EGF regulates tyrosine phosphorylation and membrane-translocation of the scaffold protein Tks5

Background

Tks5/FISH is a scaffold protein comprising of five SH3 domains and one PX domain. Tks5 is a substrate of the tyrosine kinase Src and is required for the organization of podosomes/invadopodia implicated in invasion of tumor cells. Recent data have suggested that a close homologue of Tks5, Tks4, is implicated in the EGF signaling.

Results

Here, we report that Tks5 is a component of the EGF signaling pathway. In EGF-treated cells, Tks5 is tyrosine phosphorylated within minutes and the level of phosphorylation is sustained for at least 2 hours. Using specific kinase inhibitors, we demonstrate that tyrosine phosphorylation of Tks5 is catalyzed by Src tyrosine kinase. We show that treatment of cells with EGF results in plasma membrane translocation of Tks5. In addition, treatment of cells with LY294002, an inhibitor of PI 3-kinase, or mutation of the PX domain reduces tyrosine phosphorylation and membrane translocation of Tks5.

Conclusions

Our results identify Tks5 as a novel component of the EGF signaling pathway.

     

CNK1 is a scaffold protein that regulates Src-mediated Raf-1 activation

Raf-1 is a regulator of cellular proliferation, differentiation, and apoptosis. Activation of the Raf-1 kinase activity is tightly regulated and involves targeting to the membrane by Ras and phosphorylation by various kinases, including the tyrosine kinase Src. Here we demonstrate that the connector enhancer of Ksr1, CNK1, mediates Src-dependent tyrosine phosphorylation and activation of Raf-1. CNK1 binds preactivated Raf-1 and activated Src and forms a trimeric complex. CNK1 regulates the activation of Raf-1 by Src in a concentration-dependent manner typical for a scaffold protein. Down-regulation of endogenously expressed CNK1 by small inhibitory RNA interferes with Src-dependent activation of ERK. Thus, CNK1 allows cross-talk between Src and Raf-1 and is essential for the full activation of Raf-1.     

A role for the podosome/invadopodia scaffold protein Tks5 in tumor growth in vivo

Podosomes and invadopodia are electron-dense, actin-rich protrusions located on the ventral side of the cellular membrane. They are detected in various types of normal cells, but also in human cancer cells and in Src-transformed fibroblasts. Previously we have shown that the scaffold protein Tks5 (tyrosine kinase substrate 5) co-localizes to podosomes/invadopodia in different human cancer cells and in Src-transformed NIH-3T3 cells. Upon reduced expression of Tks5 podosome formation is decreased, which leads to diminished gelatin degradation in vitro in various human cancer cell lines. It is unclear, however, whether cancer cells need podosomes for tumor growth and metastasis in vivo. To test this idea, we evaluated the ability of Src-transformed NIH-3T3 cells, showing stable reduced expression of Tks5 and podosome formation (Tks5 KD), to form subcutaneous tumors in mice. We demonstrate that decreased expression of Tks5 correlated with reduced tumor growth at this site. In addition, we generated lung metastases from these cells following tail vein injection. The lungs of mice injected i.v. with the Tks5 KD showed smaller-sized metastases, but there was no difference in the number of lesions compared to the controls, indicating that podosomes may not be required for extravasation from the blood stream into the lung parenchyma. Independent of the microenvironment however, the reduced tumor growth correlated with decreased tumor vascularization. Our data potentially implicate a novel role of podosomes as mediators of tumor angiogenesis and support further exploration of how podosome formation and Tks5 expression contribute to tumor progression.     

BAD Ser-155 phosphorylation regulates BAD/Bcl-XL interaction and cell survival

The BH3 domain of BAD mediates its death-promoting activities via heterodimerization to the Bcl-XL family of death regulators. Growth and survival factors inhibit the death-promoting activity of BAD by stimulating phosphorylation at multiple sites including Ser-112 and Ser-136. Phosphorylation at these sites promotes binding of BAD to 14-3-3 proteins, sequestering BAD away from the mitochondrial membrane where it dimerizes with Bcl-XL to exert its killing effects. We report here that the phosphorylation of BAD at Ser-155 within the BH3 domain is a second phosphorylation-dependent mechanism that inhibits the death-promoting activity of BAD. Protein kinase A, RSK1, and survival factor signaling stimulate phosphorylation of BAD at Ser-155, blocking the binding of BAD to Bcl-XL. RSK1 phosphorylates BAD at both Ser-112 and Ser-155 and rescues BAD-mediated cell death in a manner dependent upon phosphorylation at both sites.     

