[Show abstract][Hide abstract] ABSTRACT: Urothelial carcinoma is the most common type of malignancy in long-term dialysis patients and kidney transplant recipients in Taiwan. mTORCs (mammalian target of rapamycin complexes) and EGF are important in urothelial carcinoma. To identify the regulation of mTORCs upon EGF stimulation is necessary. mTOR integrates signals from growth factors via mTOR Complex 1 (mTORC1) and mTOR Complex 2 (mTORC2). The mechanism of mTORC1 action has been widely studied; however, the regulation of mTORC2 has not been well studied. Here, we demonstrate that Gab1 is an important upstream regulator in EGF-mediated activation of mTORCs. In our study, we confirm that mTORCs translocate from the cytoplasm to the plasma membrane via the PH domain of Gab1 upon EGF stimulation. Moreover, Gab1 associates with mTORCs. This association stabilizes the integrity of mTORCs and induces mTORC activity. Compared to normal bladder tissue, the expression of Gab1 and activity of mTORCs are elevated in urothelial carcinoma. Collectively, our results suggest that Gab1 is an essential regulator of the EGF-mediated mTORC pathways and may potentially be used as a biomarker for urothelial carcinoma to predict diagnosis and drug response.
[Show abstract][Hide abstract] ABSTRACT: Mitotic spindles are microtubule-based structures, but increasing evidence indicates that filamentous actin (F-actin) and F-actin-based motors are components of these structures. ADD1 (adducin-1) is an actin-binding protein that has been shown to play important roles in the stabilization of the membrane cortical cytoskeleton and cell-cell adhesions. In this study, we show that ADD1 associates with mitotic spindles and is crucial for proper spindle assembly and mitotic progression. Phosphorylation of ADD1 at Ser12 and Ser355 by cyclin-dependent kinase 1 enables ADD1 to bind to myosin-X (Myo10) and therefore to associate with mitotic spindles. ADD1 depletion resulted in distorted, elongated, and multipolar spindles, accompanied by aberrant chromosomal alignment. Remarkably, the mitotic defects caused by ADD1 depletion were rescued by reexpression of ADD1 but not of an ADD1 mutant defective in Myo10 binding. Together, our findings unveil a novel function for ADD1 in mitotic spindle assembly through its interaction with Myo10.
The Journal of Cell Biology 12/2013; 204(1). DOI:10.1083/jcb.201306083 · 9.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Podosomes are actin-based membrane protrusions that facilitate extracellular matrix degradation and invasive cell motility. Podosomes can self-organize into large rosette-like structures in Src-transformed fibroblasts, osteoclasts, and some highly invasive cancer cells. However, the mechanism of this assembly remains obscure. In this study, we show that the suppression of c-Jun N-terminal kinase (JNK) by the JNK inhibitor SP600125 or short-hairpin RNA inhibited podosome rosette formation in SrcY527F-transformed NIH3T3 fibroblasts. In addition, SrcY527F was less potent to induce podosome rosettes in JNK1-null or JNK2-null mouse embryo fibroblasts than in their wild-type counterparts. The kinase activity of JNK was essential for promoting podosome rosette formation but not for its localization to podosome rosettes. Moesin, a member of the ERM (ezrin, radixin, and moesin) protein family, was identified as a substrate of JNK. We show that the phosphorylation of moesin at Thr558 by JNK was important for podosome rosette formation in SrcY527F-transformed NIH3T3 fibroblasts. Taken together, our results unveil a novel role of JNK in podosome rosette formation by phosphorylating moesin.
[Show abstract][Hide abstract] ABSTRACT: Oncofetal protein insulin-like growth factor II mRNA-binding protein 1 (IMP1) regulates cellular proliferation and migration. Expression of IMP1 is limited to a few adult human tissues. However, it commonly expresses in a variety of cancers. Our objective was to study the regulatory mechanism of IMP1 on the cellular functions of choriocarcinoma (CC) JAR cells.
