 proteins in HeLa cells did not alter either the disassembly or the assembly of the Golgi complex following the addition or withdrawal of BFA, respectively, the ER-to-Golgi VSV-G transport or the Sar1(dn)-induced ER accumulation of Golgi proteins. Moreover, unlike Cdc42-expressing cells, the 15 degrees C-induced subcellular redistribution of the KDEL receptor remained unaltered. Only cells that constitutively express the activated Cdc42 mutant (Cdc42Q61L), or that were microinjected with activated Cdc42Q61L protein, exhibited a significant change in Golgi complex morphology. Collectively, our results demonstrate that RhoA and Rac1 are not located in the Golgi complex, nor do they directly or indirectly regulate membrane trafficking at the ER/Golgi interface. This finding, in turn, confirms that Cdc42 is the only Rho GTPase to have a specific function on the Golgi complex.     

Tenascin-C suppresses Rho activation

Cell binding to extracellular matrix (ECM) components changes cytoskeletal organization by the activation of Rho family GTPases. Tenascin-C, a developmentally regulated matrix protein, modulates cellular responses to other matrix proteins, such as fibronectin (FN). Here, we report that tenascin-C markedly altered cell phenotype on a three-dimensional fibrin matrix containing FN, resulting in suppression of actin stress fibers and induction of actin-rich filopodia. This distinct morphology was associated with complete suppression of the activation of RhoA, a small GTPase that induces actin stress fiber formation. Enforced activation of RhoA circumvented the effects of tenascin. Effects of active Rho were reversed by a Rho inhibitor C3 transferase. Suppression of GTPase activation allows tenascin-C expression to act as a regulatory switch to reverse the effects of adhesive proteins on Rho function. This represents a novel paradigm for the regulation of cytoskeletal organization by ECM.     

ADP-ribosylation of the ras-related, GTP-binding protein RhoA inhibits lymphocyte-mediated cytotoxicity.

The Rho proteins are identified as a subgroup of the Ras superfamily of low molecular weight GTP-binding proteins. We have studied the expression of these proteins in human cytotoxic natural killer cells and found that RhoA is the most abundantly expressed member of the Rho family. The Rho proteins are specific substrates for ADP-ribosylation catalyzed by the C3 exoenzyme from Clostridium botulinum. We report here that introduction of recombinant C3 in electropermeabilized natural killer cells or in cytotoxic T lymphocytes resulted in a dose-dependent inhibition of their cytolytic function. Furthermore, a single substrate is efficiently ADP-ribosylated by C3 in extracts from cytotoxic cells. Biochemical analyses indicate that this substrate is RhoA, and subcellular fractionation experiments demonstrate that it is essentially present in the cytosol of the cells. Western blot analysis, however, revealed that a small proportion of the Rho protein can be found associated with the cell membrane as well as with the cytotoxic granules. These results indicate that the low molecular weight GTP-binding protein RhoA is present in cytotoxic lymphocytes and plays a critical role in cell-mediated cytotoxicity.     

p115 Rho GTPase activating protein interacts with MEKK1

Mammalian MAP/ERK kinase kinase 1 (MEKK1) was identified as a mammalian homolog of Ste11p of the yeast pheromone-induced mating pathway. Like Ste11p, MEKK1 is a MAP3 kinase linked to at least two MAP kinase cascades and regulatory events that require cytoskeletal reorganization. MEKK1 is activated by molecules that impact cytoskeletal function. MEKK1-/-cells are defective in cell migration, demonstrating that it is required for cell motility. MEKK1 has a 1,200 residue N-terminal regulatory domain that interacts with a dozen identified proteins. Using part of the MEKK1 N-terminus in a yeast two-hybrid screen, we discovered a novel interaction with p115 Rho GTPase-activating protein (GAP). The p115 Rho GAP binds to MEKK1 in vitro and in intact cells. The p115 Rho GAP has selectivity for RhoA over other Rho family members. Expression of p115 Rho GAP reduces MEKK1-induced signaling to AP-1. The reduced activation of AP-1 is dependent on the association of MEKK1 with p115 Rho GAP, because deletion of the Rho GAP SH3 domain, which abrogates their interaction, restores the stimulatory effect of MEKK1 on AP-1 activity. Here we have identified an MEKK1 binding partner that offers a connection between this protein kinase and the machinery regulating cytoskeletal reorganization.     

