EPLIN regulates actin dynamics by cross-linking and stabilizing filaments

Epithelial protein lost in neoplasm (EPLIN) is a cytoskeleton-associated protein encoded by a gene that is down-regulated in transformed cells. EPLIN increases the number and size of actin stress fibers and inhibits membrane ruffling induced by Rac. EPLIN has at least two actin binding sites. Purified recombinant EPLIN inhibits actin filament depolymerization and cross-links filaments in bundles. EPLIN does not affect the kinetics of spontaneous actin polymerization or elongation at the barbed end, but inhibits branching nucleation of actin filaments by Arp2/3 complex. Side binding activity may stabilize filaments and account for the inhibition of nucleation mediated by Arp2/3 complex. We propose that EPLIN promotes the formation of stable actin filament structures such as stress fibers at the expense of more dynamic actin filament structures such as membrane ruffles. Reduced expression of EPLIN may contribute to the motility of invasive tumor cells.     

Essential role of Cdc42 in Ras-induced transformation revealed by gene targeting

The ras proto-oncogene is one of the most frequently mutated genes in human cancer. However, given the prevalence of activating mutations in Ras and its association with aggressive forms of cancer, attempts to therapeutically target aberrant Ras signaling have been largely disappointing. This lack of progress highlights the deficiency in our understanding of cellular pathways required for Ras-mediated tumorigenesis and suggests the importance of identifying new molecular pathways associated with Ras-driven malignancies. Cdc42 is a Ras-related small GTPase that is known to play roles in oncogenic processes such as cell growth, survival, invasion, and migration. A pan-dominant negative mutant overexpression approach to suppress Cdc42 and related pathways has previously shown a requirement for Cdc42 in Ras-induced anchorage-independent cell growth, however the lack of specificity of such approaches make it difficult to determine if effects are directly related to changes in Cdc42 activity or other Rho family members. Therefore, in order to directly and unambiguously address the role of Cdc42 in Ras-mediated transformation, tumor formation and maintenance, we have developed a model of conditional cdc42 gene in Ras-transformed cells. Loss of Cdc42 drastically alters the cell morphology and inhibits proliferation, cell cycle progression and tumorigenicity of Ras-transformed cells, while non-transformed cells or c-Myc transformed cells are largely unaffected. The loss of Cdc42 in Ras-transformed cells results in reduced Akt signaling, restoration of which could partially rescues the proliferation defects associated with Cdc42 loss. Moreover, disruption of Cdc42 function in established tumors inhibited continued tumor growth. These studies implicate Cdc42 in Ras-driven tumor growth and suggest that targeting Cdc42 is beneficial in Ras-mediated malignancies.     

Regulation of anoikis by Cdc42 and Rac1

Ras family small GTPases play a critical role in malignant transformation, and Rho subfamily members contribute significantly to this process. Anchorage-independent growth and the ability to avoid detachment-induced apoptosis (anoikis) are hallmarks of transformed epithelial cells. In this study, we have demonstrated that constitutive activation of Cdc42 inhibits anoikis in Madin-Darby canine kidney (MDCK) epithelial cells. We showed that activated Cdc42 stimulates the ERK, JNK, and p38 MAPK pathways in suspension condition; however, inhibition of these signaling does not affect Cdc42-stimulated cell survival. However, we demonstrated that inhibition of phosphatidylinositol 3-kinase (PI3K) pathway abolishes the protective effect of Cdc42 on anoikis. Taking advantage of a double regulatory expression system, we also showed that Cdc42-stimulated cell survival in suspension condition is, at least in part, mediated by Rac1. We also provide evidence for a positive feedback loop involving Rac1 and PI3K. In addition, we show that the survival functions of both constitutively active Cdc42 and Rac1 GTPases are abrogated by Latrunculin B, an actin filament-depolymerizing agent, implying an important role for the actin cytoskeleton in mediating survival signaling activated by Cdc42 and Rac1. Together, our results indicate a role for Cdc42 in anchorage-independent survival of epithelial cells. We also propose that this survival function depends on a positive feedback loop involving Rac1 and PI3K.     