IMP1 protein levels were measured in CC tissues via immunohistochemistry. Specific siRNAs were used to down-regulate gene expressions. The abilities of migration and invasion were estimated by wound-healing and Matrigel chamber assays. The profile of IMP1-binding genes was investigated with an Agilent microarray. RT-qPCR, RNA immunoprecipitation, and IMP1 rescue experiments were performed to confirm the association between IMP1 and its binding genes. Gene expression was further analyzed by using RT-PCR and Western blotting.
Strong IMP1 expressions were frequently detected in CC tissues. Knockdown of IMP1 expression in JAR cells inhibited cell migration and invasion, but did not affect cellular proliferation and morphology. Microarray and RNA-immunoprecipitation results revealed several candidate genes regulated by IMP1. Among them, ribosomal protein S6 kinase (RSK2) and protein phosphatase methylesterase 1 (PPME1) were confirmed to be down-regulated in IMP1-depleted JAR cells. Re-expression of IMP1 into the cells restored the expressions of RSK2 and PPME1. Furthermore, the depletion of RSK2 or PPME1 decreased the migration and invasion of JAR cells.
Our results suggest that IMP1 plays an essential role in the regulation of migration and invasion of human CC cells, possibly through the novel effectors RSK2 and PPME1.
[Show abstract][Hide abstract] ABSTRACT: Podosomes are actin-enriched membrane protrusions that play important roles in extracellular matrix degradation and invasive cell motility. Podosomes undergo self-assembly into large rosette-like structures in Src-transformed fibroblasts, osteoclasts, and certain highly invasive cancer cells. Several protein tyrosine kinases have been shown to be important for the formation of podosome rosettes, but little is known regarding the role of protein tyrosine phosphatases in this process. We found that knockdown of the Src homolog domain-containing phosphatase 2 (SHP2) significantly increased podosome rosette formation in Src-transformed fibroblasts. In contrast, SHP2 overexpression suppressed podosome rosette formation in these cells. The phosphatase activity of SHP2 was essential for the suppression of podosome rosette formation. SHP2 selectively suppressed the tyrosine phosphorylation of Tks5, a scaffolding protein required for podosome formation. The inhibitory effect of SHP2 on podosome rosette formation was associated with the increased activation of Rho-associated kinase (ROCK) and the enhanced polymerization of vimentin filaments. A higher content of polymerized vimentin filaments was correlated with a lower content of podosome rosettes. Taken together, our findings indicate that SHP2 serves as a negative regulator of podosome rosette formation through the dephosphorylation of Tks5 and the activation of ROCK-mediated polymerization of vimentin in Src-transformed fibroblasts.
[Show abstract][Hide abstract] ABSTRACT: Ras genes are the most common targets for somatic gain-of-function mutations in human cancers. In this study, we found a high incidence of correlation between Ras oncogenic mutations and c-Src activation in human cancer cells. We showed that oncogenic Ras induces c-Src activation mainly on the Golgi complex and endoplasmic reticulum. Moreover, we identified p120RasGAP as an effector for oncogenic Ras to activate c-Src. The recruitment of p120RasGAP to the Golgi complex by oncogenic Ras facilitated its interaction with c-Src, thereby leading to c-Src activation, and this p120RasGAP-mediated activation of c-Src was important for tumor invasion induced by oncogenic Ras. Collectively, our findings unveil a relationship between oncogenic Ras, p120RasGAP, and c-Src, suggesting a critical role for c-Src in cancers evoked by oncogenic mutations in Ras genes.
Cancer Research 03/2012; 72(9):2405-15. DOI:10.1158/0008-5472.CAN-11-3078 · 9.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cell adhesion has been shown to induce activation of certain growth factor receptors in a ligand-independent manner. However, the mechanism for such activation remains obscure.
Human epidermal carcinoma A431 cells were used as a model to examine the mechanism for adhesion-induced activation of hepatocyte growth factor receptor Met and epidermal growth factor receptor (EGFR). The cells were suspended and replated on culture dishes under various conditions. The phosphorylation of Met at Y1234/1235 and EGFR at Y1173 were used as indicators for their activation. The distribution of the receptors and lipid rafts on the plasma membrane were visualized by confocal fluorescent microscopy and total internal reflection microscopy.
