Wanjin Hong

A Role for TAZ in Migration, Invasion, and Tumorigenesis of Breast Cancer Cells

TAZ (WWTR1), identified as a 14-3-3 binding protein with a PDZ binding motif, modulates mesenchymal stem cell differentiation. We now show that TAZ plays a critical role in the migration, invasion, and tumorigenesis of breast cancer cells. TAZ is conspicuously expressed in human breast cancer cell lines in which its expression levels generally correlate with the invasiveness of cancer cells. Overexpression of TAZ in low-expressing MCF10A cells causes morphologic changes characteristic of cell transformation and promotes cell migration and invasion. Conversely, RNA interference-mediated knockdown of TAZ expression in MCF7 and Hs578T cells reduces cell migration and invasion. TAZ knockdown in MCF7 cells also retards anchorage-independent growth in soft agar and tumorigenesis in nude mice. Significantly, TAZ is overexpressed in approximately 20% of breast cancer samples. These results indicate that TAZ plays a role in the migration, invasion, and tumorigenesis of breast cancer cells and thus presents a novel target for the detection and treatment of breast cancer.

SNX3 regulates endosomal function through its PX−domain−mediated interaction with PtdIns(3)P

The sorting nexin (SNX) protein family is implicated in regulating membrane traffic, but the mechanism is still unknown. We show that SNX3 is associated with the early endosome through a novel motif (PX domain) capable of interaction with phosphatidylinositol-3-phosphate (PtdIns(3)P). Overexpression of SNX3 alters endosomal morphology and delays transport to the lysosome. Transport from the early to the recycling endosome is affected upon microinjection of SNX3 antibodies. Our results highlight a novel mechanism by which SNX proteins regulate traffic and uncover a novel class of effectors for PtdIns(3)P.

Hippo pathway-independent restriction of TAZ and YAP by angiomotin.

The Hippo pathway restricts the activity of transcriptional co-activators TAZ and YAP by phosphorylating them for cytoplasmic sequestration or degradation. In this report, we describe an independent mechanism for the cell to restrict the activity of TAZ and YAP through interaction with angiomotin (Amot) and angiomotin-like 1 (AmotL1). Amot and AmotL1 were robustly co-immunoprecipitated with FLAG-tagged TAZ, and their interaction is dependent on the WW domain of TAZ and the PPXY motif in the N terminus of Amot. Amot and AmotL1 also interact with YAP via the first WW domain of YAP. Overexpression of Amot and AmotL1 caused cytoplasmic retention of TAZ and suppressed its transcriptional outcome such as the expression of CTGF and Cyr61. Hippo refractory TAZ mutant (S89A) is also negatively regulated by Amot and AmotL1. HEK293 cells express the highest level of Amot and AmotL1 among nine cell lines examined, and silencing the expression of endogenous Amot increased the expression of CTGF and Cyr61 either at basal levels or upon overexpression of exogenous S89A. These results reveal a novel mechanism to restrict the activity of TAZ and YAP through physical interaction with Amot and AmotL1.

The Hippo Tumor Pathway Promotes TAZ Degradation by Phosphorylating a Phosphodegron and Recruiting the SCFβ-TrCP E3 Ligase

The TAZ transcription co-activator promotes cell proliferation and epithelial-mesenchymal transition. TAZ is inhibited by the Hippo tumor suppressor pathway, which promotes TAZ cytoplasmic localization by phosphorylation. We report here that TAZ protein stability is controlled by a phosphodegron recognized by the F-box protein β-TrCP and ubiquitylated by the SCF/CRL1β-TrCP E3 ligase. The interaction between TAZ and β-TrCP is regulated by the Hippo pathway. Phosphorylation of a phosphodegron in TAZ by LATS primes it for further phosphorylation by CK1ϵ and subsequent binding by β-TrCP. Therefore, the Hippo pathway negatively regulates TAZ function by both limiting its nuclear accumulation and promoting its degradation. The phosphodegron-mediated TAZ degradation plays an important role in negatively regulating TAZ biological functions.

Overexpression of E2F-1 in rat embryo fibroblasts leads to neoplastic transformation.

The transcription factor E2F has been implicated in controlling the activation of multiple genes associated with cell proliferation. E2F‐1, which is a component of E2F, can promote oncogenesis when transfected into REF cells. The transformation caused by E2F‐1 correlates with constitutive overexpression of the transgene, increased transcription of E2F‐dependent genes and the enhancement of two E2F DNA binding complexes containing the retinoblastoma susceptibility gene product (Rb) and E2F‐1. The oncogenic potential of E2F‐1 is dependent on functional DNA binding and transactivation domains but does not require the ability to interact directly with Rb. These findings provide the first direct evidence that sustained unregulated expression of E2F‐1 can lead to the loss of cell proliferation control and that E2F‐1 is a key component in cell cycle control.

