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1.  Trip12, a HECT domain E3 ubiquitin ligase, targets Sox6 for proteasomal degradation and affects fiber type-specific gene expression in muscle cells 
Skeletal Muscle  2013;3:11.
Background
A sophisticated level of coordinated gene expression is necessary for skeletal muscle fibers to obtain their unique functional identities. We have previously shown that the transcription factor Sox6 plays an essential role in coordinating muscle fiber type differentiation by acting as a transcriptional suppressor of slow fiber-specific genes. Currently, mechanisms regulating the activity of Sox6 in skeletal muscle and how these mechanisms affect the fiber phenotype remain unknown.
Methods
Yeast two-hybrid screening was used to identify binding partners of Sox6 in muscle. Small interfering RNA (siRNA)-mediated knockdown of one of the Sox6 binding proteins, Trip12, was used to determine its effect on Sox6 activity in C2C12 myotubes using quantitative analysis of fiber type-specific gene expression.
Results
We found that the E3 ligase Trip12, a HECT domain E3 ubiquitin ligase, recognizes and polyubiquitinates Sox6. Inhibiting Trip12 or the 26S proteasome activity resulted in an increase in Sox6 protein levels in C2C12 myotubes. This control of Sox6 activity in muscle cells via Trip12 ubiquitination has significant phenotypic outcomes. Knockdown of Trip12 in C2C12 myotubes led to upregulation of Sox6 protein levels and concurrently to a decrease in slow fiber-specific Myh7 expression coupled with an increased expression in fast fiber-specific Myh4. Therefore, regulation of Sox6 cellular levels by the ubiquitin-proteasome system can induce identity-changing alterations in the expression of fiber type-specific genes in muscle cells.
Conclusions
Based on our data, we propose that in skeletal muscle, E3 ligases have a significant role in regulating fiber type-specific gene expression, expanding their importance in muscle beyond their well-established role in atrophy.
doi:10.1186/2044-5040-3-11
PMCID: PMC3666947  PMID: 23663701
Sox6; Skeletal muscle; Fiber type differentiation; Trip12; E3 ubiquitin ligase; HECT domain; Ubiquitin-proteasome system
2.  TRAF-interacting protein (TRIP) negatively regulates IFN-β production and antiviral response by promoting proteasomal degradation of TANK-binding kinase 1 
The Journal of Experimental Medicine  2012;209(10):1703-1711.
TRAF-interacting protein (TRIP) negatively regulates TLR3/4- and RIG-I–induced IFN-β signaling by promoting K48-linked ubiquitination and proteasomal degradation of TBK1.
TANK-binding kinase 1 (TBK1) plays an essential role in Toll-like receptor (TLR)– and retinoic acid–inducible gene I (RIG-I)–mediated induction of type I interferon (IFN; IFN-α/β) and host antiviral responses. How TBK1 activity is negatively regulated remains largely unknown. We report that TNF receptor-associated factor (TRAF)–interacting protein (TRIP) promotes proteasomal degradation of TBK1 and inhibits TLR3/4- and RIG-I–induced IFN-β signaling. TRIP knockdown resulted in augmented activation of IFN regulatory factor 3 (IRF3) and enhanced expression of IFN-β in TLR3/4- and RIG-I–activated primary peritoneal macrophages, whereas overexpression of TRIP had opposite effects. Consistently, TRIP impaired Sendai virus (SeV) infection–induced IRF3 activation and IFN-β production and promoted vesicular stomatitis virus (VSV) replication. As an E3 ubiquitin ligase, TRIP negatively regulated the cellular levels of TBK1 by directly binding to and promoting K48-linked polyubiquitination of TBK1. Therefore, we identified TRIP as a negative regulator in TLR3/4- and RIG-I–triggered antiviral responses and suggested TRIP as a potential target for the intervention of diseases with uncontrolled IFN-β production.
doi:10.1084/jem.20120024
PMCID: PMC3457734  PMID: 22945920
3.  TRIP-1: A Regulator of Osteoblast Function 
Journal of Bone and Mineral Research  2012;27(7):1576-1584.
TGFβ receptor interacting protein-1 (TRIP-1) is an intracellular protein expressed in osteoblasts with high affinity for type 5b tartrate resistant acid phosphatase (TRAP). It is suggested that through this interaction, TRIP-1 serves as a positive regulator of TGFβ signaling and osteoblast differentiation during bone remodeling. We show here that TRIP-1 is abundant in osteoblasts in vivo and in vitro. TRIP-1 mRNA and protein expression were increased at early stages and decreased at later stages during osteoblast differentiation, suggesting a predominant role during early maturation. To investigate a role during bone remodeling, primary osteoblasts were treated with different hormones and factors that are known to affect remodeling. TRIP-1 levels were decreased with dexamethasone and increased with vitamin D3, DHT, TGFβ1 and BMP-2. Treatment with PTH and β-estradiol did not affect TRIP-1 levels. Transfected siRNA against TRIP-1 inhibited osteoblast differentiation as characterized by a decrease in alkaline phosphatase staining and enzyme activity, and decrease in the expression of collagen I, alkaline phosphatase, Runx2, osteopontin and osteocalcin. The proliferation of osteoblasts was also affected by TRIP-1 siRNA. This particular effect was defined by decreased cell number, marked reduction of cyclin D1, a 38% decrease of cells in S phase (p<0.001) and a 97% increase of cells in the G2/M phase (p<0.01) of the cell cycle. However, TRIP-1 siRNA did not induce an effect in apoptosis. Using a TGFβ luciferase reporter we found that knocking down TRIP-1 decreased by 40% percent the activation of TGFβ signaling (p<0.001). In conclusion, our characterization of TRIP-1 in osteoblasts provides the first evidence of its key role as a positive regulator of osteoblast function.