We demonstrate that Met and EGFR are constitutively activated in A431 cells, which confers proliferative and invasive potentials to the cells. The ligand-independent activation of Met and EGFR in A431 cells relies on cell adhesion to a substratum, but is independent of cell spreading, extracellular matrix proteins, and substratum stiffness. This adhesion-induced activation of Met and EGFR cannot be attributed to Src activation, production of reactive oxygen species, and the integrity of the cytoskeleton. In addition, we demonstrate that Met and EGFR are independently activated upon cell adhesion. However, partial depletion of Met and EGFR prevents their activation upon cell adhesion, suggesting that overexpression of the receptors is a prerequisite for their self-activation upon cell adhesion. Although Met and EGFR are largely distributed in 0.04% Triton-insoluble fractions (i.e. raft fraction), their activated forms are detected mainly in 0.04% Triton-soluble fractions (i.e. non-raft fraction). Upon cell adhesion, lipid rafts are accumulated at the cell surface close to the cell-substratum interface, while Met and EGFR are mostly excluded from the membrane enriched by lipid rafts.
Our results suggest for the first time that cell adhesion to a substratum may induce a polarized distribution of lipid rafts to the cell-substratum interface, which may allow Met and EGFR to be released from lipid rafts, thus leading to their activation in a ligand-independent manner.
[Show abstract][Hide abstract] ABSTRACT: Podosomes are dynamic actin-enriched membrane structures that play an important role in invasive cell motility and extracellular matrix degradation. They are often found to assemble into large rosettelike structures in highly invasive cells. However, the mechanism of this assembly remains obscure. In this study, we identified focal adhesion kinase (FAK) as a key molecule necessary for assembly. Moreover, phosphorylation of p130Cas and suppression of Rho signaling by FAK were found to be important for FAK to induce the assembly of podosome rosettes. Finally, we found that suppression of vimentin intermediate filaments by FAK facilitates the assembly of podosome rosettes. Collectively, our results strongly suggest a link between FAK, podosome rosettes, and tumor invasion and unveil a negative role for Rho signaling and vimentin filaments in podosome rosette assembly.
The Journal of Cell Biology 10/2011; 195(1):113-29. DOI:10.1083/jcb.201103016 · 9.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The F-actin binding protein adducin plays an important role in plasma membrane stability, cell motility and cell-cell junctions. In this study, we demonstrate that α-adducin is mainly localized in the nucleus of sparsely cultured epithelial cells, whereas it is localized at cell-cell junctions when the cells are grown to confluence. Disruption of cell-cell adhesions induces a nuclear translocation of α-adducin. Conversely, α-adducin is redistributed to the cytoplasm and cell-cell junctions in the process of establishing cell-cell adhesions. We identify that α-adducin contains a bipartite nuclear localization signal (NLS) in its COOH-terminal tail domain and a nuclear export signal in its neck region. The phosphorylation of α-adducin at Ser716 that is immediately adjacent to the NLS appears to antagonize the function of the NLS. Moreover, we show that depletion of α-adducin has adverse effects on cell-cell adhesions and, to our surprise, cell proliferation. The impaired cell proliferation is associated with mitotic defects characterized by disorganized mitotic spindles, aberrant chromosomal congregation/segregation and abnormal centrosomes. Taken together, our results not only reveal the mechanism for α-adducin to shuttle between the cytoplasm and nucleus, but also highlight a potential role for α-adducin in mitosis.