Cdc42 and RhoA have opposing roles in regulating membrane type 1-matrix metalloproteinase localization and matrix metalloproteinase-2 activation

Proteolysis of the basement membrane and interstitial matrix occurs early in the angiogenic process and requires matrix metalloproteinase (MMP) activity. Skeletal muscle microvascular endothelial cells exhibit robust actin stress fibers, low levels of membrane type 1 (MT1)-MMP expression, and minimal MMP-2 activation. Depolymerization of the actin cytoskeleton increases MT1-MMP expression and MMP-2 activation. Rho family GTPases are regulators of actin cytoskeleton dynamics, and their activity can be modulated in response to angiogenic stimuli such as vascular endothelial growth factor (VEGF). Therefore, we investigated their roles in MMP-2 and MT1-MMP production. Endothelial cells treated with H1152 [an inhibitor of Rho kinase (ROCK)] induced stress fiber depolymerization and an increase in cortical actin. Both MMP-2 and MT1-MMP mRNA increased, which translated into greater MMP-2 protein production and activation. ROCK inhibition rapidly increased cell surface localization of MT1-MMP and increased PI3K activity, which was required for MMP-2 activation. Constitutively active Cdc42 increased cortical actin polymerization, phosphatidylinositol 3-kinase activity, MT1-MMP cell surface localization, and MMP-2 activation similarly to inhibition of ROCK. Activation of Cdc42 was sufficient to decrease RhoA activity. Capillary sprout formation in a three-dimensional collagen matrix was increased in cultures treated with RhoAN19 or Cdc42QL and, conversely, decreased in cultures treated with dominant negative Cdc42N17. VEGF stimulation also induced activation of Cdc42 while inhibiting RhoA activity. Furthermore, VEGF-dependent activation of MMP-2 was reduced by inhibition of Cdc42. These results suggest that Cdc42 and RhoA have opposing roles in regulating cell surface localization of MT1-MMP and MMP-2 activation.     

Identification of novel, evolutionarily conserved Cdc42p-interacting proteins and of redundant pathways linking Cdc24p and Cdc42p to actin polarization in yeast

In the yeast Saccharomyces cerevisiae, Cdc24p functions at least in part as a guanine-nucleotide-exchange factor for the Rho-family GTPase Cdc42p. A genetic screen designed to identify possible additional targets of Cdc24p instead identified two previously known genes, MSB1 and CLA4, and one novel gene, designated MSB3, all of which appear to function in the Cdc24p-Cdc42p pathway. Nonetheless, genetic evidence suggests that Cdc24p may have a function that is distinct from its Cdc42p guanine-nucleotide-exchange factor activity; in particular, overexpression of CDC42 in combination with MSB1 or a truncated CLA4 in cells depleted for Cdc24p allowed polarization of the actin cytoskeleton and polarized cell growth, but not successful cell proliferation. MSB3 has a close homologue (designated MSB4) and two more distant homologues (MDR1 and YPL249C) in S. cerevisiae and also has homologues in Schizosaccharomyces pombe, Drosophila (pollux), and humans (the oncogene tre17). Deletion of either MSB3 or MSB4 alone did not produce any obvious phenotype, and the msb3 msb4 double mutant was viable. However, the double mutant grew slowly and had a partial disorganization of the actin cytoskeleton, but not of the septins, in a fraction of cells that were larger and rounder than normal. Like Cdc42p, both Msb3p and Msb4p localized to the presumptive bud site, the bud tip, and the mother-bud neck, and this localization was Cdc42p dependent. Taken together, the data suggest that Msb3p and Msb4p may function redundantly downstream of Cdc42p, specifically in a pathway leading to actin organization. From previous work, the BNI1, GIC1, and GIC2 gene products also appear to be involved in linking Cdc42p to the actin cytoskeleton. Synthetic lethality and multicopy suppression analyses among these genes, MSB, and MSB4, suggest that the linkage is accomplished by two parallel pathways, one involving Msb3p, Msb4p, and Bni1p, and the other involving Gic1p and Gic2p. The former pathway appears to be more important in diploids and at low temperatures, whereas the latter pathway appears to be more important in haploids and at high temperatures.     