Volatile anesthetics affect the morphology of rat glioma C6 cells via RhoA, ERK, and Akt activation

Treatment of rat glioma C6 cells with the beta-receptor agonist isoproterenol induces a massive increase in cAMP. Concomitantly the cells change their morphology from a fibroblast-type to an astrocyte-like (stellated) cell shape. The stellated morphology can be completely reverted by thrombin and sphingosine-1-phosphate (S-1-P) but also to a certain extent by clinical concentrations of volatile anesthetics. The anesthetic-induced reversion of the stellated cell shape seems to be mediated by a number of cellular alterations. Central to the effect is most likely a RhoA/Rho-kinase activation, but also the MAPKK/MEK and the Akt/protein kinase B pathway are activated by the anesthetics. With the use of specific inhibitors we were able to show that activation of the MAPKK/MEK pathway inhibits, whereas activation of the Akt/protein kinase B pathway stimulates the reversal of the stellated cell shape by the anesthetics. In summary, volatile anesthetics affect the morphology of rat glioma C6 cells by activation of the RhoA/Rho kinase, the MAPKK/MEK, and the Akt/protein kinase B signaling pathways.     

Targeted inactivation of the isoprenylcysteine carboxyl methyltransferase gene causes mislocalization of K-Ras in mammalian cells

After isoprenylation and endoproteolytic processing, the Ras proteins are methylated at the carboxyl-terminal isoprenylcysteine. The importance of isoprenylation for targeting of Ras proteins to the plasma membrane is well established, but the importance of carboxyl methylation, which is carried out by isoprenylcysteine carboxyl methyltransferase (Icmt), is less certain. We used gene targeting to produce homozygous Icmt knockout embryonic stem cells (Icmt-/-). Lysates from Icmt-/- cells lacked the ability to methylate farnesyl-K-Ras4B or small-molecule Icmt substrates such as N-acetyl-S-geranylgeranyl-L-cysteine. To assess the impact of absent Icmt activity on the localization of K-Ras within cells, wild-type and Icmt-/- cells were transfected with a green fluorescent protein (GFP)-K-Ras fusion construct. As expected, virtually all of the GFP-K-Ras fusion in wild-type cells was localized along the plasma membrane. In contrast, a large fraction of the fusion in Icmt-/- cells was trapped within the cytoplasm, and fluorescence at the plasma membrane was reduced. Also, cell fractionation/Western blot studies revealed that a smaller fraction of the K-Ras in Icmt-/- cells was associated with the membranes. We conclude that carboxyl methylation of the isoprenylcysteine is important for proper K-Ras localization in mammalian cells.     

Rho Family GTPase modification and dependence on CAAX motif-signaled posttranslational modification

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.     

A role for Rac3 GTPase in the regulation of autophagy

The process of autophagy is situated at the intersection of multiple cell signaling pathways, including cell metabolism, growth, and death, and hence is subject to multiple forms of regulation. We previously reported that inhibition of isoprenylcysteine carboxylmethyltransferase (Icmt), which catalyzes the final step in the post-translational prenylation of so-called CAAX proteins, results in the induction of autophagy which enhances cell death in some cancer cells. In this study, using siRNA-mediated knockdown of a group of small GTPases that are predicted Icmt substrates, we identify Rac3 GTPase as a negative regulator of the process of autophagy. Knockdown of Rac3, but not the closely related isoforms Rac1 and Rac2, results in induction of autophagy. Ectopic expression of Rac3, significantly rescues cells from autophagy and cell death induced by Icmt inhibition, strengthening the notion of an isoform-specific autophagy regulatory function of Rac3. This role of Rac3 was observed in multiple cell lines with varying Rac subtype expression profiles, suggesting its broad involvement in the process. The identification of this less-studied Rac member as a novel regulator provides new insight into autophagy and opens opportunities in identifying additional regulatory inputs of the process.     