doi:10.1002/jbmr.1611
PMCID: PMC3377841  PMID: 22460930
osteoblast; TRIP-1; TGFβ; TRAP; remodeling
4.  TRIP8b regulates HCN1 channel trafficking and gating through two distinct C-terminal interaction sites 
Hyperpolarization-activated cyclic nucleotide-regulated (HCN) channels in the brain associate with their auxiliary subunit TRIP8b (also known as PEX5R), a cytoplasmic protein expressed as a family of alternatively spliced isoforms. Recent in vitro and in vivo studies have shown that association of TRIP8b with HCN subunits both inhibits channel opening and alters channel membrane trafficking, with some splice variants increasing and others decreasing channel surface expression. Here, we address the structural bases of the regulatory interactions between mouse TRIP8b and HCN1. We find that HCN1 and TRIP8b interact at two distinct sites: an upstream site where the C-linker/cyclic nucleotide-binding domain of HCN1 interacts with an 80 amino acid domain in the conserved central core of TRIP8b, and a downstream site where the C-terminal -SNL tripeptide of the channel interacts with the tetratricopeptide repeat domain of TRIP8b. These two interaction sites play distinct functional roles in the effects of TRIP8b on HCN1 trafficking and gating. Binding at the upstream site is both necessary and sufficient for TRIP8b to inhibit channel opening. It is also sufficient to mediate the trafficking effects of those TRIP8b isoforms that downregulate channel surface expression, in combination with the trafficking motifs present in the N-terminal region of TRIP8b. In contrast, binding at the downstream interaction site serves to stabilize the C-terminal domain of TRIP8b, allowing for optimal interaction between HCN1 and TRIP8b as well as for proper assembly of the molecular complexes that mediate the effects of TRIP8b on HCN1 channel trafficking.
doi:10.1523/JNEUROSCI.5707-10.2011
PMCID: PMC3077297  PMID: 21411649
5.  TRIP-1 via AKT modulation drives lung fibroblast/myofibroblast trans-differentiation 
Respiratory Research  2014;15(1):19.
Background
Myofibroblasts are the critical effector cells in the pathogenesis of pulmonary fibrosis which carries a high degree of morbidity and mortality. We have previously identified Type II TGFβ receptor interacting protein 1 (TRIP-1), through proteomic analysis, as a key regulator of collagen contraction in primary human lung fibroblasts—a functional characteristic of myofibroblasts, and the last, but critical step in the process of fibrosis. However, whether or not TRIP-1 modulates fibroblast trans-differentiation to myofibroblasts is not known.
Methods
TRIP-1 expression was altered in primary human lung fibroblasts by siRNA and plasmid transfection. Transfected fibroblasts were then analyzed for myofibroblast features and function such as α-SMA expression, collagen contraction ability, and resistance to apoptosis.
Results
The down-regulation of TRIP-1 expression in primary human lung fibroblasts induces α-SMA expression and enhances resistance to apoptosis and collagen contraction ability. In contrast, TRIP-1 over-expression inhibits α-SMA expression. Remarkably, the effects of the loss of TRIP-1 are not abrogated by blockage of TGFβ ligand activation of the Smad3 pathway or by Smad3 knockdown. Rather, a TRIP-1 mediated enhancement of AKT phosphorylation is the implicated pathway. In TRIP-1 knockdown fibroblasts, AKT inhibition prevents α-SMA induction, and transfection with a constitutively active AKT construct drives collagen contraction and decreases apoptosis.
Conclusions
TRIP-1 regulates fibroblast acquisition of phenotype and function associated with myofibroblasts. The importance of this finding is it suggests TRIP-1 expression could be a potential target in therapeutic strategy aimed against pathological fibrosis.
doi:10.1186/1465-9921-15-19
PMCID: PMC3946032  PMID: 24528651
Type II TGFβ receptor interacting protein 1 (TRIP-1); Eukaryotic translation initiation factor-3 (eIF3); Pulmonary fibroblasts; α-smooth muscle actin (α-SMA); Fibrosis
6.  TRIP6 Enhances Lysophosphatidic Acid-induced Cell Migration by Interacting with the Lysophosphatidic Acid 2 Receptor* 
The Journal of biological chemistry  2003;279(11):10459-10468.
Lysophosphatidic acid (LPA) induces actin rearrangement, focal adhesion assembly, and cell migration through the activation of small G protein Rho and its downstream effectors. These diverse cellular responses are mediated by its associated G protein-coupled receptors. However, the mechanisms and specificity by which these LPA receptors mediate LPA actions are still poorly understood. Here we show that LPA stimulation promotes the interaction of the LPA2 receptor with a focal adhesion molecule, TRIP6 (thyroid receptor interacting protein 6)/ZRP-1 (zyxin-related protein 1). TRIP6 directly binds to the carboxyl-terminal tail of the LPA2 receptor through its LIM domains. LPA-dependent recruitment of TRIP6 to the plasma membrane promotes its targeting to focal adhesions and co-localization with actin stress fibers. In addition, TRIP6 associates with the components of focal complexes including paxillin, focal adhesion kinase, c-Src, and p130cas in an agonist-dependent manner. Overexpression of TRIP6 augments LPA-induced cell migration; in contrast, suppression of endogenous TRIP6 expression by a TRIP6-specific small interfering RNA reduces it in SKOV3 ovarian cancer cells. Strikingly, the association with TRIP6 is specific to the LPA2 receptor but not LPA1 or LPA3 receptor, indicating a specific role for TRIP6 in regulating LPA2 receptor-mediated signaling. Taken together, our results suggest that TRIP6 functions at a point of convergence between the activated LPA2 receptor and downstream signals involved in cell adhesion and migration.
doi:10.1074/jbc.M311891200
PMCID: PMC3904432  PMID: 14688263
7.  c-Src-Mediated Phosphorylation of TRIP6 Regulates Its Function in Lysophosphatidic Acid-Induced Cell Migration†  
Molecular and Cellular Biology  2005;25(14):5859-5868.
