Roles of E3 ubiquitin ligases in cell adhesion and migration

REVIEW

review

Cell Adhesion & Migration 4:1, 10-18; January/February/March 2010; © 2010 Landes Bioscience

Roles of E3 ubiquitin ligases in cell adhesion and migration Cai Huang Department of Cell and Developmental Biology; University of North Carolina at Chapel Hill; Chapel Hill, NC USA

Key words: adhesion, migration, E3 ubiquitin ligases, ubiquitination, degradation

Recent studies have demonstrated that a number of E3 ubiquitin ligases, including Cbl, Smurf1, Smurf2, HDM2, BCA2, SCFβ-TRCP and XRNF185, play important roles in cell adhesion and migration. Cbl negatively regulates cell adhesion via α integrin and Rap1 and inhibits actin polymerization by ubiquitinating mDab1 and WAVE2. Smurf1 regulates cell migration through ubiquitination of RhoA, talin head domain and hPEM2, while Smurf2 ubiquitinates Smurf1, TGFβ type I receptor and RaplB to modulate cell migration and adhesion. HDM2 negatively regulates cell migration by targeting NFAT (a transcription factor) for ubiquitination and degradation, while SCFβ-TRCP ubiquitinates Snail (a transcriptional repressor of E-cadherin) to inhibit cell migration. TRIM32 promotes cell migration through ubiquitination of Abl interactor 2 (Abi2), a tumor suppressor. RNF5 and XRNF185 modulate cell migration by ubiquitinating paxillin. Thus, these E3 ubiquitin ligases regulate cell adhesion and (or) migration through ubiquitination of their specific substrates.

Ubiquitin is an evolutionarily conserved protein that consists of 76 amino acid residues and ubiquitination is a post-translational modification process that covalently conjugates single or multiple ubiquitins to target proteins.1-3 Most ubiquitination processes can be divided into three categories based on the length and linkage of ubiquitin chains: K48-linked and K63-linked poly-ubiquitination and mono-ubiquitination.4,5 K48-linked poly-ubiquitination, where ubiquitin is conjugated to another ubiquitin on its lysine 48 residue, is degraded by the 26S proteasome.4,6 K63linked poly-ubiquitination fulfills a variety of signaling functions including protein kinase activation and DNA repair.5 Monoubiquitination usually marks membrane proteins for endocytosis and subsequent degradation in lysosomes.5 Protein ubiquitination consists of three sequential steps: activation of ubiquitin by a ubiquitin-activating enzyme (E1), transfer of ubiquitin from E1 to a ubiquitin-conjugating enzyme (E2), and conjugation of ubiquitin to target proteins by a ubiquitin ligase (E3).1,2 E3 ubiquitin ligases contain motifs for substrate recognition, are primarily responsible for recognizing specific substrate proteins, and are the key control points for protein Correspondence to: Cai Huang; Email: [email protected] Submitted: 04/30/09; Accepted: 08/18/09 Previously published online: www.landesbioscience.com/journals/celladhesion/article/9834

10

ubiquitination.7-9 So far, approximately 500 E3 ubiquitin ligase genes have been identified in humans. The majority of these ubiquitin ligases can be divided into two categories based on specific structural motifs: (1) those possessing the HECT (homologous to the E6-AP carboxyl terminus) domain; (2) those containing the RING (really interesting new gene)-finger domain.10,11 The roles of E3 ubiquitin ligases in regulating cell proliferation, apoptosis, cancer invasion and neurodegenerative diseases have been extensively investigated and reviewed.12-16 However, their roles in cell adhesion and migration, processes that play pivotal roles in wound healing, embryonic development, cancer invasion and inflammation, are only beginning to be recognized. Indeed, more and more evidence indicates that E3 ubiquitin ligases participate in the regulation of cell adhesion and migration. Here, I summarize the roles of E3 ubiquitin ligases in cell matrix-adhesion and migration, emphasizing their mechanisms of action and highlighting future directions. Cbl The Cbl (Casitas B-lineage Lymphoma) family of ubiquitin ligases in mammals contains three members: c-Cbl, Cbl-b, and Cbl-c, encoded by three homologous genes (Fig. 1).17-19 C-Cbl encodes a 120 kDa cytoplasmic protein, featuring a tyrosine kinase binding (TKB) domain in the amino terminal portion, a C3HC4 Zincbinding RING finger domain, a proline-rich region and a leucine zipper domain in the carboxyl terminal portion.20,21 The C3HC4 Zinc-binding RING finger domain mediates the E3 ubiquitin ligase activity, while all other domains function as protein-binding modules. Cbl-b and c-Cbl have similar domain structures, but their sequences between the proline-rich regions and leucine zipper domains show no significant homology.18 Cbl-c retains the TKB domain, the C3HC4 RING finger domain and very short proline-rich region, but lacks the other carboxyl terminal portion.19 Both c-Cbl and Cbl-b are ubiquitously expressed in mammalian cells. They localize in the cytoplasm and are recruited to the plasma membrane under certain circumstances.22 Cbl-b also associates with the lipid raft.23 C-Cbl is present in the thymus, spleen, testis, lung, heart and brain, as well as T- and B-cells, but is highly expressed in the thymus and testis.17 Cbl-b is ubiquitously expressed in diversified tissues, including the spleen, testis, ovary, placenta, heart, thymus, prostate, kidney, brain, lung, liver, skeletal muscle and pancreas, but the spleen has the highest

Cell Adhesion & Migration Volume 4 Issue 1

REVIEW

review

Figure 1. Domain structures of the Cbl family members in mammals. Both c-Cbl and Cbl-b have a tyrosine kinase binding (TKB) domain, a C3HC4 Zinc-binding RING finger (RF) domain, a proline-rich region and a leucine zipper (LZ) domain. A ubiquitin-associated (UBA) domain overlaps with the LZ domain. Cbl-c has a TKB domain, a C3HC4 RF domain and a very short proline-rich region. C-Cbl has four major tyrosine phosphorylation sites: Tyr371, Tyr700, Tyr731 and Tyr774, whereas Cbl-b is phosphorylated at Tyr655 and Tyr709. Currently identified major binding partners for each domain (or region) are also listed.

level of expression.18 Cbl-c is expressed primarily in the epithelial cells of the small intestine, colon, prostate, adrenal gland, and salivary gland, but not in many tissues that express c-Cbl and Cbl-b, such as the spleen and thymus.19 C-Cbl undergoes tyrosine phosphorylation upon growth factor and cytokine stimulation as well as T-cell receptor (TCR) ligation.24-33 C-Cbl is a substrate for several protein tyrosine kinases, including Src, Syk, Yes, Fyn and Abl.34-41 In vitro, Fyn predominantly phosphorylates Tyr731 and Abl phosphorylates Tyr700, whereas Syk phosphorylates Tyr700, Tyr731 and Tyr774. In v-Abl-transformed cells, c-Cbl is phosphorylated at Tyr700 and Tyr774. Overexpression of various Src family protein tyrosine kinases (PTKs) phosphorylates Tyr700, Tyr731 and Tyr774 without apparent specificity.37 In T cells, c-Cbl is phosphorylated at Tyr700, Tyr731 and Tyr774 upon TCR ligation.37 In adipocytes, c-Cbl is phosphorylated at Tyr371, Tyr700 and Tyr774 in response to insulin stimulation.42 Thus, Tyr371, Tyr700, Tyr731 and Tyr774 are the major tyrosine phosphorylation sites identified in c-Cbl (Fig. 1). Like c-Cbl, Cbl-b also becomes tyrosine phosphorylated in response to insulin stimulation and TCR engagement.22,43 In adipocytes and Jurkat cells, Cbl-b is phosphorylated at Tyr 655 and Tyr709 upon TCR stimulation (Fig. 1). However, the phosphorylation of Cbl-b is much weaker as compared to that of c-Cbl upon insulin stimulation and TCR engagement.22,44