Compartmentalized Ras proteins transform NIH 3T3 cells with different efficiencies

Ras GTPases were long thought to function exclusively from the plasma membrane (PM). However, a current model suggests that Ras proteins can compartmentalize to regulate different functions, and an oncogenic H-Ras mutant that is restricted to the endomembrane can still transform cells. In this study, we demonstrated that cells transformed by endomembrane-restricted oncogenic H-Ras formed tumors in nude mice. To define downstream targets of endomembrane Ras pathways, we analyzed Cdc42, which concentrates in the endomembrane and has been shown to act downstream of Ras in Schizosaccharomyces pombe. Our data show that cell transformation induced by endomembrane-restricted oncogenic H-Ras was blocked when Cdc42 activity was inhibited. Moreover, H-Ras formed a complex with Cdc42 on the endomembrane, and this interaction was enhanced when H-Ras was GTP bound or when cells were stimulated by growth factors. H-Ras binding evidently induced Cdc42 activation by recruiting and/or activating Cdc42 exchange factors. In contrast, when constitutively active H-Ras was restricted to the PM by fusing to a PM localization signal from the Rit GTPase, the resulting protein did not detectably activate Cdc42 although it activated Raf-1 and efficiently induced hallmarks of Ras-induced senescence in human BJ foreskin fibroblasts. Surprisingly, PM-restricted oncogenic Ras when expressed alone could only weakly transform NIH 3T3 cells; however, when constitutively active Cdc42 was coexpressed, together they transformed cells much more efficiently than either one alone. These data suggest that efficient cell transformation requires Ras proteins to interact with Cdc42 on the endomembrane and that in order for a given Ras protein to fully transform cells, multiple compartment-specific Ras pathways need to work cooperatively.     

Cdc42 and Ras cooperate to mediate cellular transformation by intersectin-L

Cdc42, a Ras-related GTP-binding protein, has been implicated in the regulation of the actin cytoskeleton, membrane trafficking, cell-cycle progression, and malignant transformation. We have shown previously that a Cdc42 mutant (Cdc42(F28L)), capable of spontaneously exchanging GDP for GTP (referred to as "fast-cycling"), transformed NIH 3T3 cells because of its ability to interfere with epidermal growth factor receptor (EGFR)-Cbl interactions and EGFR down-regulation. To further examine the link between the hyperactivation of Cdc42 and its ability to alter EGFR signaling and thereby cause cellular transformation, we examined the effects of expressing different forms of the Cdc42-specific guanine nucleotide exchange factor, intersectin-L, in fibroblasts. Full-length intersectin-L exhibited little ability to stimulate nucleotide exchange on Cdc42, whereas a truncated version that contained five Src homology 3 (SH3) domains, the Dbl and pleckstrin homology domains (DH and PH domains, respectively), and a C2 domain (designated as SH3A-C2) showed modest guanine nucleotide exchange factor activity, whereas a form containing just the DH, PH, and C2 domains (DH-C2) strongly activated Cdc42. However, DH-C2 showed little ability to stimulate growth in low serum or colony formation in soft agar, whereas SH3A-C2 gave rise to a much stronger stimulation of cell growth in low serum and was highly effective in stimulating colony formation. Moreover, although SH3A-C2 strongly transformed fibroblasts, it differed from the actions of the Cdc42(F28L) mutant, as SH3A-C2 showed little ability to alter EGFR levels or the lifetime of EGF-coupled signaling through ERK. Rather, we found that SH3A-C2 exhibited strong transforming activity through its ability to mediate cooperation between Ras and Cdc42.     

The actin-binding and bundling protein,EPLIN, is required for cytokinesis

Cytokinesis involves two phases: 1) membrane ingression followed by 2) membrane abscission. The ingression phase generates a cleavage furrow and this requires co-operative function of the actin-myosin II contractile ring and septin filaments. We demonstrate that the actin-binding protein, EPLIN, locates to the cleavage furrow during cytokinesis and this is possibly via association with the contractile ring components, myosin II, and the septin, Sept2. Depletion of EPLIN results in formation of multinucleated cells and this is associated with inefficient accumulation of active myosin II (MRLCS19) and Sept2 and their regulatory small GTPases, RhoA and Cdc42, respectively, to the cleavage furrow during the final stages of cytokinesis. We suggest that EPLIN may function during cytokinesis to maintain local accumulation of key cytokinesis proteins at the furrow.     