G-CSFR ubiquitination critically regulates myeloid cell survival and proliferation

The granulocyte colony-stimulating factor receptor (G-CSFR) is a critical regulator of granulopoiesis. Mutations in the G-CSFR in patients with severe congenital neutropenia (SCN) transforming to acute myelogenous leukemia (AML) have been shown to induce hypersensitivity and enhanced growth responses to G-CSF. Recent studies have demonstrated the importance of the ubiquitin/proteasome system in the initiation of negative signaling by the G-CSFR. To further investigate the role of ubiquitination in regulating G-CSFR signaling, we generated a mutant form of the G-CSFR (K762R/G-CSFR) which abrogates the attachment of ubiquitin to the lysine residue at position 762 of the G-CSFR that is deleted in the Delta716 G-CSFR form isolated from patients with SCN/AML. In response to G-CSF, mono-/polyubiquitination of the G-CSFR was impaired in cells expressing the mutant K762R/G-CSFR compared to cells transfected with the WT G-CSFR. Cells stably transfected with the K762R/G-CSFR displayed a higher proliferation rate, increased sensitivity to G-CSF, and enhanced survival following cytokine depletion, similar to previously published data with the Delta716 G-CSFR mutant. Activation of the signaling molecules Stat5 and Akt were also increased in K762R/G-CSFR transfected cells in response to G-CSF, and their activation remained prolonged after G-CSF withdrawal. These results indicate that ubiquitination is required for regulation of G-CSFR-mediated proliferation and cell survival. Mutations that disrupt G-CSFR ubiquitination at lysine 762 induce aberrant receptor signaling and hyperproliferative responses to G-CSF, which may contribute to leukemic transformation.     

Laminin 5 regulates polycystic kidney cell proliferation and cyst formation

Renal cyst formation is the hallmark of autosomal dominant polycystic kidney disease (ADPKD). ADPKD cyst-lining cells have an increased proliferation rate and are surrounded by an abnormal extracellular matrix (ECM). We have previously shown that Laminin 5 (Ln-5, a alpha(3)beta(3)gamma(2) trimer) is aberrantly expressed in the pericystic ECM of ADPKD kidneys. We report that ADPKD cells in primary cultures produce and secrete Ln-5 that is incorporated to the pericystic ECM in an in vitro model of cystogenesis. In monolayers, purified Ln-5 induces ERK activation and proliferation of ADPKD cells, whereas upon epidermal growth factor stimulation blocking endogenously produced Ln-5 with anti-gamma(2) chain antibody reduces the sustained ERK activation and inhibits proliferation. In three-dimensional gel culture, addition of purified Ln-5 stimulates cell proliferation and cyst formation, whereas blocking endogenous Ln-5 strongly inhibits cyst formation. Ligation of alpha(6)beta(4) integrin, a major Ln-5 receptor aberrantly expressed by ADPKD cells, induces beta(4) integrin phosphorylation, ERK activation, cell proliferation, and cyst formation. These findings indicate that Ln-5 is an important regulator of ADPKD cell proliferation and cystogenesis and suggest that Ln-5 gamma(2) chain and Ln-5-alpha(6)beta(4) integrin interaction both contribute to these phenotypic changes.     

Terf/TRIM17 stimulates degradation of kinetochore protein ZWINT and regulates cell proliferation

Terf/TRIM17 is a tripartite motif protein that has been originally isolated from testis. Terf has been characterized to exhibit an E3 ubiquitin ligase activity and to undergo self-ubiquitination. The cellular function of terf and its substrates, however, remain elusive. In the present study, we performed a yeast two-hybrid screening assay using terf as bait and identified a positive clone coding for ZW10 interacting protein (ZWINT), a known component of the kinetochore complex required for the mitotic spindle checkpoint. Immunoprecipitation and western blot analyses showed that terf interacted with ZWINT and that overexpression of terf caused down-regulation of protein levels of ZWINT in mammalian cells. In addition, the coiled-coil domain of terf was required for the interaction with ZWINT. In a cell growth assay, stable transfection with terf decreased proliferation of MCF7 breast cancer cells. In contrast, the growth rate of MCF7 cells was increased by stable expression of ZWINT. Specific siRNAs targeting terf and ZWINT dampened these negative and positive effects of terf and ZWINT on cell proliferation, respectively. These results suggest that the E3 ubiquitin ligase terf causes protein degradation of ZWINT and negatively regulates cell proliferation.     