[Show abstract][Hide abstract] ABSTRACT: Tumor metastasis might be evoked in response to microenvironmental stress, such as a shortage of oxygen. Although the cellular response to hypoxia has been well established, we know little about how tumors adapt themselves to deprivation of growth factor. Protein kinase Cdelta (PKCdelta), a stress-sensitive protein kinase, has been implicated in tumor progression. In this study, we demonstrate that elevated expression of PKCdelta in Madin-Darby canine kidney cells induces a scatter response upon serum starvation, a condition that mimics growth-factor deprivation. Serum starvation stimulates the catalytic activity and Y311 phosphorylation of PKCdelta through reactive oxygen species (ROS) and the Src family kinases. Mutation of PKCdelta at Y311 and Y322, both of which are phosphorylation sites for Src, impairs its activation and ability to promote cell scattering upon serum deprivation. Once activated by ROS, PKCdelta itself activates ROS production at least partially through NADPH oxidase. In addition, the c-Jun N-terminal kinase is identified as a crucial downstream mediator of ROS and PKCdelta for induction of cell scattering upon serum deprivation. We demonstrate that the C1B domain of PKCdelta is essential not only for its localization at the Golgi complex, but also for its activation and ability to induce cell scattering upon serum deprivation. Finally, depletion of PKCdelta in human bladder carcinoma T24 cells restores their cell-cell contacts, which thereby reverses a scattered growth pattern to an epithelial-like growth pattern. Collectively, our results suggest that elevated expression of PKCdelta might facilitate the scattering of cells in order to escape stress induced by growth-factor deprivation.
[Show abstract][Hide abstract] ABSTRACT: Protein kinase C (PKC) delta, a member of the novel PKC subfamily, has been shown to have an important role in cell proliferation, differentiation, apoptosis and cell motility. In this study, we investigated the effect of green fluorescent protein (GFP)-PKCdelta and GFP-PKCalpha on cell-cell junctions of Madin-Darby canine kidney (MDCK) cells and found that only GFP-PKCdelta suppressed the homophilic interactions between the ectodomains of E-cadherins, accompanied by a weaker cell-cell adhesion. The kinase-deficient mutant of GFP-PKCdelta retained its localization at cell-cell junctions but failed to suppress the function of E-cadherin. In addition, we demonstrated that the hinge region (residues 280-347) that links the regulatory domain and the catalytic domain of PKCdelta is essential for both its kinase activity and the targeting of cell-cell junctions. A PKCdelta mutant with the deletion of amino acids 280-323 within the hinge region, which is catalytically active but defective in the targeting of cell-cell junctions, failed to suppress the function of E-cadherin. Moreover, expression of GFP-PKCdelta in MDCK cells expedited the detachment of cells from their neighbors and facilitated cell scatter induced by hepatocyte growth factor. By contrast, the GFP-PKCdelta mutants including the kinase-deficient mutant and the truncated mutant lacking residues 280-323 suppressed hepatocyte-growth-factor-induced cell scattering. Finally, siRNA-mediated knockdown of endogenous PKCdelta in MDCK cells was found to delay the onset of cell-cell detachment and cell scattering induced by hepatocyte growth factor. Taken together, our results demonstrate that the catalytic activity of PKCdelta and its localization to cell-cell junctions are necessary for PKCdelta to suppress the function of E-cadherin, which thereby facilitates scattering of epithelial cells in response to extracellular cues.
[Show abstract][Hide abstract] ABSTRACT: We have identified a novel protein, protein phosphatase 1 F-actin cytoskeleton targeting subunit (phostensin). This protein is encoded by KIAA1949 and was found to associate with protein phosphatase 1 (PP1) in the yeast two-hybrid assay, co-immunoprecipitation, and GST pull-down assay. Northern blot analysis revealed that phostensin mRNA was predominantly distributed in leukocytes and spleen, and phostensin protein was present in crude extracts of human peripheral leukocytes. Immunofluorescence microscopic analysis revealed that the phostensin/PP1 complex was conspicuously localized with the actin cytoskeleton at the cell periphery in Madin-Darby canine kidney (MDCK) epithelial cells. Taken together, our data shows that phostensin targets PP1 to F-actin cytoskeleton. The phostensin/PP1 complex may play a vital role in modulation of actin rearrangements.