TRIP6 (thyroid receptor-interacting protein 6), also known as ZRP-1 (zyxin-related protein 1), is a member of the zyxin family that has been implicated in cell motility. Previously we have shown that TRIP6 binds to the LPA2 receptor and associates with several components of focal complexes in an agonist-dependent manner and, thus, enhances lysophosphatidic acid (LPA)-induced cell migration. Here we further report that the function of TRIP6 in LPA signaling is regulated by c-Src-mediated phosphorylation of TRIP6 at the Tyr-55 residue. LPA stimulation induces tyrosine phosphorylation of endogenous TRIP6 in NIH 3T3 cells and c-Src-expressing fibroblasts, which is virtually eliminated in Src-null fibroblasts. Strikingly, both phosphotyrosine-55 and proline-58 residues of TRIP6 are required for Crk binding in vitro and in cells. Mutation of Tyr-55 to Phe does not alter the ability of TRIP6 to localize at focal adhesions or associate with actin. However, it abolishes the association of TRIP6 with Crk and p130cas in cells and significantly reduces the function of TRIP6 to promote LPA-induced ERK activation. Ultimately, these signaling events control TRIP6 function in promoting LPA-induced morphological changes and cell migration.
doi:10.1128/MCB.25.14.5859-5868.2005
PMCID: PMC1168818  PMID: 15988003
8.  Functional characterization of Trip10 in cancer cell growth and survival 
Background
The Cdc42-interacting protein-4, Trip10 (also known as CIP4), is a multi-domain adaptor protein involved in diverse cellular processes, which functions in a tissue-specific and cell lineage-specific manner. We previously found that Trip10 is highly expressed in estrogen receptor-expressing (ER+) breast cancer cells. Estrogen receptor depletion reduced Trip10 expression by progressively increasing DNA methylation. We hypothesized that Trip10 functions as a tumor suppressor and may be involved in the malignancy of ER-negative (ER-) breast cancer. To test this hypothesis and evaluate whether Trip10 is epigenetically regulated by DNA methylation in other cancers, we evaluated DNA methylation of Trip10 in liver cancer, brain tumor, ovarian cancer, and breast cancer.
Methods
We applied methylation-specific polymerase chain reaction and bisulfite sequencing to determine the DNA methylation of Trip10 in various cancer cell lines and tumor specimens. We also overexpressed Trip10 to observe its effect on colony formation and in vivo tumorigenesis.
Results
We found that Trip10 is hypermethylated in brain tumor and breast cancer, but hypomethylated in liver cancer. Overexpressed Trip10 was associated with endogenous Cdc42 and huntingtin in IMR-32 brain tumor cells and CP70 ovarian cancer cells. However, overexpression of Trip10 promoted colony formation in IMR-32 cells and tumorigenesis in mice inoculated with IMR-32 cells, whereas overexpressed Trip10 substantially suppressed colony formation in CP70 cells and tumorigenesis in mice inoculated with CP70 cells.
Conclusions
Trip10 regulates cancer cell growth and death in a cancer type-specific manner. Differential DNA methylation of Trip10 can either promote cell survival or cell death in a cell type-dependent manner.
doi:10.1186/1423-0127-18-12
PMCID: PMC3044094  PMID: 21299869
9.  INHIBITION OF TRIP1/S8/hSUG1, A COMPONENT OF THE HUMAN 19S PROTEASOME, ENHANCES MITOTIC APOPTOSIS INDUCED BY SPINDLE POISONS 
Molecular cancer therapeutics  2006;5(1):29-38.
Mitotic spindle poisons (e.g. Taxol, vinblastine), used as chemotherapy drugs, inhibit mitotic spindle function, activate the mitotic spindle checkpoint, arrest cells in mitosis, and then cause cell death by mechanisms that are poorly understood. By expression cloning we identified a truncated version of human TRIP1 (also known as S8, hSug1), an AAA (ATPases Associated with diverse cellular Activities) family ATPase subunit of the 19S proteasome regulatory complex, as an enhancer of spindle poison-mediated apoptosis. Stable expression of the truncated TRIP1/S8/hSug1 in HeLa cells (OP-TRIP1(88-406)) resulted in a decrease of measurable cellular proteasome activity, indicating that OP-had a dominant-negative effect on proteasome function. OP-TRIP1(88-406) revealed an increased apoptotic response after treatment with spindle poisons or with proteasome inhibitors. The increased apoptosis coincided with a significant decrease in expression of BubR1, a kinase required for activation and maintenance of the mitotic spindle checkpoint in response to treatment with spindle poisons. SiRNA-mediated knockdown of TRIP1/S8/hSug1 resulted in a reduction of general proteasome activity, and an increase in mitotic index. The siRNA treatment also caused increased cell death after spindle poison treatment. These results indicate that inhibition of TRIP1/S8/hSug1 function by expression of a truncated version of the protein or by siRNA-mediated suppression enhances cell death in response to spindle poison treatment. Current proteasome inhibitor drugs in trial as anticancer agents target elements of the 20S catalytic subcomplex. Our results suggest that targeting the ATPase subunits in 19S regulatory complex in the proteasome may enhance the anti-tumor effects of spindle poisons.
doi:10.1158/1535-7163.MCT-05-0126
PMCID: PMC1630635  PMID: 16432160
spindle; microtubule; proteasome; TRIP1/Sug1; Taxol
10.  TRIP8b Is Required for Maximal Expression of HCN1 in the Mouse Retina 
PLoS ONE  2014;9(1):e85850.
Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are cation-selective channels present in retina, brain and heart. The activity of HCN channels contributes to signal integration, cell excitability and pacemaker activity. HCN1 channels expressed in photoreceptors participate in keeping light responses transient and are required for normal mesopic vision. The subcellular localization of HCN1 varies among cell types. In photoreceptors HCN1 is concentrated in the inner segments while in other retinal neurons, HCN1 is evenly distributed though the cell. This is in contrast to hippocampal neurons where HCN1 is concentrated in a subset of dendrites. A key regulator of HCN1 trafficking and activity is tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b). Multiple splice isoforms of TRIP8b are expressed throughout the brain and can differentially regulate the surface expression and activity of HCN1. The purpose of the present study was to determine which isoforms of TRIP8b are expressed in the retina and to test if loss of TRIP8b alters HCN1 expression or trafficking. We found that TRIP8b colocalizes with HCN1 in multiple retina neurons and all major splice isoforms of TRIP8b are expressed in the retina. Photoreceptors express three different isoforms. In TRIP8b knockout mice, the ability of HCN1 to traffic to the surface of retinal neurons is unaffected. However, there is a large decrease in the total amount of HCN1. We conclude that TRIP8b in the retina is needed to achieve maximal expression of HCN1.
doi:10.1371/journal.pone.0085850
PMCID: PMC3883711  PMID: 24409334
11.  The Adaptor Protein TRIP6 Antagonizes Fas-Induced Apoptosis but Promotes Its Effect on Cell Migration ▿  
Molecular and Cellular Biology  2010;30(23):5582-5596.
