Phosphorylation of E-cadherin at threonine 790 by protein kinase Cδ reduces ß-catenin binding and suppresses the function of E-cadherin.

Proper control of cell-cell adhesion is crucial for embryogenesis and tissue homeostasis. In this study, we show that protein kinase C (PKC)δ, a member of the novel PKC subfamily, localizes at cell-cell contacts of epithelial cells through its C2-like domain in an F-actin-dependent manner. Upon hepatocyte growth factor stimulation, PKCδ is phosphorylated and activated by Src, which then phosphorylates E-cadherin at Thr790. Phosphorylation of E-cadherin at Thr790 diminishes its interaction with ß-catenin and impairs the homophilic interaction between the ectodomains of E-cadherin. The suppression of PKCδ by its dominant-negative mutants or specific short-hairpin RNA inhibits the disruption of cell-cell adhesions induced by hepatocyte growth factor. Elevated PKCδ expression in cancer cells is correlated with increased phosphorylation of E-cadherin at Thr790, reduced binding of E-cadherin to ß-catenin, and poor homophilic interaction between E-cadherin. Analysis of surgical specimens confirmed that PKCδ is overexpressed in cervical cancer tissues, accompanied by increased phosphorylation of E-cadherin at Thr790. Together, our findings unveil a negative role for PKCδ in cell-cell adhesion through phosphorylation of E-cadherin.

Gab1 is essential for membrane translocation, activity and integrity of mTORCs after EGF stimulation in urothelial cell carcinoma.

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.

Adducin-1 is essential for mitotic spindle assembly through its interaction with myosin-X.

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.

p120RasGAP-mediated activation of c-Src is critical for oncogenic Ras to induce tumor invasion.

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.

Elevated expression of protein kinase C delta induces cell scattering upon serum deprivation.

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.

Crosstalk between hepatocyte growth factor and integrin signaling pathways.

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.

Blockade of v-Src-stimulated tumor formation by the Src homology 3 domain of Crk-associated substrate (Cas).

Crk-associated substrate (Cas) is highly phosphorylated by v-Src and plays a critical role in v-Src-induced cell transformation. In this study, we found that the Src homology (SH) 3 domain of Cas blocked v-Src-stimulated anchorage-independent cell growth, Matrigel invasion, and tumor growth in nude mice. Biochemical analysis revealed that the Cas SH3 domain selectively inhibited v-Src-stimulated activations of AKT and JNK, but not ERK and STAT3. Attenuation of the AKT pathway by the Cas SH3 domain rendered v-Src-transformed cells susceptible to apoptosis. Inhibition of the JNK pathway by the Cas SH3 domain led to suppression of v-Src-stimulated invasion. Taken together, our results indicate that the Cas SH3 domain has an anti-tumor function, which severely impairs the transforming potential of v-Src.

Src phosphorylates Grb2-associated binder 1 upon hepatocyte growth factor stimulation.

Grb2-associated binder 1 (Gab1) is known to play an important role in hepatocyte growth factor (HGF) signaling, which rapidly becomes tyrosine-phosphorylated upon HGF stimulation. In this study, we found that the tyrosine phosphorylation of Gab1 in the cells derived from Src/Yes/Fyn null mouse embryos was approximately 40% lower than that in their wild type counterparts upon HGF stimulation. Increased expression of wild-type Src enhanced HGF-induced phosphorylation of Gab1, and, in contrast, expression of the Src kinase-deficient mutant or treatment of the specific Src inhibitor PP1 suppressed it. Expression of a constitutively active Src mutant (Y527F) or oncogenic v-Src led to a prominent increase in Gab1 phosphorylation independent of HGF stimulation. Moreover, Src interacted with Gab1 via both its Src homology 2 and 3 domains and was capable of phosphorylating purified Gab1 in vitro. Finally, the increased phosphorylation of Gab1 by Src selectively potentiated HGF-induced activation of ERK and AKT. Taken together, our results establish a new role for Src in HGF-induced Gab1 phosphorylation.

Synergistic effect of focal adhesion kinase overexpression and hepatocyte growth factor stimulation on cell transformation.

Although an elevated level of focal adhesion kinase (FAK) has been observed in a variety of invasive human tumors, forced expression of FAK alone in cultured cells does not cause them to exhibit transformed phenotypes. Therefore, the role of FAK in oncogenic transformation remains unclear. In this study, we have demonstrated that FAK overexpression in Madin-Darby canine kidney epithelial cells rendered them susceptible to transformation by hepatocyte growth factor (HGF). Using various FAK mutants, we found that the simultaneous bindings of Src and p130(cas) were required for FAK to potentiate cell transformation. Expression of FAK-related nonkinase, kinase-deficient Src, or the Src homology 3 domain of p130(cas), which respectively serve as dominant negative versions of FAK, Src, and p130(cas), apparently reversed the transformed phenotypes of FAK-overexpressed cells upon HGF stimulation. Moreover, FAK overexpression was able to enhance HGF-elicited signals, leading to sustained activation of ERK, JNK, and AKT, which could be prevented by the expression of the Src homology 3 domain of p130(cas). Taken together, our results indicate that the synergistic effect of FAK overexpression and HGF stimulation leads to cell transformation and implicate a critical role of p130(cas) in this process.