www.landesbioscience.com

C-Cbl interacts with a great number of proteins. This topic was extensively reviewed recently.45,46 Briefly, c-Cbl interacts with c-Met, EGFR, PDGFR, Src and Syk through its TKB domain, and binds CrkL, Fyn, Grb2, Lyn, PI 3-kinase, PLCγ, Src and Syk via its proline-rich region (Fig. 1). The phosphotyrosine residues at the C-terminal portion are associated with Abl, CrkII, CrkL, Fyn, PI 3-kinase, Src and vav, whereas the RING finger domain recruits E2 ubiquitin-conjugating enzymes, such as, Ubc4 and UbcH5. In addition, many proteins including c-Kit, FGFR, integrin, paxillin, PKCα/θ, SHIP2 and talin has been shown to associate with c-Cbl, but the nature of the interaction remains to be determined. Similar to c-Cbl, Cbl-b also interacts with many proteins, including c-Kit, c-Met, CrkL, EGFR, Grb2, PI 3-kinase, PKCθ, PLCγ, Syk and vav (Fig. 1).43,47-53 Since the domain structures between c-Cbl and Cbl-b are so similar, it is speculated that Cbl-b may bind many other binding partners of c-Cbl as well. On the other hand, Cbl-b shows different binding capacities toward some binding partners than c-Cbl. For example, Cbl-b but not c-Cbl binds ubiquitin;54 Also, c-Cbl, but not Cbl-b, forms a complex with PI 3-kinase in BCR-Abl-transformed cells.53 Cbl can function as a signaling adaptor by forming complexes via its various domains. In fact, many signaling processes regulated by Cbl are mediated through its signaling adaptor function. For example, the Tyr731 of c-Cbl provides a docking site for the

Cell Adhesion & Migration

11

Figure 2. Cbl regulates cell adhesion and migration. Cbl targets α5 integrin, CrkL and FAK for ubiquitination thus suppressing integrin activation and cell adhesion. It also negatively regulates actin polymerization by ubiquitinating mDab1 and Wave2 and downregulates growth factor receptors, such as EGFR, PDGFR, Met and Kit, by ubiquitinating the receptors to induce cell polarization.

SH2 domain of the p85 subunit of PI 3-kinase, thus recruiting PI 3-kinase to the membrane.55,56 In addition to this role, Cbl is an E3 ubiquitin ligase that fulfills its roles by ubiquitinating its binding partners. Here, I summarize its roles in regulating cell adhesion and migration (Fig. 2). Cbl negatively regulates cell adhesions. The involvement of Cbl in negatively regulating cell adhesions has been demonstrated in several publications. Cbl inhibits cell adhesion by ubiquitinating α5 integrin in osteoblasts,57 and suppresses T cell adhesion by ubiquitinating FAK or inhibiting Rap1 activation.58 By contrast, the macrophages from Cbl-b (-/-) mice exhibit increased recruitment in thioglycollate-induced peritonitis and mononuclear phagocytes derived from Cbl-b (-/-) bone marrow show increased adhesion to endothelial cells.59 C-Cbl and α5 integrin. Alpha5 integrin co-immunoprecipitates with c-Cbl and it also co-localizes with c-Cbl at the leading edge of membrane ruffles, indicating that α5 integrin is a c-Cbl-binding protein. Binding of c-Cbl to α5 integrin results in its ubiquitination and degradation.57 In human calvarial osteoblasts expressing activated FGFR2, FGF stimulation causes α5 integrin ubiquitination and degradation and reduces the adhesions of osteoblasts on fibronectin. However, expressing a c-Cbl mutant lacking the RING finger domain or a c-Cbl mutant with mutation in the PTB domain restores α5 integrin levels. Also, lactacystin, a potent proteasome inhibitor, restores α5 integrin levels in FGF-stimulated osteoblasts. In addition, exogenous expression of α5 integrin rescues cell attachment. Thus, c-Cblmediated ubiquitination and degradation of α5 integrin reduce the adhesion of the osteoblasts to fibronectin.57 Cbl and Rap1. Rap1 mediates integrin activation through RIAM,60,61 while the activity of Rap1 is stimulated by C3G, a guanine nucleotide exchange factor that is activated by CrkL interaction.62 Shao et al.58 reported that Cbl promotes CrkL ubiquitination, which in turn inhibited the interaction of CrkL with C3G, thus suppressing Rap1 activation; Cbl deficiency caused an increase in the activity of C3G, Rap1 activation, and integrin-mediated cell adhesion in Cbl (-/-) thymocytes. Cbl-b

12

deficiency also promotes cell adhesion and cell surface binding to ICAM-1 and enhanced clustering of LFA-1 in Cbl-b(-/-) T cells in response to TCR stimulation.59 However, the underlying molecular mechanisms remain to be elucidated. Cbl and FAK. Cbl ubiquitinates FAK in a STAP-2-dependent manner and inhibits T cell adhesion to fibronectin. STAP-2 (signal-transducing adaptor protein-2) is a signaling adaptor protein that interacts with FAK and Cbl. Sekine et al.63 showed that STAP-2 recruits FAK and Cbl together and mediates the ubiquitination of FAK by Cbl, thus inhibiting cell adhesion to fibronectin. Overexpression of STAP-2 causes a dramatic decrease in the levels of FAK, whereas STAP-2 (-/-) T cells have increased FAK levels. Furthermore, STAP-2 (-/-) splenocytes or T cells exhibit enhanced cell adhesion to fibronectin after PMA treatment, whereas overexpression of STAP-2 inhibits integrin-mediated T cell adhesion to fibronectin.63 Cbl regulates actin polymerization. Cbl and mDab1. Cbl ubiquitinates phosphorylated mDab1 (mouse Disabled homologue 1), thus inhibiting mDab1-induced actin polymerization and filopodium formation. mDab1 binds and activates N-WASP (neuronal Wiskott–Aldrich syndrome protein) directly, inducing actin polymerization through the Arp2/3 (actin-related protein 2/3) complex and filapodium formation. Suetsugu et al.64 reported that mDab1 is ubiquitinated in a Cbl-dependent manner after it is phosphorylated by Fyn; Fyn activation inhibits mDab1-induced filopodium formation. These results suggest that Cbl and Fyn negatively regulate mDab1-N-WASP-mediated actin polymerization. This pathway may regulate actin dynamics during cell migration. Cbl and WAVE2. Cbl ubiquitinates WAVE2 to inhibit actin polymerization and lamellipodium formation. WAVE2, a member of the WASP and WAVE family proteins, has a WAVE homology/Scar homology domain (WHD/SHD), a basic region, a proline-rich region and a VCA region.65 Upon Rac activation, WAVE2 forms a complex with Rac and IRSp53 and then binds G-actin and Arp2/3, thus inducing actin polymerization and lamellipodium formation.65 On the other hand, Cestra et al.66


are regulators of cell migration, currently there are no direct links between the ubiquitination of these PTKs and cell migration. Smurfs