The Fas/CD95 receptor mediates apoptosis but is also capable of triggering nonapoptotic signals. However, the mechanisms that selectively regulate these opposing effects are not yet fully understood. Here we demonstrate that the activation of Fas or stimulation with lysophosphatidic acid (LPA) induces cytoskeletal reorganization, leading to the association of Fas with actin stress fibers and the adaptor protein TRIP6. TRIP6 binds to the cytoplasmic juxtamembrane domain of Fas and interferes with the recruitment of FADD to Fas. Furthermore, through physical interactions with NF-κB p65, TRIP6 regulates nuclear translocation and the activation of NF-κB upon Fas activation or LPA stimulation. As a result, TRIP6 antagonizes Fas-induced apoptosis and further enhances the antiapoptotic effect of LPA in cells that express high levels of TRIP6. On the other hand, TRIP6 promotes Fas-mediated cell migration in apoptosis-resistant glioma cells. This effect is regulated via the Src-dependent phosphorylation of TRIP6 at Tyr-55. As TRIP6 is overexpressed in glioblastomas, this may have a significant impact on enhanced NF-κB activity, resistance to apoptosis, and Fas-mediated cell invasion in glioblastomas.
doi:10.1128/MCB.00134-10
PMCID: PMC2976429  PMID: 20876301
12.  Alternatively spliced isoforms of TRIP8b differentially control h channel trafficking and function 
Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels (h channels) are the molecular basis for the current, Ih, which contributes crucially to intrinsic neuronal excitability. The subcellular localization and biophysical properties of h channels govern their function, but the mechanisms controlling these characteristics, and especially the potential role of auxiliary subunits or other binding proteins remain unclear.
We focused on TRIP8b, an h channel-interacting protein that colocalizes with HCN1 in cortical and hippocampal pyramidal neuron dendrites, and found that it exists in multiple alternative splice variants with distinct effects on h channel trafficking and function. The developmentally-regulated splice variants of TRIP8b all shared dual, C-terminus-located interaction sites with HCN1. When coexpressed with HCN1 in heterologous cells individual TRIP8b isoforms similarly modulated gating of Ih, causing a hyperpolarizing shift in voltage-dependence of channel activation, but differentially upregulated or downregulated Ih current density and HCN1 surface expression. In hippocampal neurons, coexpression of TRIP8b isoforms with HCN1 produced isoform-specific changes of HCN1 localization. Interestingly, the TRIP8b isoforms most abundant in the brain are those predicted to enhance h channel surface expression. Indeed, shRNA knockdown of TRIP8b in hippocampal neurons significantly reduced native Ih. Thus, although TRIP8b exists in multiple splice isoforms, our data suggest that the predominant role of this protein in brain is to promote h channel surface expression and enhance Ih. Because Ih expression is altered in models of several diseases, including temporal lobe epilepsy, TRIP8b may play a role in both normal neuronal function and in aberrant neuronal excitability associated with neurological disease.
doi:10.1523/JNEUROSCI.0856-09.2009
PMCID: PMC2730639  PMID: 19439603
Ion Channel; Ih; Hyperpolarization-activated cyclic nucleotide-gated channel; Tetratricopeptide repeat; Peroxin 5; dendrite; trafficking; chaperone; scaffold
13.  Deletion of the HCN channel auxiliary subunit TRIP8b impairs hippocampal Ih localization and function and promotes antidepressant behavior in mice 
Output properties of neurons are greatly shaped by voltage-gated ion channels, whose biophysical properties and localization within axodendritic compartments serve to significantly transform the original input. The hyperpolarization-activated current, Ih, is mediated by HCN channels and plays a fundamental role in influencing neuronal excitability by regulating both membrane potential and input resistance. In neurons such as cortical and hippocampal pyramidal neurons, the subcellular localization of HCN channels plays critical functional role, yet mechanisms controlling HCN channel trafficking are not fully understood. Because ion channel function and localization are often influenced by interacting proteins, we generated a knockout mouse lacking the HCN channel auxiliary subunit, TRIP8b. Eliminating expression of TRIP8b dramatically reduced Ih expression in hippocampal pyramidal neurons. Loss of Ih-dependent membrane voltage properties was attributable to reduction of HCN channels on the neuronal surface, and there was a striking disruption of the normal expression pattern of HCN channels in pyramidal neuron dendrites. In heterologous cells and neurons, absence of TRIP8b increased HCN subunit targeting to and degradation by lysosomes. Mice lacking TRIP8b demonstrated motor learning deficits and enhanced resistance to multiple tasks of behavioral despair with high predictive validity for antidepressant efficacy. We observed similar resistance to behavioral despair in distinct mutant mice lacking HCN1 or HCN2. These data demonstrate that interaction with the auxiliary subunit TRIP8b is a major mechanism underlying proper expression of HCN channels and Ih in vivo, and suggest that targeting Ih may provide a novel approach to treatment of depression.
doi:10.1523/JNEUROSCI.0936-11.2011
PMCID: PMC3169171  PMID: 21593326
TRIP8b; HCN channels; Ih; depression; knockout mouse; hippocampus
14.  PTPL1/FAP-1 Negatively Regulates TRIP6 Function in Lysophosphatidic Acid-induced Cell Migration*,S 
The Journal of biological chemistry  2007;282(33):24381-24387.