Rab40–Cullin5 complex regulates EPLIN and actin cytoskeleton dynamics during cell migration

Rab40b is a SOCS box–containing protein that regulates the secretion of MMPs to facilitate extracellular matrix remodeling during cell migration. Here, we show that Rab40b interacts with Cullin5 via the Rab40b SOCS domain. We demonstrate that loss of Rab40b–Cullin5 binding decreases cell motility and invasive potential and show that defective cell migration and invasion stem from alteration to the actin cytoskeleton, leading to decreased invadopodia formation, decreased actin dynamics at the leading edge, and an increase in stress fibers. We also show that these stress fibers anchor at less dynamic, more stable focal adhesions. Mechanistically, changes in the cytoskeleton and focal adhesion dynamics are mediated in part by EPLIN, which we demonstrate to be a binding partner of Rab40b and a target for Rab40b–Cullin5-dependent localized ubiquitylation and degradation. Thus, we propose a model where Rab40b–Cullin5-dependent ubiquitylation regulates EPLIN localization to promote cell migration and invasion by altering focal adhesion and cytoskeletal dynamics.     

Inducible clustering of membrane-targeted SH3 domains of the adaptor protein Nck triggers localized actin polymerization

BACKGROUND: SH2/SH3 adaptor proteins play a critical role in tyrosine kinase signaling pathways, regulating essential cell functions by increasing the local concentration or altering the subcellular localization of downstream effectors. The SH2 domain of the Nck adaptor can bind tyrosine-phosphorylated proteins, while its SH3 domains can modulate actin polymerization by interacting with effectors such as WASp/Scar family proteins. Although several studies have implicated Nck in regulating actin polymerization, its role in living cells is not well understood. RESULTS: We used an antibody-based system to experimentally modulate the local concentration of Nck SH3 domains on the plasma membrane of living cells. Clustering of fusion proteins containing all three Nck SH3 domains induced localized polymerization of actin, including the formation of actin tails and spots, accompanied by general cytoskeletal rearrangements. All three Nck SH3 domains were required, as clustering of individual SH3 domains or a combination of the two N-terminal Nck SH3 domains failed to promote significant local polymerization of actin in vivo. Changes in actin dynamics induced by Nck SH3 domain clustering required the recruitment of N-WASp, but not WAVE1, and were unaffected by downregulation of Cdc42. CONCLUSIONS: We show that high local concentrations of Nck SH3 domains are sufficient to stimulate localized, Cdc42-independent actin polymerization in living cells. This study provides strong evidence of a pivotal role for Nck in directly coupling ligand-induced tyrosine phosphorylation at the plasma membrane to localized changes in organization of the actin cytoskeleton through a signaling pathway that requires N-WASp.     

Schizosaccharomyces pombe Pxl1 is a paxillin homologue that modulates Rho1 activity and participates in cytokinesis

Schizosaccharomyces pombe Rho GTPases regulate actin cytoskeleton organization and cell integrity. We studied the fission yeast gene SPBC4F6.12 based on its ability to suppress the thermosensitivity of cdc42-1625 mutant strain. This gene, named pxl1(+), encodes a protein with three LIM domains that is similar to paxillin. Pxl1 does not interact with Cdc42 but it interacts with Rho1, and it negatively regulates this GTPase. Fission yeast Pxl1 forms a contractile ring in the cell division region and deletion of pxl1(+) causes a delay in cell-cell separation, suggesting that it has a function in cytokinesis. Pxl1 N-terminal region is required and sufficient for its localization to the medial ring, whereas the LIM domains are necessary for its function. Pxl1 localization requires actin polymerization and the actomyosin ring, but it is independent of the septation initiation network (SIN) function. Moreover, Pxl1 colocalizes and interacts with Myo2, and Cdc15, suggesting that it is part of the actomyosin ring. Here, we show that in cells lacking Pxl1, the myosin ring is not correctly assembled and that actomyosin ring contraction is delayed. Together, these data suggest that Pxl1 modulates Rho1 GTPase signaling and plays a role in the formation and contraction of the actomyosin ring during cytokinesis.     