Figure 3. Domain structures of Smurf1 and Smurf2. Smurf1 has an amino terminal C2 domain, two WW domains and a catalytic carboxyl terminal HECT domain. A nuclear export signal (NES) sequence (I606GGLDKIDL614) localizes in the carboxyl terminus of Smurf1. Smurf2 has an amino terminal C2 domain, three WW domains and a catalytic carboxyl terminal HECT domain.

demonstrated that WAVE2 is a substrate for Cbl and is ubiquitinated by Cbl when it is phosphorylated by Abl. Thus, ubiquitination of WAVE2 by Cbl may provide a mechanism for negatively regulating actin polymerization and lamellipodium dynamics during cell migration. Cbl regulates cell migration by downregulating growth factor receptors. Growth factors induce directional migration by promoting cell polarization, which requires cells to regulate their growth factor receptors temporally and spatially. Studies by Jékely et al.67 showed that Cbl modulates the spatial distribution of growth factor receptors in cells through ubiquitinating the receptors, thus promoting cell polarization; cells deficient in Cbl had severe migration defects,67 indicating that Cbl is required for cell migration. Cbl and EGFR. EGFR is a substrate for Cbl.49,68-70 SHIP2, an enzyme that dephosphorylates phosphatidylinositol 3,4,5-trisphosphate, interacts with Cbl and has an inhibitory effect on Cbl.71 SHIP2 knockdown in HeLa cells promotes Cbl-mediated EGFR ubiquitination and degradation.71 SHIP2 knockdown also inhibits cell adhesions and spreading, but it is unknown whether this inhibition is caused by Cbl. Cbl and PDGFR. PDGFR is also a substrate for Cbl. Overexpression of Cbl promotes PDGF-induced ubiquitination and degradation of the PDGFR, whereas a point mutation (G306E) that inactivates the tyrosine kinase binding domain of Cbl abolishes Cbl’s ability to induce ubiquitination and degradation of the PDGFR.72,73 These results indicate that Cbl negatively regulates PDGF signaling. Cbl and Met. Met, a receptor tyrosine kinase, is the receptor for hepatocyte growth factor/scatter factor (HGF). HGF induces c-Cbl-mediated ubiquitination of Met, while a Met mutant that is not ubiquitinated by Cbl promotes its oncogenic activation,48,74,75 indicating that c-Cbl is a negative regulator of HGF/ Met signaling. Cbl and Kit. Kit is a receptor tyrosine kinase and the receptor for stem cell factor (SCF). SCF stimulates Kit-mediated phosphorylation of Cbl, which in turn causes ubiquitination and degradation of KIT, leading to downregulation of KIT signaling.47,76 Cbl downregulates non-receptor protein tyrosine kinases (PTKs). Cbl ubiquitinates a great number of non-receptor PTKs,35,77-79 including Syk, Fyn, Src and Abl. Although these PTKs


Smurf (Smad ubiquitin regulatory factor) 1 and 2 are members of the Nedd4 family ubiquitin ligases. These enzymes feature an amino terminal C2 domain and 2–3 WW domains that are responsible for cellular localization and substrate recognition, and a catalytic carboxyl terminal HECT domain (Fig. 3).80 Smurf1 is localized in both the nucleus and the cytoplasm. A nuclear export signal sequence in the carboxyl terminus of Smurf1 is responsible for its cytoplasmic distribution.81 Smurf2 is predominantly a nuclear protein, but it can form a complex with Smad7, resulting in its export and recruitment to the activated TGF receptor.82 Smurfs were originally identified as regulators of TGFβ family signaling,83,84 but recent studies show that they regulate a wide range of signaling networks including the Wnt signaling pathway.85 TGFβ family signaling regulates a wide range of biological processes including cell growth, differentiation and development.80,86,87 Signaling is initiated with the activation of Type I and II serine/ threonine receptor kinases through dimerization of TGFβ receptors, which phosphorylate the receptor-regulated Smads (R-Smads) 1, 2, 3, 5 and 8. The phosphorylated Smads then interact with the common Smad, Smad4, resulting in translocation to the nucleus and transcription initiation.88 Smurf1 negatively regulates bone morphogenetic protein signaling by ubiquitinating R-Smad 1 and 5.89,90 Smurf1 also regulates transcription by ubiquitinating Runx2, a bone-specific transcription factor.91 On the other hand, Smurf2 inhibits TGFβ signaling mainly through binding and ubiquitinating R-Smad 2 and TGFβ receptor.92,93 Besides the canonical roles in cell growth and differentiation, accumulating evidence indicates that Smurfs play key roles in regulating cell adhesion and migration (Fig. 4). First, Smurf1 is localized to lamellipodia and filopodia and a fraction of Smurf1 is also localized to focal adhesions.94,95 Second, Smurfs ubiquitinate a number of molecules that regulate cell adhesion and migration, such as RhoA, Rap1, talin head and hPEM-2.94-97 Third, exogenous expression of Smurf1 promotes protrusive activity;94 overexpression of Smurf2 promotes the migration of a human trophoblast cell line.93 Fourth, inhibition of Smurf1 by Smurf1C699A, a ligase-dead mutant or siRNA abrogates protrusive activity and cell migration;94 also, inhibition of Smurf2 by Smurf2C716G, a ligase-dead mutant or siRNA induces cell rounding and inhibits cell migration and metastasis in cancer cell lines.98 These results collectively implicate Smurfs in the regulation of cell adhesion and migration. Smurf1 and RhoA. The discovery of Smurf1-mediated RhoA ubiquitination represents the first direct evidence for the role of Smurf in cell migration.94 RhoA plays a pivotal role in cell migration by regulating stress fiber and focal adhesion formation.99 In migratory cells, RhoA activity is low at the leading edge and higher at the rear and sides.100 Wang et al.94 reported that Smurf1 is localized to lamellipodia and targets RhoA there for ubiquitination and degradation, thus inhibiting stress fiber formation and facilitating protrusive activity and cell migration.94 However,


13

Figure 4. Smurfs regulate cell adhesion and migration. Smurf1 ubiquitinates RhoA and hPEM-2, thus inhibiting actin polymerization and regulating cell protrusions. It also regulates focal adhesion dynamics and cell migration through targeting talin head domain for ubiquitination and degradation. Smurf2 mediates Rap1B ubiquitination and is implicated in integrin suppression, and promotes cell polarization and migration by ubiquitinating TGFβR. In addition, it may ubiquitinate Smurf1.