The LIM domain-containing TRIP6 (Thyroid Hormone Receptor-interacting Protein 6) is a focal adhesion molecule known to regulate lysophosphatidic acid (LPA)-induced cell migration through interaction with the LPA2 receptor. LPA stimulation targets TRIP6 to the focal adhesion complexes and promotes c-Src-dependent phosphorylation of TRIP6 at Tyr-55, which creates a docking site for the Crk Src homology 2 domain, thereby promoting LPA-induced morphological changes and cell migration. Here we further demonstrate that a switch from c-Src-mediated phosphorylation to PTPL1/Fas-associated phosphatase-1-dependent dephosphorylation serves as an inhibitory feedback control mechanism of TRIP6 function in LPA-induced cell migration. PTPL1 dephosphorylates phosphotyrosine 55 of TRIP6 in vitro and inhibits LPA-induced tyrosine phosphorylation of TRIP6 in cells. This negative regulation requires a direct protein-protein interaction between these two molecules and the phosphatase activity of PTPL1. In contrast to c-Src, PTPL1 prevents TRIP6 turnover at the sites of adhesions. As a result, LPA-induced association of TRIP6 with Crk and the function of TRIP6 to promote LPA-induced morphological changes and cell migration are inhibited by PTPL1. Together, these results reveal a novel mechanism by which PTPL1 phosphatase plays a counteracting role in regulating TRIP6 function in LPA-induced cell migration.
doi:10.1074/jbc.M701499200
PMCID: PMC3923842  PMID: 17591779
15.  Mechanism of ubiquitin ligation and lysine prioritization by a HECT E3 
eLife  2013;2:e00828.
Ubiquitination by HECT E3 enzymes regulates myriad processes, including tumor suppression, transcription, protein trafficking, and degradation. HECT E3s use a two-step mechanism to ligate ubiquitin to target proteins. The first step is guided by interactions between the catalytic HECT domain and the E2∼ubiquitin intermediate, which promote formation of a transient, thioester-bonded HECT∼ubiquitin intermediate. Here we report that the second step of ligation is mediated by a distinct catalytic architecture established by both the HECT E3 and its covalently linked ubiquitin. The structure of a chemically trapped proxy for an E3∼ubiquitin-substrate intermediate reveals three-way interactions between ubiquitin and the bilobal HECT domain orienting the E3∼ubiquitin thioester bond for ligation, and restricting the location of the substrate-binding domain to prioritize target lysines for ubiquitination. The data allow visualization of an E2-to-E3-to-substrate ubiquitin transfer cascade, and show how HECT-specific ubiquitin interactions driving multiple reactions are repurposed by a major E3 conformational change to promote ligation.
DOI: http://dx.doi.org/10.7554/eLife.00828.001
eLife digest
Ubiquitin is a small protein that can be covalently linked to other, ‘target’, proteins in a cell to influence their behavior. Ubiquitin can be linked to its targets either as single copies or as polyubiquitin chains in which several ubiquitin molecules are bound end-on-end to each other, with one end of the chain attached to the target protein. A multi-step cascade involving enzymes known as E1, E2, and E3 adds ubiquitin to its targets. These enzymes function in a manner like runners in a relay, with ubiquitin a baton that is passed from E1 to E2 to E3 to the target.
The E3 enzyme is a ligase that catalyzes the formation of a new chemical bond between a ubiquitin and its target. There are approximately 600 different E3 enzymes in human cells that regulate a wide variety of target proteins. A major class of E3 enzymes, called HECT E3s, attaches ubiquitin to its targets in a unique two-step mechanism: the E2 enzymes covalently link a ubiquitin to a HECT E3 to form a complex that subsequently transfers the ubiquitin to its target protein. The ubiquitin is typically added to a particular amino acid, lysine, on the target protein, but the details of how HECT E3s execute this transfer are not well understood. To address this issue, Kamadurai et al. investigate how Rsp5, a HECT E3 ligase in yeast, attaches ubiquitin to a target protein called Sna3.
All HECT E3s have a domain—the HECT domain—that catalyzes the transfer of ubiquitin to its target protein. This domain consists of two sub-structures: the C-lobe, which can receive ubiquitin from E2 and then itself become linked to ubiquitin, and the N-lobe. These lobes were previously thought to adopt various orientations relative to each other to deliver ubiquitin to sites on different target proteins (including to multiple lysines on a single target protein). Unexpectedly, Kamadurai et al. find that in order to transfer the ubiquitin to Sna3, Rsp5 adopts a discrete HECT domain architecture that creates an active site in which parts of the C-lobe and the N-lobe, which are normally separated, are brought together with a ubiquitin molecule. This architecture also provides a mechanism that dictates which substrate lysines can be ubiquitinated based on how accessible they are to this active site.
The same regions of Rsp5 transfer ubiquitin to targets other than Sna3, suggesting that a uniform mechanism—which Kamadurai et al. show is conserved in two related human HECT E3 ligases—might transfer ubiquitin to all its targets. These studies therefore represent a significant step toward understanding how a major class of E3 enzymes modulates the functions of their targets.
DOI: http://dx.doi.org/10.7554/eLife.00828.002
doi:10.7554/eLife.00828
PMCID: PMC3738095  PMID: 23936628
ubiquitin; HECT; E3 ligase; E2 conjugating enzyme; NEDD4; Rsp5; S. cerevisiae
16.  TRIP-Br2 promotes oncogenesis in nude mice and is frequently overexpressed in multiple human tumors 
Background
Members of the TRIP-Br/SERTAD family of mammalian transcriptional coregulators have recently been implicated in E2F-mediated cell cycle progression and tumorigenesis. We, herein, focus on the detailed functional characterization of the least understood member of the TRIP-Br/SERTAD protein family, TRIP-Br2 (SERTAD2).
Methods
Oncogenic potential of TRIP-Br2 was demonstrated by (1) inoculation of NIH3T3 fibroblasts, which were engineered to stably overexpress ectopic TRIP-Br2, into athymic nude mice for tumor induction and (2) comprehensive immunohistochemical high-throughput screening of TRIP-Br2 protein expression in multiple human tumor cell lines and human tumor tissue microarrays (TMAs). Clinicopathologic analysis was conducted to assess the potential of TRIP-Br2 as a novel prognostic marker of human cancer. RNA interference of TRIP-Br2 expression in HCT-116 colorectal carcinoma cells was performed to determine the potential of TRIP-Br2 as a novel chemotherapeutic drug target.