Nuclear translocation of LIM kinase mediates Rho-Rho kinase regulation of cyclin D1 expression

We previously reported that the Rho-Rho kinase pathway controls cyclin D1 expression by preventing its early G1 phase induction in response to Rac and/or Cdc42, thus increasing its dependence on ERK signaling and actin stress fiber formation. We now show that the Rho kinase effector LIM kinase is responsible for this effect. Surprisingly, inhibition of Rac-dependent cyclin D1 expression by LIM kinase is independent of both cofilin phosphorylation and actin polymerization. Instead, specific mutation of its nuclear localization and export sequences showed that LIM kinase acts in the nucleus to suppress Rac/Cdc42-dependent cyclin D1 expression. Our results therefore describe an unexpected role for LIM kinase that requires nuclear translocation. The effect of nuclear LIM kinase on cyclin D1 expression ultimately regulates the duration of G1 phase and the degree to which G1 phase progression depends on actin stress fiber formation and imposition of cellular tension.     

Regulation of phosphoinositide levels by the phospholipid transfer protein Sec14p controls Cdc42p/p21-activated kinase-mediated cell cycle progression at cytokinesis

Sec14p is an essential phosphatidylcholine/phosphatidylinositol transfer protein with a well-described role in the regulation of Golgi apparatus-derived vesicular transport in yeast. Inactivation of the CDP-choline pathway for phosphatidylcholine synthesis allows cells to survive in the absence of Sec14p function through restoration of Golgi vesicular transport capability. In this study, Saccharomyces cerevisiae cells containing a SEC14 temperature-sensitive allele along with an inactivated CDP-choline pathway were transformed with a high-copy-number yeast genomic library. Genes whose increased expression inhibited cell growth in the absence of Sec14p function were identified. Increasing levels of the Rho GTPase Cdc42p and its direct effector kinases Cla4p and Ste20p prevented the growth of cells lacking Sec14p and CDP-choline pathway function. Growth suppression was accompanied by an increase in large and multiply budded cells. This effect on polarized cell growth did not appear to be due to an inability to establish cell polarity, since both the actin cytoskeleton and localization of the septin Cdc12p were unaffected by increased expression of Cdc42p, Cla4p, or Ste20p. Nuclei were present in both the mother cell and the emerging bud, consistent with Sec14p regulation of the cell cycle subsequent to anaphase but prior to cytokinesis/septum breakdown. Increased expression of phosphatidylinositol 4-kinases and phosphatidylinositol 4-phosphate 5-kinase prevented growth arrest by CDC42, CLA4, or STE20 upon inactivation of Sec14p function. Sec14p regulation of phosphoinositide levels affects cytokinesis at the level of the Cdc42p/Cla4p/Ste20p signaling cascade.     

The Cdc42p GTPase is targeted to the site of cell division in the fission yeast Schizosaccharomyces pombe

The Rho-family GTPase Cdc42p regulates many aspects of cell polarity and growth in eukaryotic cells, including the organization of the actin cytoskeleton. To further examine Cdc42p function in the fission yeast Schizosaccharomyces pombe, a functional green fluorescent protein (GFP)-Cdc42p fusion protein was generated. GFP-Cdc42p was observed at the medial region of the cell at the cell-division site early in cytokinesis and remained there through cell separation, and was also localized to the periphery of the cell and to internal membranes. Unexpectedly, treatment with the actin-depolymerizing drug latrunculin-A disrupted the medial region targeting pattern, and cells deficient in the actin-binding proteins tropomyosin and profilin also did not exhibit medial GFP-Cdc42p staining. In addition, medial GFP-Cdc42p localization was eliminated in a number of cytokinesis mutants, including strains defective in assembling the medial actinomyosin ring, medial ring contraction, and septum assembly. GFP-Cdc42p targeting was less affected in mutants that formed misplaced or multiple septa. These results suggest that the localization of Cdc42p at the cell-division site was dependent upon the actin cytoskeleton and that Cdc42p may function in the interdependent processes of cytokinesis and septation.