Glomerulocystic kidney disease in mice with a targeted inactivation of Wwtr1

Wwtr1 is a widely expressed 14-3-3-binding protein that regulates the activity of several transcription factors involved in development and disease. To elucidate the physiological role of Wwtr1, we generated Wwtr1−/− mice by homologous recombination. Surprisingly, although Wwtr1 is known to regulate the activity of Cbfa1, a transcription factor important for bone development, Wwtr1−/− mice show only minor skeletal defects. However, Wwtr1−/− animals present with renal cysts that lead to end-stage renal disease. Cysts predominantly originate from the dilation of Bowmans spaces and atrophy of glomerular tufts, reminiscent of glomerulocystic kidney disease in humans. A smaller fraction of cysts is derived from tubules, in particular the collecting duct (CD). The corticomedullary accumulation of cysts also shows similarities with nephronophthisis. Cells lining the cysts carry fewer and shorter cilia and the expression of several genes associated with glomerulocystic kidney disease (Ofd1 and Tsc1) or encoding proteins involved in cilia structure and/or function (Tg737, Kif3a, and Dctn5) is decreased in Wwtr1−/− kidneys. The loss of cilia integrity and the down-regulation of Dctn5, Kif3a, Pkhd1 and Ofd1 mRNA expression can be recapitulated in a renal CD epithelial cell line, mIMCD3, by reducing Wwtr1 protein levels using siRNA. Thus, Wwtr1 is critical for the integrity of renal cilia and its absence in mice leads to the development of renal cysts, indicating that Wwtr1 may represent a candidate gene for polycystic kidney disease in humans.

A role for oxysterol-binding protein-related protein 5 in endosomal cholesterol trafficking.

ORP5 works together with Niemann Pick C-1 to facilitate exit of cholesterol from endosomes and lysosomes.

TEADs Mediate Nuclear Retention of TAZ to Promote Oncogenic Transformation

The transcriptional coactivators YAP and TAZ are downstream targets inhibited by the Hippo tumor suppressor pathway. The expression level of TAZ is recently shown to be elevated in invasive breast cancer cells and some primary breast cancers. TAZ is important for breast cancer cell migration, invasion, and tumorigenesis, but the underlying mechanism is not defined. In this study, we show that TAZ interacts with TEAD transcriptional factors. Knockdown of TEADs suppresses TAZ-mediated oncogenic transformation of MCF10A cells. Uncoupling TAZ from Hippo regulation by S89A mutation enhances its transforming ability. Several residues located in the N-terminal region of TAZ are identified to be important for interaction with TEADs, and these same residues are equally important for TAZ to transform MCF10A cells. Mechanistically, TAZ mutants defective in interaction with TEADs fail to accumulate in the nucleus. Live cell imaging of enhanced green fluorescent protein-TAZ and its mutant defective in TEAD interaction suggests that TEAD interaction mediates nuclear retention. These results reveal a novel mechanism for TEADs to regulate nuclear retention and thus the transforming ability of TAZ.

New sorting nexin (SNX27) and NHERF specifically interact with the 5-HT4(a) receptor splice variant: roles in receptor targeting

The 5-hydroxytryptamine type 4 receptor (5-HT4R) is involved in learning, feeding, respiratory control and gastrointestinal transit. This receptor is one of the G-protein-coupled receptors for which alternative mRNA splicing generates the most variants that differ in their C-terminal extremities. Some 5-HT4R variants (a, e and f) express canonical PDZ ligands at their C-termini. Here, we have examined whether some mouse 5-HT4R variants associate with specific sets of proteins, using a proteomic approach based on peptide-affinity chromatography, two-dimensional electrophoresis and mass spectrometry. We have identified ten proteins that interact specifically with the 5-HT4(a)R and three that only associate with the 5-HT4(e)R. Most of them are PDZ proteins. Among the proteins that associated specifically with the 5-HT4(a)R variant, NHERF greatly modified its subcellular localization. Moreover, NHERF recruited the 5-HT4(a)R to microvilli, where it localized with activated ezrin, consistent with the role of 5-HT4(a)R in cytoskeleton remodelling. The 5-HT4(a)R also interacted with both the constitutive and inducible (upon methamphetamine treatment) forms of the recently cloned sorting nexin 27 (SNX27a and b, respectively). We found that SNX27a redirected part of 5-HT4(a)R to early endosomes. The interaction of the 5-HT4R splice variants with distinct sets of PDZ proteins might specify their cellular localization as well as their signal transduction properties.

The mammalian homolog of yeast Sec13p is enriched in the intermediate compartment and is essential for protein transport from the endoplasmic reticulum to the Golgi apparatus.

The role of COPII components in endoplasmic reticulum (ER)-Golgi transport, first identified in the yeast Saccharomyces cerevisiae, has yet to be fully characterized in higher eukaryotes. A human cDNA whose predicted amino acid sequence showed 70% similarity to the yeast Sec13p has previously been cloned. Antibodies raised against the human SEC13 protein (mSEC13) recognized a cellular protein of 35 kDa in both the soluble and membrane fractions. Like the yeast Sec13p, mSEC13 exist in the cytosol in both monomeric and higher-molecular-weight forms. Immunofluorescence microscopy localized mSEC13 to the characteristic spotty ER-Golgi intermediate compartment (ERGIC) in cells of all species examined, where it colocalized well with the KDEL receptor, an ERGIC marker, at 15 degrees C. Immunoelectron microscopy also localized mSEC13 to membrane structures close to the Golgi apparatus. mSEC13 is essential for ER-to-Golgi transport, since both the His6-tagged mSEC13 recombinant protein and the affinity-purified mSEC13 antibody inhibited the transport of restrictive temperature-arrested vesicular stomatitis virus G protein from the ER to the Golgi apparatus in a semi-intact cell assay. Moreover, cytosol immunodepleted of mSEC13 could no longer support ER-Golgi transport. Transport could be restored in a dose-dependent manner by a cytosol fraction enriched in the high-molecular-weight mSEC13 complex but not by a fraction enriched in either monomeric mSEC13 or recombinant mSEC13. As a putative component of the mammalian COPII complex, mSEC13 showed partially overlapping but mostly different properties in terms of localization, membrane recruitment, and dynamics compared to that of beta-COP, a component of the COPI complex.