excess Smurf-mediated RhoA degradation impairs cell migration. For example, gene silencing of synaptopodin, an actinassociated protein that competitively inhibits Smurf1-mediated ubiquitination of RhoA, induces the loss of stress fibers and nonpolarized filopodium formation and inhibits cell migration.101 Thus, Smurf1-mediated ubiquitination of RhoA is responsible for precise temporal and spatial regulation of RhoA, which is required for optimal cell migration. Smurf1 and talin head domain. Talin is an actin and β-integrin tail-binding protein that regulates integrin activation and focal adhesion turnover.102-104 Talin is cleaved into a ~47-kDa head domain and a ~190-kDa rod domain by calpain.105 The talin head (TH) domain localizes to focal adhesions and stimulates integrin activation.105,106 Smurf1 binds to TH and mediates TH ubiquitination and degradation, thus leading to focal adhesion disassembly.95 This action of Smurf1 is inhibited by Cdk5, a regulator of cell migration and cancer metastasis. Cdk5 phosphorylates TH at Ser425 and abrogates its binding to Smurf1, thus inhibiting TH ubiquitination and focal adhesion disassembly.95 Thus, precise coordination of the roles of Smurf1 and Cdk5 is required for optimal cell migration. Smurf1 and hEMP-2. hPEM-2, a guanine nucleotide exchange factor for Cdc42, is also a substrate of Smurf1.97 Smurf1 binds the PH domain of hPEM-2 via its C2 domain and induces hPEM-2 ubiquitination and degradation in cells. Thus, Smurf1-mediated ubiquitination of hPEM-2 may play a role in the spatiotemporal regulation of Cdc42 during cell migration. Smurf2 and Smurf1. Fukunaga et al.107 reported that Smurf2 binds Smurf1 and induces its ubiquitination and degradation, but Smurf1 fails to induce degradation of Smurf2. They also showed that knockdown of Smurf2 in human breast cancer MDA-MB-231 cells causes an increase in the Smurf1 protein level, and promotes cell migration in vitro and bone metastasis in vivo. However, this result is inconsistent with studies showing that Smurf2 knockdown inhibits the migration and metastasis of

14

MDA-MB-231 cells,98 and indicates that further investigation is required to clarify the role of Smurf2 in cell migration. Smurf2 and TGFβ type I receptor. Trophoblast cell migration and invasion are key steps during embryo implantation. These are regulated by Smurf2-mediated degradation of TGFβ type I receptor (TGFβR I).93 Overexpression of Smurf2 in a human trophoblast cell line leads to TGFβR I degradation, and enhances cell migration and invasion. In contrast, Smurf2 knockdown using siRNA causes a significant increase in TGFβR I levels and inhibits the migration of trophoblast cells. Smurf2 and Rap1B. Rap1 is a small GTPase that regulates integrin activation, cell migration, cell polarity and differentiation.108,109 Rap1B is a substrate for Smurf2. Smurf2 ubiquitinates inactive Rap1B and mediates its degradation in proteasomes.96 Smurf2-mediated degradation of Rap1B is essential for neuronal polarity. Because of the role of Rap1 in cell migration, Smurf2mediated Rap1B ubiquitination may regulate cell migration. HDM2 HDM2 is the human counterpart of Mdm2 (murine double minute 2), which encodes a 90-kDa protein. Hdm2 is overexpressed in a wide range of human cancers, including breast cancers, esophageal cancers, lung cancers, and malignant melanomas.110 HDM2 is a RING finger ubiquitin ligase that mediates the ubiquitination of p53, a classic tumor suppressor.111-113 It has been reported that HDM2 also targets NFAT (nuclear factor of activated T cells) for ubiquitination and degradation.114 NFAT is a transcription factor that is emerging as a novel positive regulator of cell migration and cancer invasion.114-116 It is downstream of the PI 3-kinase-Akt-HDM2 pathway.114 Stimulation of the PI 3-kinase pathway results in sequential activation of Akt and then HDM2, which in turn mediates NFAT ubiquitination and degradation, thus inhibiting the migration of cancer cells.114


BCA2 The BCA2 protein is a RING finger E3 ubiquitin ligase and is overexpressed in human breast tumors. Wild-type BCA2 significantly promotes the migration of MCF-7 cells in wound healing assays, whereas mutants that are deficient in autoubiquitination (RING mutation or NH2-terminal lysine mutations) significantly inhibit migration.117 SCFβ-TRCP SCFβ-TRCP is a ubiquitin ligase complex that consists of Skp1, Cul1 and the F-box protein β-TRCP and it is responsible for mediating phosphorylation dependent ubiquitination of a number of protein substrates, including IκBα, WEE1, catenin and Snail.118 Snail is a key transcription repressor of E-cadherin and is essential for epithelial-mesenchymal transition (EMT), which is critical for epithelial cell migration and cancer metastasis.119,120 Snail is phosphorylated by GSK-3 and subsequently is targeted for ubiquitination and degradation by SCFβ-TRCP, whereas that the small C-terminal domain phosphatase (SCP), a specific phosphatase for Snail, induces Snail dephosphorylation and inhibits Snail ubiquitination and degradation, thus suppressing E-cadherin expression and promoting cell migration.121,122 The NFκB pathway also abrogates Snail ubiquitination and degradation through COP9 signalosome 2-mediated inhibition of SCFβ-TRCP, thus promoting cell migration.123 TRIM32 Tripartite motif protein 32 (TRIM32) is a RING domain E3 ubiquitin ligase that is highly expressed in human head and neck squamous cell carcinoma.124 It features a RING and a B-box finger domain, a coiled-coil domain at the N-terminus and multiple NHL domains at the C-terminus.125 TRIM32 binds to and ubiquitinates Abl interactor 2 (Abi2), a tumor suppressor and a cell migration inhibitor.126 Overexpression of TRIM32 causes Abi2 degradation and enhances cell growth and cell migration, whereas a dominant-negative mutant of TRIM32 without the RING domain inhibits Abi2 degradation. XRNF185 and RNF5

1. 2. 3.

4.

Finley D, Chau V. Ubiquitination. Annu Rev Cell Biol 1991; 7:25-69. Weissman AM. Regulating protein degradation by ubiquitination. Immunol Today 1997; 18:189-98. Wilkinson KD. Ubiquitination and deubiquitination: Targeting of proteins for degradation by the proteasome. Semin Cell Dev Biol 2000; 11:141-8. Pickart CM. Ubiquitin in chains. Trends Biochem Sci 2000; 25:544-8.


Conclusions and Future Prospects E3 ubiquitin ligases are emerging as important regulators of cell adhesion and migration. They regulate cell adhesion and migration through ubiquitinating specific substrates, such as adhesion molecules, actin polymerization regulators, small GTPases, protein kinases, growth factor receptors and transcription factors. These ubiquitin ligases may temporally and spatially regulate their substrates, thus promoting cell polarization, such as Smurf1-mediated ubiquitination of RhoA. They may also regulate the levels of cell migration promoters, thus modulating the migration of cells, such as SCFβ-TRCP-mediated Snail ubiquitination. These ligases also target many molecules that regulate cell adhesion and migration for ubiquitination and degradation, but direct links between the ubiquitination of these molecules and cell migration remain to be elucidated. Future studies will focus on the roles and the underlying mechanisms of different E3 ubiquitin ligases in cell adhesion and migration. It is of great importance to identify other E3 ubiquitin ligases and novel substrates that regulate cell adhesion and migration. How ubiquitin ligases temporally and spatially regulate their substrates and whether this kind of regulation participates in cycling processes such as focal adhesion assembly/disassembly during cell migration is another key issue. Eventually, we would like to understand how E3 ubiquitin ligases coordinate other signaling pathways that may regulate both inhibition and acceleration of cell migration. Acknowledgements

XRNF185 is a Xenopus RING finger domain ubiquitin ligase. It shares 91% homology with human RNF185. XRNF185 binds and ubiquitinates paxillin,127 a signaling adaptor and a focal adhesion-associated protein implicated in regulating focal adhesion dynamics and cell migration.128,129 It enhances the turnover References

of paxillin at focal adhesions and promotes mesodermal cell migration during gastrulation.127 RNF5, a RING finger domain ubiquitin ligase, was originally identified as a regulator of growth and development of Caenorhabditis elegans.130 The human homologue of RNF5 interacts with the NH2-terminus of paxillin and mediates its ubiquitination.131 RNF5 requires intact RING and C-terminal domains to mediate paxillin ubiquitination. However, instead of inducing paxillin degradation, RNF5-mediated ubiquitination causes a translocation of paxillin from focal adhesions to the cytoplasm. Overexpression of RNF5 inhibits cell migration in wound healing assays.131

5. 6.