Biochemical and Biophysical Research Communications 06/2007; 356(3):594-8. DOI:10.1016/j.bbrc.2007.03.026 · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Protein kinase Cdelta (PKCdelta) has been implicated to play a crucial role in cell proliferation, differentiation and apoptosis. In this study, we have investigated the role of PKCdelta in cell motility using Madin-Darby canine kidney cells. Overexpression of PKCdelta promoted membrane protrusions, concomitant with increased cell motility. By contrast, suppression of PKCdelta expression by RNA interference inhibited cell motility. Moreover, a fraction of PKCdelta was detected at the edge of membrane protrusions in which it colocalized with adducin, a membrane skeletal protein whose phosphorylation state is important for remodeling of the cortical actin cytoskeleton. Elevated expression of PKCdelta correlated with increased phosphorylation of adducin at Ser726 in intact cells. In vitro, PKCdelta, but not PKCalpha, directly phosphorylated the Ser726 of adducin. Finally, we demonstrated that overexpression of both adducin and PKCdelta could generate a synergistic effect on promoting cell spreading and cell migration. Our results support a positive role for PKCdelta in cell motility and strongly suggest a link between PKCdelta activity, adducin phosphorylation and cell motility.
[Show abstract][Hide abstract] ABSTRACT: Over-expression of hydantoinase from Agrobacterium radiobacter NRRL B11291 (HDTar) results in the formation of insoluble aggregates in Escherichia coli. As previously reported, recombinant HDTar could be obtained in a homogeneous form using one chromatographic step. However, soluble proteins are required for the pre-treatment in several steps before proceeding to the chromatographic purification step. In this study, we reported a method based on artificial oil bodies (AOBs) to obtain homologous HDTar from its insoluble form in one step. By linkage of HDTar to intein-oleosin gene fusion, the tripartite fusion protein was over-expressed as aggregates in E. coli. Upon sonication, the mixture comprising plant oil and the insoluble fusion protein was readily assembled into AOBs. Further induction for peptide cleavage mediated by intein, the bound HDTar was liberated from AOBs, and the protein free of fusion tags was then recovered. As a result, refolded HDTar was amplified by over 300-fold. Obviously, this simplified method provides an efficient way to obtain HDTar with high yield and high purity.
Protein Expression and Purification 04/2007; 52(1):14-8. DOI:10.1016/j.pep.2006.07.008 · 1.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aristolochic acid (AA) has been demonstrated to play a causal role in Chinese herbs nephropathy. However, the detailed mechanism for AA to induce apoptosis of renal tubular cells remains obscure. In this study, we show that AA evokes a rapid rise in the intracellular Ca(2+) concentration of renal tubular cells through release of intracellular endoplasmic reticulum Ca(2+) stores and influx of extracellular Ca(2+), which in turn causes endoplasmic reticulum stress and mitochondria stress, resulting in activation of caspases and finally apoptosis. Ca(2+) antagonists, including calbindin-D(28k) (an intracellular Ca(2+) buffering protein) and BAPTA-AM (a cell-permeable Ca(2+) chelator), are capable of ameliorating endoplasmic reticulum stress and mitochondria stress, and thereby enhance the resistance of the cells to AA. Moreover, we show that overexpression of the anti-apoptotic protein Bcl-2 in combination with BAPTA-AM treatment can provide renal tubular cells with almost full protection against AA-induced cytotoxicity. In conclusion, our results demonstrate an impact of AA to intracellular Ca(2+) concentration and its link with AA-induced cytotoxicity.