Results
Overexpression of TRIP-Br2 is sufficient to transform murine fibroblasts and promotes tumorigenesis in nude mice. The transformed phenotype is characterized by deregulation of the E2F/DP-transcriptional pathway through upregulation of the key E2F-responsive genes CYCLIN E, CYCLIN A2, CDC6 and DHFR. TRIP-Br2 is frequently overexpressed in both cancer cell lines and multiple human tumors. Clinicopathologic correlation indicates that overexpression of TRIP-Br2 in hepatocellular carcinoma is associated with a worse clinical outcome by Kaplan-Meier survival analysis. Small interfering RNA-mediated (siRNA) knockdown of TRIP-Br2 was sufficient to inhibit cell-autonomous growth of HCT-116 cells in vitro.
Conclusion
This study identifies TRIP-Br2 as a bona-fide protooncogene and supports the potential for TRIP-Br2 as a novel prognostic marker and a chemotherapeutic drug target in human cancer.
doi:10.1186/1479-5876-7-8
PMCID: PMC2671481  PMID: 19152710
17.  Disruption of the COP9 Signalosome Csn2 Subunit in Mice Causes Deficient Cell Proliferation, Accumulation of p53 and Cyclin E, and Early Embryonic Death 
Molecular and Cellular Biology  2003;23(19):6790-6797.
Csn2 (Trip15/Cops2/Alien) encodes the second subunit of the COP9 signalosome (CSN), an eight-subunit heteromeric complex homologous to the lid subcomplex of the 26S proteasome. CSN is a regulator of SCF (Skp1-cullin-F-box protein)ubiquitin ligases, mostly through the enzymatic activity that deconjugates the ubiquitin-like protein Nedd8 from the SCF Cul1 component. In addition, CSN associates with protein kinase activities targeting p53, c-Jun, and IκB for phosphorylation. Csn2 also interacts with and regulates a subset of nuclear hormone receptors and is considered a novel corepressor. We report that targeted disruption of Csn2 in mice caused arrest of embryo development at the peri-implantation stage. Csn2−/− blastocysts failed to outgrow in culture and exhibited a cell proliferation defect in inner cell mass, accompanied by a slight decrease in Oct4. In addition, lack of Csn2 disrupted the CSN complex and resulted in a drastic increase in cyclin E, supporting a role for CSN in cooperating with the SCF-ubiquitin-proteasome system to regulate protein turnover. Furthermore, Csn2−/− embryos contained elevated levels of p53 and p21, which may contribute to premature cell cycle arrest of the mutant.
doi:10.1128/MCB.23.19.6790-6797.2003
PMCID: PMC193936  PMID: 12972599
18.  HUWE1 and TRIP12 Collaborate in Degradation of Ubiquitin-Fusion Proteins and Misframed Ubiquitin 
PLoS ONE  2012;7(11):e50548.
In eukaryotic cells an uncleavable ubiquitin moiety conjugated to the N-terminus of a protein signals the degradation of the fusion protein via the proteasome-dependent ubiquitin fusion degradation (UFD) pathway. In yeast the molecular mechanism of the UFD pathway has been well characterized. Recently the human E3 ubiquitin-protein ligase TRIP12 was connected with the UFD pathway, but little is otherwise known about this system in mammalian cells. In the present work, we utilized high-throughput imaging on cells transfected with a targeted siRNA library to identify components involved in degradation of the UFD substrate UbG76V-YFP. The most significant hits from the screen were the E3 ubiquitin-protein ligase HUWE1, as well as PSMD7 and PSMD14 that encode proteasome subunits. Accordingly, knock down of HUWE1 led to an increase in the steady state level and a retarded degradation of the UFD substrate. Knock down of HUWE1 also led to a stabilization of the physiological UFD substrate UBB+1. Precipitation experiments revealed that HUWE1 is associated with both the UbG76V-YFP substrate and the 26S proteasome, indicating that it functions late in the UFD pathway. Double knock down of HUWE1 and TRIP12 resulted in an additive stabilization of the substrate, suggesting that HUWE1 and TRIP12 function in parallel during UFD. However, even when both HUWE1 and TRIP12 are downregulated, ubiquitylation of the UFD substrate was still apparent, revealing functional redundancy between HUWE1, TRIP12 and yet other ubiquitin-protein ligases.
doi:10.1371/journal.pone.0050548
PMCID: PMC3507821  PMID: 23209776
19.  Arkadia Enhances Nodal/TGF-β Signaling by Coupling Phospho-Smad2/3 Activity and Turnover 
PLoS Biology  2007;5(3):e67.
Regulation of transforming growth factor-β (TGF-β) signaling is critical in vertebrate development, as several members of the TGF-β family have been shown to act as morphogens, controlling a variety of cell fate decisions depending on concentration. Little is known about the role of intracellular regulation of the TGF-β pathway in development. E3 ubiquitin ligases target specific protein substrates for proteasome-mediated degradation, and several are implicated in signaling. We have shown that Arkadia, a nuclear RING-domain E3 ubiquitin ligase, is essential for a subset of Nodal functions in the embryo, but the molecular mechanism of its action in embryonic cells had not been addressed. Here, we find that Arkadia facilitates Nodal signaling broadly in the embryo, and that it is indispensable for cell fates that depend on maximum signaling. Loss of Arkadia in embryonic cells causes nuclear accumulation of phospho-Smad2/3 (P-Smad2/3), the effectors of Nodal signaling; however, these must be repressed or hypoactive as the expression of their direct target genes is reduced or lost. Molecular and functional analysis shows that Arkadia interacts with and ubiquitinates P-Smad2/3 causing their degradation, and that this is via the same domains required for enhancing their activity. Consistent with this dual function, introduction of Arkadia in homozygous null (−/−) embryonic stem cells activates the accumulated and hypoactive P-Smad2/3 at the expense of their abundance. Arkadia−/− cells, like Smad2−/− cells, cannot form foregut and prechordal plate in chimeras, confirming this functional interaction in vivo. As Arkadia overexpression never represses, and in some cells enhances signaling, the degradation of P-Smad2/3 by Arkadia cannot occur prior to their activation in the nucleus. Therefore, Arkadia provides a mechanism for signaling termination at the end of the cascade by coupling degradation of P-Smad2/3 with the activation of target gene transcription. This mechanism can account for achieving efficient and maximum Nodal signaling during embryogenesis and for rapid resetting of target gene promoters allowing cells to respond to dynamic changes in extracellular signals.