7.

I thank Drs. Ken Jacobson, Mark Ginsberg, Zenon Rajfur and Ms. Michelle Itano for their critical reading of this manuscript. Supported by a Cell Migration Consortium grant (NIH GM64346) to M.G. and K.J. and a Ruth L. Kirschstein National Research Service Award (F32 HL08321) to C.H.

Sun L, Chen ZJ. The novel functions of ubiquitination in signaling. Curr Opin Cell Biol 2004; 16:119-26. Thrower JS, Hoffman L, Rechsteiner M, Pickart CM. Recognition of the polyubiquitin proteolytic signal. EMBO J 2000; 19:94-102. Jackson PK, Eldridge AG, Freed E, Furstenthal L, Hsu JY, Kaiser BK, et al. The lore of the rings: Substrate recognition and catalysis by ubiquitin ligases. Trends Cell Biol 2000; 10:429-39.


8.

Crews CM. Feeding the machine: Mechanisms of proteasome-catalyzed degradation of ubiquitinated proteins. Curr Opin Chem Biol 2003; 7:534-9. 9. Deshaies RJ, Joazeiro CAP. Ring domain e3 ubiquitin ligases. Annu Rev Biochem 2009; 78:399-434. 10. Pickart CM. Mechanisms underlying ubiquitination. Annu Rev Biochem 2001; 70:503-33. 11. Ardley HCRP. E3 ubiquitin ligases. Essays Biochem 2005; 41:15-30.

15

12. Alves-Rodrigues A, Gregori L, Figueiredo-Pereira ME. Ubiquitin, cellular inclusions and their role in neurodegeneration. Trends Neurosci 1998; 21:516-20. 13. Krek W. Proteolysis and the g1-s transition: The scf connection. Curr Opin Genet Dev 1998; 8:36-42. 14. Sumara I, Maerki S, Peter M. E3 ubiquitin ligases and mitosis: Embracing the complexity. Trends Cell Biol 2008; 18:84-94. 15. Bernassola F, Karin M, Ciechanover A, Melino G. The hect family of e3 ubiquitin ligases: Multiple players in cancer development. Cancer Cell 2008; 14:10-21. 16. Zhang H-G, Wang J, Yang X, Hsu H-C, Mountz JD. Regulation of apoptosis proteins in cancer cells by ubiquitin. Oncogene 2004; 23:2009-15. 17. Langdon WY, Hyland CD, Grumont RJ, Morse HC. The c-cbl proto-oncogene is preferentially expressed in thymus and testis tissue and encodes a nuclear-protein. J Virol 1989; 63:5420-4. 18. Keane MM, Riverolezcano OM, Mitchell JA, Robbins KC, Lipkowitz S. Cloning and characterization of cblb—a sh3 binding-protein with homology to the c-cbl protooncogene. Oncogene 1995; 10:2367-77. 19. Keane MM, Ettenberg SA, Nau MM, Banerjee P, Cuello M, Penninger J, et al. Cbl-3: A new mammalian cbl family protein. Oncogene 1999; 18:3365-75. 20. Joazeiro CAnP, Wing SS, Huang H-k, Leverson JD, Hunter T, Liu Y-C. The tyrosine kinase negative regulator c-cbl as a ring-type, e2-dependent ubiquitin-protein ligase. Science 1999; 286:309-12. 21. Lupher ML Jr, Andoniou CE, Bonita D, Miyake S, Band H. Molecules in focus: The c-cbl oncoprotein. Int J Biochem Cell Biol 1998; 30:439-44. 22. Liu J, DeYoung SM, Hwang JB, O’Leary EE, Saltiel AR. The roles of cbl-b and c-cbl in insulin-stimulated glucose transport. J Biol Chem 2003; 278:36754-62. 23. Qu X, Sada K, Kyo S, Maeno K, Miah SMS, Yamamura H. Negative regulation of fc{epsilon}ri-mediated mast cell activation by a ubiquitin-protein ligase cbl-b. Blood 2004; 103:1779-86. 24. Galisteo ML, Dikic I, Batzer AG, Langdon WY, Schlessinger J. Tyrosine phosphorylation of the c-cbl proto-oncogene protein product and association with epidermal growth factor (egf ) receptor upon egf stimulation. J Biol Chem 1995; 270:20242-5. 25. Wisniewski D, Strife A, Clarkson B. C-kit ligand stimulates tyrosine phosphorylation of the c-cbl protein in human hematopoietic cells. Leukemia 1996; 10:1436-42. 26. Ribon V, Saltiel AR. Insulin stimulates tyrosine phosphorylation of the proto-oncogene product of c-cbl in 3t3-l1 adipocytes. Biochem J 1997; 324: 839-45. 27. Odai H, Sasaki K, Iwamatsu A, Hanazono Y, Tanaka T, Mitani K, et al. The proto-oncogene product c-cbl becomes tyrosine phosphorylated by stimulation with gm-csf or epo and constitutively binds to the sh3 domain of grb2/ash in human hematopoietic cells. J Biol Chem 1995; 270:10800-5. 28. Meisner H, Czech MP. Coupling of the proto-oncogene product c-cbl to the epidermal growth factor receptor. J Biol Chem 1995; 270:25332-5. 29. Fukazawa T, Miyake S, Band V, Band H. Tyrosine phosphorylation of cbl upon epidermal growth factor (egf ) stimulation and its association with egf receptor and downstream signaling proteins. J Biol Chem 1996; 271:14554-9. 30. Barber DL, Mason JM, Fukazawa T, Reedquist KA, Druker BJ, Band H, et al. Erythropoietin and interleukin-3 activate tyrosine phosphorylation of cbl and association with crk adaptor proteins. Blood 1997; 89:3166-74. 31. Gesbert F, Garbay C, Bertoglio J. Interleukin-2 stimulation induces tyrosine phosphorylation of p120-cbl and crkl and formation of multimolecular signaling complexes in t lymphocytes and natural killer cells. J Biol Chem 1998; 273:3986-93.