FAK regulates intestinal epithelial cell survival and proliferation during mucosal wound healing

Background

Following damage to the intestinal epithelium, restoration of epithelial barrier integrity is triggered by a robust proliferative response. In other tissues, focal adhesion kinase (FAK) regulates many of the cellular processes that are critical for epithelial homeostasis and restitution, including cell migration, proliferation and survival. However, few studies to date have determined how FAK contributes to mucosal wound healing in vivo.

Methodology and Principal Findings

To examine the role of FAK in intestinal epithelial homeostasis and during injury, we generated intestinal epithelium (IE)-specific conditional FAK knockout mice. Colitis was induced with dextran-sulfate-sodium (DSS) and intestinal tissues were analyzed by immunohistochemistry and immunoblotting. While intestinal development occurred normally in mice lacking FAK, FAK-deficient animals were profoundly susceptible to colitis. The loss of epithelial FAK resulted in elevated p53 expression and an increased sensitivity to apoptosis, coincident with a failure to upregulate epithelial cell proliferation. FAK has been reported to function as a mechanosensor, inducing cyclin D1 expression and promoting cell cycle progression under conditions in which tissue/matrix stiffness is increased. Collagen deposition, a hallmark of inflammatory injury resulting in increased tissue rigidity, was observed in control and FAK knockout mice during colitis. Despite this fibrotic response, the colonic epithelium in FAK-deficient mice exhibited significantly reduced cyclin D1 expression, suggesting that proliferation is uncoupled from fibrosis in the absence of FAK. In support of this hypothesis, proliferation of Caco-2 cells increased proportionally with matrix stiffness in vitro only under conditions of normal FAK expression; FAK depleted cells exhibited reduced proliferation concomitant with attenuated cyclin D1 expression.

Conclusions

In the colon, FAK functions as a regulator of epithelial cell survival and proliferation under conditions of mucosal injury and a mechanosensor of tissue compliance, inducing repair-driven proliferation in the colonic epithelium through upregulation of cyclin D1.     

The scaffold protein c-Jun NH2-terminal kinase-associated leucine zipper protein regulates cell migration through interaction with the G protein G(alpha 13)

Scaffold proteins for MAP kinase (MAPK) signalling modules play an important role in the specific and efficient signal transduction of the relevant MAPK cascades. Here, we investigated the function of the scaffolding protein c-Jun NH(2)-terminal kinase (JNK)-associated leucine zipper protein (JLP) by depleting it in cultured cells using a short hairpin RNA (shRNA) against human JLP. HeLa and DLD-1 cells stably expressing the shRNA showed a defect in cell migration. The re-expression of full-length shRNA-resistant mouse JLP rescued the impaired cell migration of the JLP-depleted HeLa cells; whereas, a C-terminal deletion mutant of mouse JLP, which failed to bind the G protein G(alpha13), showed little or no effect on the cell migration defect. Furthermore, although a constitutively active G(alpha13) enhanced the migration of control HeLa cells, the G(alpha13)-induced cell migration was significantly suppressed in the JLP-depleted HeLa cells. Taken together, these results suggest that JLP regulates cell migration through an interaction with G(alpha13).     

BEX2与INI1/hSNF5蛋白的相互作用及其在细胞周期中的功能鉴定

BEX2(Brain expressed X-linked protein 2)分子量约为13 kDa,高表达于人的脑和睾丸中。据报道,该蛋白在胚胎发育中表达量变化巨大,提示该蛋白可能在胚胎发育中具有重要作用,但迄今为止,其功能知之甚少。文章应用酵母双杂交系统,以BEX2为"诱饵"蛋白,筛选发现INI1/hSNF5是BEX2的一个结合蛋白。INI1/hSNF5是SWI/SNF染色质重塑复合体的核心组分,是"表观遗传"分子机制中的重要成员。文章通过体外GST Pull-down实验验证,BEX2与INI1/hSNF5之间的相互作是直接并且特异的。通过进一步的缺失突变分析,表明INI1/hSNF5的两个保守的反向重复序列是BEX2的结合区域。该区域是SNF5与多种蛋白相互作用的结构平台。亚细胞定位分析显示BEX2与INI1/hSNF5都主要集中分布于细胞核,这表明二者之间的相互作用可能参与基因表达调控的过程。文章进一步对此相互作用的功能进行了探讨,发现BEX2通过与INI1/hSNF5的相互作用从而影响细胞周期。     

Cdk5 Modulation of mitogen-activated protein kinase signaling regulates neuronal survival