[Show abstract][Hide abstract] ABSTRACT: Overproduction of heterologous proteins in Escherichia coli commonly results in the formation of insoluble aggregates. Prior to the purification and immobilization of insoluble aggregates, an indispensable need calls for their renaturation. However, this process is usually laborious, inefficient, and costly. In this study, a new method based on artificial oil bodies (AOBs) was developed to complete protein refolding and immobilization in one step. To illustrate the proposed method, the d-hydantoinase gene fused to the C terminus of oleosin, a storage protein of plant oil body, was constructed and overexpressed in E. coli. As a result, the hybrid protein was largely produced in the form of inclusion bodies, and the protein yield reached 30% of total cell protein content. Without the use of denaturant, the mixture containing the insoluble fusion protein and plant oil was subjected to sonication to form AOBs. Subsequent analysis by the protease accessibility and enzyme activity assay confirmed the presence of active d-hydantoinase on the surfaces of AOBs. In comparison with its free counterpart, the immobilized d-hydantoinase exhibited higher tolerance to heat. Furthermore, the immobilized enzyme could be reused for seven cycles to give a conversion yield exceeding 80% when applied in a bioconversion process. It clearly indicates that the AOBs-based system is featured with simplicity as well as efficiency and is practically useful for direct immobilization of enzymes with low solubility.
[Show abstract][Hide abstract] ABSTRACT: Focal adhesion kinase (FAK) has been implicated to be a point of convergence of integrin and growth factor signaling pathways. Here we report that FAK directly interacts with the hepatocyte growth factor receptor c-Met. Phosphorylation of c-Met at Tyr-1349 and, to a lesser extent, Tyr-1356 is required for its interaction with the band 4.1 and ezrin/radixin/moesin homology domain (FERM domain) of FAK. The F2 subdomain of the FAK FERM domain alone is sufficient for Met binding, in which a patch of basic residues (216KAKTLRK222) are critical for the interaction. Met-FAK interaction leads to FAK activation and subsequent contribution to hepatocyte growth factor-induced cell motility and cell invasion. Our results provide evidence that constitutive Met-FAK interaction may be a critical determinant for tumor cells to acquire invasive potential.
[Show abstract][Hide abstract] ABSTRACT: Most types of normal cells require integrin-mediated attachment to extracellular matrix to be able to respond to growth factor stimulation for proliferation and survival. Therefore, a consensus that integrins are close collaborators with growth factors in signal transduction has gradually emerged. Some integrins and growth factor receptors appear to be normally in relatively close proximity, which can be induced to form complexes upon cell adhesion or growth factor stimulation. Moreover, since integrins and growth factor receptors share many common elements in their signaling pathways, it is clear tzhat there are many opportunities for integrin signals to modulate growth factor signals and vice versa. Increasing evidence indicates that integrins can crosstalk with receptor tyrosine kinases in a cell- and integrin-type-dependent manner through a variety of specific mechanisms. This review is intended specifically for summarizing recent progress uncovering how the hepatocyte growth factor receptor c-Met coordinates with integrins to transmit signals.
[Show abstract][Hide abstract] ABSTRACT: Nattokinase, a serine protease, and pronattokinase, when expressed in Escherichia coli, formed insoluble aggregates without enzymatic activity. For functional expression and purification, nattokinase or pronattokinase was first overexpressed in E. coli as an insoluble recombinant protein linked to the C terminus of oleosin, a structural protein of seed oil bodies, by an intein fragment. Artificial oil bodies were reconstituted with triacylglycerol, phospholipid, and the insoluble recombinant protein thus formed. Soluble nattokinase was subsequently released through self-splicing of intein induced by temperature alteration, with the remaining oleosin-intein residing in oil bodies and the leading propeptide of pronattokinase, when present, spontaneously cleaved in the process. Active nattokinase with fibrinolytic activity was harvested by concentrating the supernatant. Nattokinase released from oleosin-intein-pronattokinase exhibited 5 times higher activity than that released from oleosin-intein-nattokinase, although the production yields were similar in both cases. Furthermore, active nattokinase could be harvested in the same system by fusing pronattokinase to the N terminus of oleosin via a different intein linker, with self-splicing induced by 1,4-dithiothreitol. These results have shown a great potential of this system for bacterial expression and purification of functional recombinant proteins.
Journal of Agricultural and Food Chemistry 07/2005; 53(12):4799-804. DOI:10.1021/jf050264a · 2.91 Impact Factor