16

32. Kontani K, Kukimoto I, Nishina H, Hoshino S-i, Hazeki O, Kanaho Y, et al. Tyrosine phosphorylation of the c-cbl proto-oncogene product mediated by cell surface antigen cd38 in hl-60 cells. J Biol Chem 1996; 271:1534-7. 33. Donovan J, Wange R, Langdon W, Samelson L. The protein product of the c-cbl protooncogene is the 120-kda tyrosine-phosphorylated protein in jurkat cells activated via the t cell antigen receptor. J Biol Chem 1994; 269:22921-4. 34. Tanaka S, Amling M, Neff L, Peyman A, Uhlmann E, Levy JB, et al. C-cbl is downstream of c-src in a signalling pathway necessary for bone resorption. Nature 1996; 383:528-31. 35. Yokouchi M, Kondo T, Sanjay A, Houghton A, Yoshimura A, Komiya S, et al. Src-catalyzed phosphorylation of c-cbl leads to the interdependent ubiquitination of both proteins. J Biol Chem 2001; 276:3518593. 36. Ota Y, Beitz LO, Scharenberg AM, Donovan JA, Kinet JP, Samelson LE. Characterization of cbl tyrosine phosphorylation and a cbl-syk complex in rbl-2 h3 cells. J Exp Med 1996; 184:1713-23. 37. Feshchenko EA, Langdon WY, Tsygankov AY. Fyn, yes, and syk phosphorylation sites in c-cbl map to the same tyrosine residues that become phosphorylated in activated t cells. J Biol Chem 1998; 273:8323-31. 38. Hunter S, Burton EA, Wu SC, Anderson SM. Fyn associates with cbl and phosphorylates tyrosine 731 in cbl, a binding site for phosphatidylinositol 3-kinase. J Biol Chem 1999; 274:2097-106. 39. Miyoshi-Akiyama T, Aleman LM, Smith JM, Adler CE, Mayer BJ. Regulation of cbl phosphorylation by the abl tyrosine kinase and the nck sh2/sh3 adaptor. Oncogene 2001; 20:4058-69. 40. Andoniou CE, Thien CBF, Langdon WY. The two major sites of cbl tyrosine phosphorylation in abltransformed cells select the crkl sh2 domain. Oncogene 1996; 12:1981-9. 41. Grossmann AH, Kolibaba KS, Willis SG, Corbin AS, Langdon WS, Deininger MWN, et al. Catalytic domains of tyrosine kinases determine the phosphorylation sites within c-cbl. FEBS Lett 2004; 577:555-62. 42. Liu J, Kimura A, Baumann CA, Saltiel AR. Aps facilitates c-cbl tyrosine phosphorylation and glut4 translocation in response to insulin in 3t3-l1 adipocytes. Mol Cell Biol 2002; 22:3599-609. 43. Elly CWS, Zhang Z, Rosnet O, Lipkowitz S, Altman A, Liu YC. Tyrosine phosphorylation and complex formation of cbl-b upon t cell receptor stimulation. Oncogene 1999; 18:1147-56. 44. Bachmaier K, Krawczyk C, Kozieradzki I, Kong Y-Y, Sasaki T, Oliveira-dos-Santos A, et al. Negative regulation of lymphocyte activation and autoimmunity by the molecular adaptor cbl-b 2000; 403:211-6. 45. Tsygankov AY, Teckchandani AM, Feshchenko EA, Swaminathan G. Beyond the ring: Cbl proteins as multivalent adapters. Oncogene 2001; 20:6382-402. 46. Schmidt MHH, Dikic I. The cbl interactome and its functions. Nature Rev Mol Cell Biol 2005; 6:907-18. 47. Zeng S, Xu Z, Lipkowitz S, Longley JB. Regulation of stem cell factor receptor signaling by cbl family proteins (cbl-b/c-cbl). Blood 2005; 105:226-32. 48. Peschard P, Fournier TM, Lamorte L, Naujokas MA, Band H, Langdon WY, et al. Mutation of the c-cbl tkb domain binding site on the met receptor tyrosine kinase converts it into a transforming protein. Mol Cell 2001; 8:995-1004. 49. Ettenberg SA, Keane MM, Nau MM, Frankel M, Wang LM, Pierce JH, et al. Cbl-b inhibits epidermal growth factor receptor signaling. Oncogene 1999; 18:1855-66. 50. Fang D, Wang H-Y, Fang N, Altman Y, Elly C, Liu Y-C. Cbl-b, a ring-type e3 ubiquitin ligase, targets phosphatidylinositol 3-kinase for ubiquitination in t cells. J Biol Chem 2001; 276:4872-8.

51. Yasuda T, Tezuka T, Maeda A, Inazu T, Yamanashi Y, Gu H, et al. Cbl-b positively regulates btk-mediated activation of phospholipase c-{gamma}2 in b cells. J Exp Med 2002; 196:51-63. 52. Gruber T, Hermann-Kleiter N, Hinterleitner R, Fresser F, Schneider R, Gastl G, et al. Pkc-{theta} modulates the strength of t cell responses by targeting cbl-b for ubiquitination and degradation. Sci Signal 2009; 2:30. 53. Sattler MPY, Quinnan LR, Verma S, Malouf NA, Husson H, Salgia R, et al. Differential expression and signaling of cbl and cbl-b in bcr/abl transformed cells. Oncogene 2002; 21:1423-33. 54. Davies GC, Ettenberg SA, Coats AO, Mussante M, Ravichandran S, Collins J, et al. Cbl-b interacts with ubiquitinated proteins; differential functions of the uba domains of c-cbl and cbl-b. Oncogene 2004; 23:710415. 55. Beckwith M, Jorgensen G, Longo D. The protein product of the proto-oncogene c-cbl forms a complex with phosphatidylinositol 3-kinase p85 and cd19 in anti-igm-stimulated human b-lymphoma cells. Blood 1996; 88:3502-7. 56. Ueno H, Sasaki K, Honda H, Nakamoto T, Yamagata T, Miyagawa K, et al. C-cbl is tyrosine-phosphorylated by interleukin-4 and enhances mitogenic and survival signals of interleukin-4 receptor by linking with the phosphatidylinositol 3'-kinase pathway. Blood 1998; 91:46-53. 57. Kaabeche K, Guenou H, Bouvard D, Didelot N, Listrat A, Marie PJ. Cbl-mediated ubiquitination of {alpha}5 integrin subunit mediates fibronectin-dependent osteoblast detachment and apoptosis induced by fgfr2 activation. J Cell Sci 2005; 118:1223-32. 58. Shao Y, Elly C, Liu YC. Negative regulation of rap1 activation by the cbl e3 ubiquitin ligase. EMBO Rep 2003; 4:425-31. 59. Choi EY, Orlova VV, Fagerholm SC, Nurmi SM, Zhang L, Ballantyne CM, et al. Regulation of lfa-1dependent inflammatory cell recruitment by cb1-b and 14-3-3 proteins. Blood 2008; 111:3607-14. 60. Lafuente EM, van Puijenbroek AAFL, Krause M, Carman CV, Freeman GJ, Berezovskaya A, et al. Riam, an ena/vasp and profilin ligand, interacts with rap1-gtp and mediates rap1-induced adhesion. Dev Cell 2004; 7:585-95. 61. Han JW, Lim CJ, Watanabe N, Soriani A, Ratnikov B, Calderwood DA, et al. Reconstructing and deconstructing agonist-induced activation of integrin alpha iib beta3. Curr Biol 2006; 16:1796-806. 62. Bos J. All in the family? New insights and questions regarding interconnectivity of ras, rap1 and ral. EMBO J 1998; 17:6776-82. 63. Sekine Y, Tsuji S, Ikeda O, Sugiyma K, Oritani K, Shimoda K, et al. Signal-transducing adaptor protein-2 regulates integrin-mediated t cell adhesion through protein degradation of focal adhesion kinase. J Immunol 2007; 179:2397-407. 64. Suetsugu S, Tezuka T, Morimura T, Hattori M, Mikoshiba K, Yamamoto T, et al. Regulation of actin cytoskeleton by mdab1 through n-wasp and ubiquitination of mdab1. Biochem J 2004; 384:1-8. 65. Takenawa T, Miki H. Wasp and wave family proteins: Key molecules for rapid rearrangement of cortical actin filaments and cell movement. J Cell Sci 2001; 114:1801-9. 66. Cestra G, Toomre D, Chang S, De Camilli P. The abl/ arg substrate argbp2/nargbp2 coordinates the function of multiple regulatory mechanisms converging on the actin cytoskeleton. Proc Natl Acad Sci USA 2005; 102:1731-6. 67. Jékely G, Sung H-H, Luque CM, Rørth P. Regulators of endocytosis maintain localized receptor tyrosine kinase signaling in guided migration. Dev Cell 2005; 9:197-207.