Author Summary
In development, cells respond to secreted signals (called morphogens) by turning on or off sets of target genes. How does gene activity adjust quickly in response to rapidly changing extracellular signals? This should require efficient removal of old/used signaling effectors (signal-activated transcription factors) from the promoters of target genes to allow new ones to assume control. We previously discovered Arkadia, an E3 ubiquitin ligase, and showed that it is an essential factor for normal development. (Ubiquitin ligases trigger the addition of ubiquitin residues to proteins, typically marking them for degradation.) Here, we show that Arkadia is required for high activity of the major signaling pathway, TGF-β/Nodal. Arkadia has a dual role to degrade Smads, the TGF-β signaling effectors, and enhance their transcriptional activity. This coupling of degradation with activation provides a mechanism to ensure that only effectors “in use” are degraded, allowing the new ones to proceed. It is possible that very similar mechanisms operate in other pathways to establish dynamic regulation and efficient signaling, while their failure may be associated with developmental abnormalities and disease, including cancer.
Arkadia enhances TGF-β family activity by degrading its inhibitory Smads but also stimulating transcription of phospho-Smads.
doi:10.1371/journal.pbio.0050067
PMCID: PMC1808117  PMID: 17341133
20.  TRIP6 Regulates p27KIP1 To Promote Tumorigenesis 
Molecular and Cellular Biology  2013;33(7):1394-1409.
TRIP6 is an adaptor protein that regulates cell motility and antiapoptotic signaling. Although it has been implicated in tumorigenesis, the underlying mechanism remains largely unknown. Here we provide evidence that TRIP6 promotes tumorigenesis by serving as a bridge to promote the recruitment of p27KIP1 to AKT in the cytosol. TRIP6 regulates the membrane translocation and activation of AKT and facilitates AKT-mediated recognition and phosphorylation of p27KIP1 specifically at T157, thereby promoting the cytosolic mislocalization of p27KIP1. This is required for p27KIP1 to enhance lysophosphatidic acid (LPA)-induced ovarian cancer cell migration. TRIP6 also promotes serum-induced reduction of nuclear p27KIP1 expression levels through Skp2-dependent and -independent mechanisms. Consequently, knockdown of TRIP6 in glioblastoma or ovarian cancer xenografts restores nuclear p27KIP1 expression and impairs tumor proliferation. As TRIP6 is upregulated in gliomas and its levels correlate with poor clinical outcomes in a dose-dependent manner, it may represent a novel prognostic marker and therapeutic target in gliomas.
doi:10.1128/MCB.01149-12
PMCID: PMC3624266  PMID: 23339869
21.  Mammalian SWI/SNF-A Subunit BAF250/ARID1 Is an E3 Ubiquitin Ligase That Targets Histone H2B▿  
Molecular and Cellular Biology  2010;30(7):1673-1688.
The mammalian SWI/SNF chromatin-remodeling complex facilitates DNA access by transcription factors and the transcription machinery. The characteristic member of human SWI/SNF-A is BAF250/ARID1, of which there are two isoforms, BAF250a/ARID1a and BAF250b/ARID1b. Here we report that BAF250b complexes purified from mammalian cells contain elongin C (Elo C), a BC box binding component of an E3 ubiquitin ligase. BAF250b was found to have a BC box motif, associate with Elo C in a BC box-dependent manner, and, together with cullin 2 and Roc1, assemble into an E3 ubiquitin ligase. The BAF250b BC box mutant protein was unstable in vivo and was autoubiquitinated in a manner similar to that for the VHL BC box mutants. The discovery that BAF250 is part of an E3 ubiquitin ligase adds an enzymatic function to the chromatin-remodeling complex SWI/SNF-A. The immunopurified BAF250b E3 ubiquitin ligase was found to target histone H2B at lysine 120 for monoubiquitination in vitro. To date, all H2B monoubiquitination was attributed to the human homolog of yeast Bre1 (RNF20/40). Mutation of Drosophila osa, the homolog of BAF250, or depletion of BAF250 by RNA interference (RNAi) in cultured human cells resulted in global decreases in monoubiquitinated H2B, implicating BAF250 in the cross talk of histone modifications.
doi:10.1128/MCB.00540-09
PMCID: PMC2838063  PMID: 20086098
22.  An active triple-catalytic hybrid enzyme engineered by linking COX-1 to prostacyclin synthase that can constantly biosynthesize prostacyclin, the vascular protector 
The FEBS journal  2008;275(23):5820-5829.
SUMMARY
It remains a challenge to create the stable and long-term expression (in human cell lines) of a previously engineered hybrid enzyme (Trip-cat enzyme-2, [Ruan, KH., et al. (2006) Biochemistry 45, 14003 – 14011]), which links cyclooxygenase-2 (COX-2) to prostacyclin (PGI2) synthase (PGIS) for the direct conversion of arachidonic acid into PGI2 through the enzyme’s triple-catalytic (Trip-cat) functions. The stable up-regulation of the biosynthesis of the vascular protector, PGI2, in cells is an ideal model for the prevention and treatment of thromboxane A2 (TXA2)-mediated thrombosis and vasoconstriction, both of which cause stroke, myocardial infarction, and hypertension. Here, we report another engineering of the Trip-cat enzyme, in which human COX isoform-1 (COX-1), which has a different C-terminal sequence than COX-2, was linked to PGIS and called the Trip-cat Enzyme-1. Transient expression of the recombinant Trip-cat Enzyme-1 in HEK293 cells demonstrated a 3–5-fold higher expression capacity and better PGI2-synthesizing activity when compared to that of the previously engineered Trip-cat Enzyme-2. Furthermore, an HEK293 cell line that can stably express the active new Trip-cat Enzyme-1 and constantly synthesize the bioactive PGI2 was established by a screening approach. In addition, the stable HEK293 cell line, with a constant production of PGI2, revealed strong anti-platelet aggregation properties through its unique dual functions (increasing PGI2 production while decreasing TXA2 production) in the TXA2 synthase (TXAS)-rich plasma. This study has optimized the active Trip-cat enzyme engineering, allowing it to become the first to stably up-regulate PGI2 biosynthesis in a human cell line, which provides a basis for developing a PGI2-producing therapeutic cell line against vascular diseases.
doi:10.1111/j.1742-4658.2008.06703.x
PMCID: PMC3038792  PMID: 19021758
23.  Mouse TRIP13/PCH2 Is Required for Recombination and Normal Higher-Order Chromosome Structure during Meiosis 
PLoS Genetics  2010;6(8):e1001062.