Cdk5, a cyclin-dependent kinase, is critical for neuronal development, neuronal migration, cortical lamination, and survival. Its survival role is based, in part, on "cross-talk" interactions with apoptotic and survival signaling pathways. Previously, we showed that Cdk5 phosphorylation of mitogen-activated protein kinase kinase (MEK)1 inhibits transient activation induced by nerve growth factor (NGF) in PC12 cells. To further explore the nature of this inhibition, we studied the kinetics of NGF activation of extracellular signal-regulated kinase (Erk)1/2 in cortical neurons with or without roscovitine, an inhibitor of Cdk5. NGF alone induced an Erk1/2-transient activation that peaked in 15 min and declined rapidly to baseline. Roscovitine, alone or with NGF, reached peak Erk1/2 activation in 30 min that was sustained for 48 h. Moreover, the sustained Erk1/2 activation induced apoptosis in cortical neurons. Significantly, pharmacological application of the MEK1 inhibitor PD98095 to roscovitine-treated cortical neurons prevented apoptosis. These results were also confirmed by knocking down Cdk5 activity in cortical neurons with Cdk5 small interference RNA. Apoptosis was correlated with a significant shift of phosphorylated tau and neurofilaments from axons to neuronal cell bodies. These results suggest that survival of cortical neurons is also dependent on tight Cdk5 modulation of the mitogen-activated protein kinase signaling pathway.     

Total ERK1/2 activity regulates cell proliferation

Regulating ERK activity is essential for normal cell proliferation to occur. In mammals and most vertebrates ERK activity is provided by ERK1 and ERK2 that are highly similar, ubiquitously expressed and share activators and substrates. By combining single and double silencings of ERK1 and ERK2 we recently demonstrated that the apparent dominant role of ERK2 to regulate cell proliferation was due to its markedly higher expression level than ERK1. The contribution of ERK1 was revealed when ERK2 activation was clamped to avoid compensating over-activation of ERK2. We found no evidences in the literature for insulated isoform-specific modules in the Ras/Raf/MEK signaling cascade that could activate specifically ERK1 or ERK2. Obviously in frogs all signal integration and fine modulation provided by three Ras and three Raf isoforms is conducted by only one MEK and one ERK isoform. In mammals, ERK1 and ERK2 display similar specific activities and are activated respectively to their expression levels. After integrating signals from Ras, Raf and MEK isoforms, ERK1 and ERK2 regulate positively cell proliferation according to their expression levels.     

CH-ILKBP regulates cell survival by facilitating the membrane translocation of protein kinase B/Akt

Cell survival depends on proper propagation of protective signals through intracellular signaling intermediates. We report here that calponin homology domain-containing integrin-linked kinase (ILK)-binding protein (CH-ILKBP), a widely expressed adaptor protein localized at plasma membrane-actin junctions, is essential for transmission of survival signals. Cells that are depleted of CH-ILKBP undergo extensive apoptosis despite the presence of cell-extracellular matrix contacts and soluble growth factors. The activating phosphorylation of protein kinase B (PKB/Akt), a key regulator of apoptosis, is impaired in the absence of CH-ILKBP. Importantly, loss of CH-ILKBP prevents the membrane translocation of PKB/Akt. Furthermore, forced membrane targeting of PKB/Akt bypasses the requirement of CH-ILKBP for the activating phosphorylation of PKB/Akt, suggesting that CH-ILKBP is required for the membrane translocation but not the subsequent phosphorylation of PKB/Akt. Finally, we show that loss of CH-ILKBP is also required for the full activation of extracellular signal-regulated kinase (ERK)1/2. However, restoration of the PKB/Akt activation is sufficient for protection of cells from apoptosis induced by the depletion of CH-ILKBP despite the persistent suppression of the ERK1/2 activation. Thus, CH-ILKBP is an important component of the prosurvival signaling pathway functioning primarily by facilitating the membrane translocation of PKB/Akt and consequently the activation of PKB/Akt in response to extracellular survival signals.     

The Golgi protein GM130 regulates centrosome morphology and function

The Golgi apparatus (GA) of mammalian cells is positioned in the vicinity of the centrosome, the major microtubule organizing center of the cell. The significance of this physical proximity for organelle function and cell cycle progression is only beginning to being understood. We have identified a novel function for the GA protein, GM130, in the regulation of centrosome morphology, position and function during interphase. RNA interference-mediated depletion of GM130 from five human cell lines revealed abnormal interphase centrosomes that were mispositioned and defective with respect to microtubule organization and cell migration. When GM130-depleted cells entered mitosis, they formed multipolar spindles, arrested in metaphase, and died. We also detected aberrant centrosomes during interphase and multipolar spindles during mitosis in ldlG cells, which do not contain detectable GM130. Although GA proteins have been described to regulate mitotic centrosomes and spindle formation, this is the first report of a role for a GA protein in the regulation of centrosomes during interphase.