68. Umebayashi K, Stenmark H, Yoshimori T. Ubc4/5 and c-cbl continue to ubiquitinate egf receptor after internalization to facilitate polyubiquitination and degradation. Mol Biol Cell 2008; 19:3454-62. 69. Stang E, Blystad FD, Kazazic M, Bertelsen V, Brodahl T, Raiborg C, et al. Cb1-dependent ubiquitination is required for progression of egf receptors into clathrincoated pits. Mol Biol Cell 2004; 15:3591-604. 70. Thien CBF, Walker F, Langdon WY. Ring finger mutations that abolish c-cbl-directed polyubiquitination and downregulation of the egf receptor are insufficient for cell transformation. Mol Cell 2001; 7:35565. 71. Prasad NK, Decker SJ. Sh2-containing 5'-inositol phosphatase, ship2, regulates cytoskeleton organization and ligand-dependent downregulation of the epidermal growth factor receptor. J Biol Chem 2005; 280:1312936. 72. Miyake S, Lupher ML, Druker B, Band H. The tyrosine kinase regulator cbl enhances the ubiquitination and degradation of the platelet-derived growth factor receptor alpha. Proc Natl Acad Sci USA 1998; 95:7927-32. 73. Miyake S, Mullane-Robinson KP, Lill NL, Douillard P, Band H. Cbl-mediated negative regulation of plateletderived growth factor receptor-dependent cell proliferation. A critical role for cbl tyrosine kinase-binding domain. J Biol Chem 1999; 274:16619-28. 74. Taher TEI, Tjin EPM, Beuling EA, Borst J, Spaargaren M, Pals ST. C-cbl is involved in met signaling in b cells and mediates hepatocyte growth factor-induced receptor ubiquitination. J Immunol 2002; 169:3793800. 75. Abella JV, Peschard P, Naujokas MA, Lin T, Saucier C, Urbe S, et al. Met/hepatocyte growth factor receptor ubiquitination suppresses transformation and is required for hrs phosphorylation. Mol Cell Biol 2005; 25:9632-45. 76. Masson K, Heiss E, Band H, Ronnstrand L. Direct binding of cbl to tyr(568) and tyr(936) of the stem cell factor receptor/c-kit is required for ligand-induced ubiquitination, internalization and degradation. Biochem J 2006; 399:59-67. 77. Andoniou CE, Lill NL, Thien CB, Lupher ML, Ota S, Bowtell DDL, et al. The cbl proto-oncogene product negatively regulates the src-family tyrosine kinase fyn by enhancing its degradation. Mol Cell Biol 2000; 20:851-67. 78. Soubeyran P, Barac A, Szymkiewicz I, Dikic I. Cblargbp2 complex mediates ubiquitination and degradation of c-abl. Biochem J 2003; 370:29-34. 79. Paolini R, Molfetta R, Beitz LO, Zhang J, Scharenberg AM, Piccoli M, et al. Activation of syk tyrosine kinase is required for c-cbl-mediated ubiquitination of fcepsilon ri and syk in rbl cells. J Biol Chem 2002; 277:369407. 80. Zhu HT, Kavsak P, Abdollah S, Wrana JL, Thomsen GH. A smad ubiquitin ligase targets the bmp pathway and affects embryonic pattern formation. Nature 1999; 400:687-93. 81. Tajima Y, Goto K, Yoshida M, Shinomiya K, Sekimoto T, Yoneda Y, et al. Chromosomal region maintenance 1 (crm1)-dependent nuclear export of smad ubiquitin regulatory factor 1 (smurf1) is essential for negative regulation of transforming growth factor-beta signaling by smad7. J Biol Chem 2003; 278:10716-21. 82. Kavsak P, Rasmussen RK, Causing CG, Bonni S, Zhu H, Thomsen GH, et al. Smad7 binds to smurf2 to form an e3 ubiquitin ligase that targets the tgf[beta] receptor for degradation. Molecular Cell 2000; 6:1365-75. 83. Izzi L, Attisano L. Regulation of the tgf beta signalling pathway by ubiquitin-mediated degradation. Oncogene 2004; 23:2071-8. 84. Arora K, Warrior R. A new smurf in the village. Dev Cell 2001; 1:441-2. 85. Narimatsu M, Bose R, Pye M, Zhang L, Miller B, Ching P, et al. Regulation of planar cell polarity by smurf ubiquitin ligases. Cell 2009; 137:295-307.


86. Moustakas A, Pardali K, Gaal A, Heldin C-H. Mechanisms of tgf-[beta] signaling in regulation of cell growth and differentiation. Immunol Lett 2002; 82:8591. 87. Wu MY, Hill CS. Tgf[beta] superfamily signaling in embryonic development and homeostasis. Dev Cell 2009; 16:329-43. 88. Clarke DC, Liu X. Decoding the quantitative nature of tgf[beta]/smad signaling. Trends Cell Biol 2008; 18:430-42. 89. Sapkota G, Alarcón C, Spagnoli FM, Brivanlou AH, Massagué J. Balancing bmp signaling through integrated inputs into the smad1 linker. Mol Cell 2007; 25:441-54. 90. Ying S-X, Hussain ZJ, Zhang YE. Smurf1 facilitates myogenic differentiation and antagonizes the bone morphogenetic protein-2-induced osteoblast conversion by targeting smad5 for degradation. J Biol Chem 2003; 278:39029-36. 91. Shen R, Chen M, Wang YJ, Kaneki H, Xing LP, O’Keefe RJ, et al. Smad6 interacts with runx2 and mediates smad ubiquitin regulatory factor 1-induced runx2 degradation. J Biol Chem 2006; 281:3569-76. 92. Lin X, Liang M, Feng X-H. Smurf2 is a ubiquitin e3 ligase mediating proteasome-dependent degradation of smad2 in transforming growth factor-beta signaling. J Biol Chem 2000; 275:36818-22. 93. Yang Q, Chen S-P, Zhang X-P, Wang H, Zhu C, Lin H-Y. Smurf2 participates in human trophoblast cell invasion by inhibiting tgf{beta} type i receptor. J Histochem Cytochem 2009; 57:605-12. 94. Wang HR, Zhang Y, Ozdamar B, Ogunjimi AA, Alexandrova E, Thomsen GH, et al. Regulation of cell polarity and protrusion formation by targeting rhoa for degradation. Science 2003; 302:1775-9. 95. Huang C, Rajfur Z, Yousefi N, Chen ZZ, Jacobson K, Ginsberg MH. Talin phosphorylation by cdk5 regulates smurf1-mediated talin head ubiquitylation and cell migration. Nat Cell Biol 2009; 11:404-624. 96. Schwamborn JC, Muller M, Becker AHM, Puschel AW. Ubiquitination of the gtpase rap1b by the ubiquitin ligase smurf2 is required for the establishment of neuronal polarity. EMBO J 2007; 26: 1410-22. 97. Yamaguchi K, Ohara O, Ando A, Nagase T. Smurf1 directly targets hpem-2, a gef for cdc42, via a novel combination of protein interaction modules in the ubiquitin-proteasome pathway. Biol Chem 2008; 389:405-13. 98. Jin C, Yang Y-a, Anver MR, Morris N, Wang X, Zhang YE. Smad ubiquitination regulatory factor 2 promotes metastasis of breast cancer cells by enhancing migration and invasiveness. Cancer Res 2009; 69:735-40. 99. Chrzanowska-Wodnicka M, Burridge K. Rhostimulated contractility drives the formation of stress fibers and focal adhesions. J Cell Biol 1996; 133:140315. 100. Ridley AJ, Schwartz MA, Burridge K, Firtel RA, Ginsberg MH, Borisy G, et al. Cell migration: Integrating signals from front to back. Science 2003; 302:1704-9. 101. Asanuma K, Yanagida-Asanuma E, Faul C, Tomino Y, Kim K, Mundel P. Synaptopodin orchestrates actin organization and cell motility via regulation of rhoa signalling. Nat Cell Biol 2006; 8:485-91. 102. Wegener KL, Partridge AW, Han J, Pickford AR, Liddington RC, Ginsberg MH, et al. Structural basis of integrin activation by talin. Cell 2007; 128: 171-82. 103. Calderwood DA. Talin controls integrin activation. Biochem Soc Trans 2004; 32:434-7. 104. Franco SJ, Rodgers MA, Perrin BJ, Han JW, Bennin DA, Critchley DR, et al. Calpain-mediated proteolysis of talin regulates adhesion dynamics. Nat Cell Biol 2004; 6:977-83. 105. Nuckolls GH, Turner CE, Burridge K. Functionalstudies of the domains of talin. J Cell Biol 1990; 110:1635-44.