Collapsin response mediator protein switches RhoA and Rac1 morphology in N1E-115 neuroblastoma cells and is regulated by Rho kinase

The formation and directional guidance of neurites involves dynamic regulation of Rho family GTPases. Rac and Cdc42 promote neurite outgrowth, whereas Rho activation causes neurite retraction. Here we describe a role for collapsin response mediator protein (Crmp-2), a neuronal protein implicated in axonal outgrowth and a component of the semaphorin 3A pathway, in switching GTPase signaling when expressed in combination with either dominant active Rac or Rho. In neuroblastoma N1E-115 cells, co-expression of Crmp-2 with dominant active RhoA V14 induced Rac morphology, cell spreading and ruffling (and the formation of neurites). Conversely, co-expression of Crmp-2 with dominant active Rac1 V12 inhibited Rac morphology, and in cells already expressing Rac1 V12, Crmp-2 caused localized peripheral collapse, involving Rho (and Cdc42) activation. Rho kinase was a pivotal regulator of Crmp-2; Crmp-2 phosphorylation was required for Crmp-2/Rac1 V12 inhibition, but not Crmp-2/RhoA V14 induction, of Rac morphology. Thus Crmp-2, regulated by Rho kinase, promotes outgrowth and collapse in response to active Rho and Rac, respectively, reversing their usual morphological effects and providing a mechanism for dynamic modulation of growth cone guidance.     

Serine phosphorylation negatively regulates RhoA in vivo

Previous work indicates that RhoA phosphorylation on Ser188 by cAMP or cGMP-dependent kinases inhibits its activity. However, these studies lacked the possibility to directly study phosphorylated RhoA activity in vivo. Therefore, we created RhoA proteins containing phosphomimetic residues in place of the cAMP/cGMP-dependent kinase phosphorylation site. RhoA phosphorylation or phosphomimetic substitution did not affect Rho guanine nucleotide exchange factor, GTPase activating protein, or geranylgeranyl transferase activity in vitro but promoted binding to the Rho guanine-dissociation inhibitor as measured by exchange factor competition assays. The in vitro similarities between RhoA phosphomimetic proteins and phosphorylated RhoA allowed us to study function of phosphorylated RhoA in vivo. RhoA phosphomimetic proteins display depressed GTP loading when transiently expressed in NIH 3T3 cells. Stable-expressing RhoA and RhoA(S188A) clones spread significantly slower than mock-transfected or RhoA(S188E) clones. RhoA(S188A) clones were protected from the morphological effects of a cAMP agonist, whereas phosphomimetic clones exhibit stress fiber disassembly similar to control cells. Together, these data provide in vivo evidence that addition of a charged group to Ser188 upon phosphorylation negatively regulates RhoA activity and indicates that this occurs through enhanced Rho guanine-dissociation inhibitor interaction rather than direct perturbation of guanine nucleotide exchange factor, GTPase activating protein, or geranylgeranyl transferase activity.     

Rho GTPases and their regulators in neuronal functions and development

Neurons are specialized cell types which send out processes in order to communicate with other cells, which can be immediate neighbors or whose cell bodies are far distant. Neuronal morphology as in all cells is determined in large part through the regulation of the cytoskeleton. One of the key regulators of the actin cytoskeleton is the Rho family of GTPases. The Rho GTPases function as molecular switches to turn on or off downstream biochemical pathways depending on the stimuli. Their activities and their regulation are controlled by many other proteins such as the guanine nucleotide exchange factors and the GTPase-activating proteins. The activities of some of the Rho family members are reported to be antagonistic to one another. In general, Rac and Cdc42 promote neurite outgrowth while RhoA stimulates retraction. The balance of these opposing activities of the different Rho GTPases is crucial for the morphology and function of the neurons.     

Mechanism of isoprenylcysteine carboxyl methylation from the crystal structure of the integral membrane methyltransferase ICMT

The posttranslational modification of C-terminal CAAX motifs in proteins such as Ras, most Rho GTPases, and G protein γ subunits, plays an essential role in determining their subcellular localization and correct biological function. An integral membrane methyltransferase, isoprenylcysteine carboxyl methyltransferase (ICMT), catalyzes the final step of CAAX processing after prenylation of the cysteine residue and endoproteolysis of the -AAX motif. We have determined the crystal structure of a prokaryotic ICMT ortholog, revealing a markedly different architecture from conventional methyltransferases that utilize S-adenosyl-L-methionine (SAM) as a cofactor. ICMT comprises a core of five transmembrane α helices and a cofactor-binding pocket enclosed within a highly conserved C-terminal catalytic subdomain. A tunnel linking the reactive methyl group of SAM to the inner membrane provides access for the prenyl lipid substrate. This study explains how an integral membrane methyltransferase achieves recognition of both a hydrophilic cofactor and a lipophilic prenyl group attached to a polar protein substrate.