Accurate chromosome segregation during meiosis requires that homologous chromosomes pair and become physically connected so that they can orient properly on the meiosis I spindle. These connections are formed by homologous recombination closely integrated with the development of meiosis-specific, higher-order chromosome structures. The yeast Pch2 protein has emerged as an important factor with roles in both recombination and chromosome structure formation, but recent analysis suggested that TRIP13, the mouse Pch2 ortholog, is not required for the same processes. Using distinct Trip13 alleles with moderate and severe impairment of TRIP13 function, we report here that TRIP13 is required for proper synaptonemal complex formation, such that autosomal bivalents in Trip13-deficient meiocytes frequently displayed pericentric synaptic forks and other defects. In males, TRIP13 is required for efficient synapsis of the sex chromosomes and for sex body formation. Furthermore, the numbers of crossovers and chiasmata are reduced in the absence of TRIP13, and their distribution along the chromosomes is altered, suggesting a role for TRIP13 in aspects of crossover formation and/or control. Recombination defects are evident very early in meiotic prophase, soon after DSB formation. These findings provide evidence for evolutionarily conserved functions for TRIP13/Pch2 in both recombination and formation of higher order chromosome structures, and they support the hypothesis that TRIP13/Pch2 participates in coordinating these key aspects of meiotic chromosome behavior.
Author Summary
Meiosis is the specialized cell division that gives rise to reproductive cells such as sperm and eggs. During meiosis in most organisms, genetic information is exchanged between homologous maternal and paternal chromosomes through the process of homologous recombination. This recombination forms connections between homologous chromosomes that allow them to segregate accurately when the meiotic cell divides. Recombination defects can result in reproductive cells with abnormal chromosome numbers, which are a major cause of developmental disorders and spontaneous abortions in humans. Meiotic recombination is tightly controlled such that each pair of chromosomes undergoes at least one crossover recombination event despite a low average number of crossovers per chromosome. Recombination is coordinated with the development of specialized, meiosis-specific chromosome structures that stabilize pairing interactions between homologous maternal and paternal chromosomes. We show here that the mouse TRIP13 protein is required for normal execution of many aspects of meiotic recombination and chromosome structure development that it was not previously known to influence. Intriguingly, many of these new roles appear to parallel known functions of a homologous protein from budding yeast, called Pch2. These findings thus indicate that TRIP13/Pch2 functions are more widely conserved throughout evolution than thought before.
doi:10.1371/journal.pgen.1001062
PMCID: PMC2920839  PMID: 20711356
24.  Association of adaptor protein TRIP8b with clathrin 
Journal of neurochemistry  2011;118(6):988-998.
TRIP8b is a brain-specific hydrophilic cytosolic protein that contains tetratricopeptide repeats (TPRs). Previous studies revealed interaction of this protein via its TPR-containing domain with Rab8b small GTPase, HCN channels and G protein-coupled receptor CIRL. We identified clathrin as a major component of eluates from the TRIP8b affinity matrix. In the present study, by in vitro binding analysis we demonstrate a direct interaction between clathrin and TRIP8b. The clathrin-binding site was localized in the N-terminal (non-TPR containing) part of the TRIP8b molecule that contains two short motifs involved in the clathrin binding. In transfected HEK293 cells, co-expression of HCN1 with TRIP8b resulted in translocation of the channels from the cell surface to large intracellular puncta where both TRIP8b and clathrin were concentrated. These puncta co-localized partially with an early endosome marker and strongly overlapped with lysosome staining reagent. When HCN1 was co-expressed with a clathrin-non-binding mutant of TRIP8b, clathrin did not translocate to HCN1 and TRIP8b-containing puncta, suggesting that TRIP8b interacts with HCN and clathrin independently. We found TRIP8b present in the fraction of clathrin-coated vesicles purified from brain tissues. Stripping the clathrin coat proteins from the vesicles with Tris alkaline buffer resulted in concomitant release of TRIP8b. Our data suggest complex regulatory functions of TRIP8b in neuronal endocytosis through independent interaction with membrane proteins and components of the clathrin coat.
doi:10.1111/j.1471-4159.2011.07384.x
PMCID: PMC3166428  PMID: 21749376
TRIP8b; clathrin; CCV; endocytosis; HCN1
25.  TRIP8b splice forms cooperate to regulate the localization and expression of HCN1 channels in CA1 pyramidal neurons 
Neuron  2011;70(3):495-509.
Summary
HCN1 channel subunits, which contribute to the hyperpolarization-activated cation current (Ih), are selectively targeted to distal apical dendrites of hippocampal CA1 pyramidal neurons. Here we addressed the importance of the brain-specific auxiliary subunit of HCN1, TRIP8b, in regulating HCN1 expression and localization. More than ten N-terminal splice variants of TRIP8b exist in brain and exert distinct effects on HCN1 trafficking when overexpressed. We found that isoform-wide disruption of the TRIP8b/HCN1 interaction caused HCN1 to be mistargeted throughout CA1 somatodendritic compartments. In contrast, HCN1 was targeted normally to CA1 distal dendrites in a TRIP8b knockout mouse that selectively lacked exons 1b and 2. Of the two remaining hippocampal TRIP8b isoforms, TRIP8b(1a-4) promoted HCN1 surface expression in dendrites whereas TRIP8b(1a) suppressed HCN1 misexpression in axons. Thus, proper subcellular localization of HCN1 depends on its differential additive and subtractive sculpting by two isoforms of a single auxiliary subunit gene.
doi:10.1016/j.neuron.2011.03.023
PMCID: PMC3107038  PMID: 21555075

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