106. Calderwood DA, Zent R, Grant R, Rees DJG, Hynes RO, Ginsberg MH. The talin head domain binds to integrin beta subunit cytoplasmic tails and regulates integrin activation. J Biol Chem 1999; 274:28071-4. 107. Fukunaga E, Inoue Y, Komiya S, Horiguchi K, Goto K, Saitoh M, et al. Smurf2 induces ubiquitin-dependent degradation of smurf1 to prevent migration of breast cancer cells. J Biol Chem 2008; 283:35660-7. 108. Bos JL, de Rooij J, Reedquist KA. Rap1 signalling: Adhering to new models. Nat Rev Mol Cell Biol 2001; 2:369-77. 109. Hattori M, Minato N. Rap1 gtpase: Functions, regulation, and malignancy. J Biochem 2003; 134:479-84. 110. Momand J, Jung D, Wilczynski S, Niland J. The mdm2 gene amplification database. Nucl Acids Res 1998; 26:3453-9. 111. Tao W, Levine AJ. Nucleocytoplasmic shuttling of oncoprotein hdm2 is required for hdm2-mediated degradation of p53. Proc Natl Acad Sci USA 1999; 96:3077-80. 112. Fang S, Jensen JP, Ludwig RL, Vousden KH, Weissman AM. Mdm2 is a ring finger-dependent ubiquitin protein ligase for itself and p53. J Biol Chem 2000; 275:8945-51. 113. Haupt Y, Maya R, Kazaz A, Oren M. Mdm2 promotes the rapid degradation of p53. Nature 1997; 387:2969. 114. Yoeli-Lerner M, Yiu GK, Rabinovitz I, Erhardt P, Jauliac S, Toker A. Akt blocks breast cancer cell motility and invasion through the transcription factor nfat. Mol Cell 2005; 20:539-50. 115. Jauliac S, Lopez-Rodriguez C, Shaw LM, Brown LF, Rao A, Toker A. The role of nfat transcription factors in integrin-mediated carcinoma invasion. Nat Cell Biol 2002; 4:540-4. 116. Corral RS, Iniguez MA, Duque J, Lopez-Perez R, Fresno M. Bombesin induces cyclooxygenase-2 expression through the activation of the nuclear factor of activated t cells and enhances cell migration in caco-2 colon carcinoma cells. Oncogene 2006; 26:958-69. 117. Amemiya Y, Azmi P, Seth A. Autoubiquitination of bca2 ring e3 ligase regulates its own stability and affects cell migration. Mol Cancer Res 2008; 6:1385-96. 118. Frescas D, Pagano M. Deregulated proteolysis by the f-box proteins skp2 and β-trcp: Tipping the scales of cancer. Nat Rev Cancer 2008; 8:438-49. 119. Peinado H, Olmeda D, Cano A. Snail, zeb and bhlh factors in tumour progression: An alliance against the epithelial phenotype? Nat Rev Cancer 2007; 7:41528. 120. Becker KF, Rosivatz E, Blechschmidt K, Kremmer E, Sarbia M, Hofler H. Analysis of the e-cadherin repressor snail in primary human cancers. Cells Tissues Organs 2007; 185:204-12. 121. Zhou BP, Deng J, Xia W, Xu J, Li YM, Gunduz M, et al. Dual regulation of snail by gsk-3[beta]-mediated phosphorylation in control of epithelial-mesenchymal transition. Nat Cell Biol 2004; 6:931-40. 122. Wu Y, Evers BM, Zhou BP. Small c-terminal domain phosphatase enhances snail activity through dephosphorylation. J Biol Chem 2009; 284:640-8. 123. Wu Y, Deng J, Rychahou PG, Qiu S, Evers BM, Zhou BP. Stabilization of snail by nf-[kappa]b is required for inflammation-induced cell migration and invasion. Cancer Cell 2009; 15:416-28. 124. Horn EJ, Albor A, Liu YG, El-Hizawi S, Vanderbeek GE, Babcock M, et al. Ring protein trim32 associated with skin carcinogenesis has anti-apoptotic and e3-ubiquitin ligase properties. Carcinogenesis 2004; 25:157-67. 125. Reymond A, Meroni G, Fantozzi A, Merla G, Cairo S, Luzi L, et al. The tripartite motif family identifies cell compartments. EMBO J 2001; 20:2140-51. 126. Kano S, Miyajima N, Fukuda S, Hatakeyama S. Tripartite motif protein 32 facilitates cell growth and migration via degradation of abl-interactor 2. Cancer Res 2008; 68:5572-80.

17

127. Iioka H, Iemura S-i, Natsume T, Kinoshita N. Wnt signalling regulates paxillin ubiquitination essential for mesodermal cell motility. Nat Cell Biol 2007; 9:81321. 128. Huang C, Rajfur Z, Borchers C, Schaller MD, Jacobson K. Jnk phosphorylates paxillin and regulates cell migration. Nature 2003; 424:219-23. 129. Huang C, Jacobson K, Schaller MD. Map kinases and cell migration. J Cell Sci 2004; 117:4619-28. 130. Kyushiki H, Kuga Y, Suzuki M, Takahashi E, Horie M. Cloning, expression and mapping of a novel ring-finger gene (rnf5), a human homologue of a putative zincfinger gene from Caenorhabditis elegans. Cytogenet Cell Genet 1997; 79:114-7. 131. Didier C, Broday L, Bhoumik A, Israeli S, Takahashi S, Nakayama K, et al. Rnf5, a ring finger protein that regulates cell motility by targeting paxillin ubiquitination and altered localization. Mol Cell Biol 2003; 23:5331-45.

18
