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Direct interaction of focal adhesion kinase (FAK) with Met is required for FAK to promote hepatocyte growth factor-induced cell invasion.

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.

SUBMITTER: Chen SY

PROVIDER: S-EPMC1489146 | biostudies-literature | 2006 Jul

REPOSITORIES: biostudies-literature

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Direct interaction of focal adhesion kinase (FAK) with Met is required for FAK to promote hepatocyte growth factor-induced cell invasion.

Chen Shu-Yi SY Chen Hong-Chen HC

Molecular and cellular biology 20060701 13

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 ...[more]

PMID: 16782899

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Project description:Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that plays an important role in survival signaling. FAK has been shown to be overexpressed in breast cancer tumors at early stages of tumorigenesis.To study the direct effect of FAK on breast tumorigenesis, we developed Tet-ON (tetracycline-inducible) system of MCF-7 breast cancer cells stably transfected with FAK or dominant-negative, C-terminal domain of FAK (FAK-CD), and also FAKsiRNA with silenced FAK MCF-7 stable cell line. Increased expression of FAK in isogenic Tet-inducible MCF-7 cells caused increased cell growth, adhesion and soft agar colony formation in vitro, while expression of dominant-negative FAK inhibitor caused inhibition of these cellular processes. To study the role of induced FAK and FAK-CD in vivo, we inoculated these Tet-inducible cells in nude mice to generate tumors in the presence or absence of doxycycline in the drinking water. FAKsiRNA-MCF-7 cells were also injected into nude mice to generate xenograft tumors.Induction of FAK resulted in significant increased tumorigenesis, while induced FAK-CD resulted in decreased tumorigenesis. Taq Man Low Density Array assay demonstrated specific induction of FAKmRNA in MCF-7-Tet-ON-FAK cells. DMP1, encoding cyclin D binding myb-like protein 1 was one of the genes specifically affected by Tet-inducible FAK or FAK-CD in breast xenograft tumors. In addition, silencing of FAK in MCF-7 cells with FAK siRNA caused increased cell rounding, decreased cell viability in vitro and inhibited tumorigenesis in vivo. Importantly, Affymetrix microarray gene profiling analysis using Human Genome U133A GeneChips revealed >4300 genes, known to be involved in apoptosis, cell cycle, and adhesion that were significantly down- or up-regulated (p < 0.05) by FAKsiRNA.Thus, these data for the first time demonstrate the direct effect of FAK expression and function on MCF-7 breast cancer tumorigenesis in vivo and reveal specific expression of genes affected by silencing of FAK.

Project description:PurposeRegulation of lens development involves an intricate interplay between growth factor (e.g. FGF and TGFbeta) and extracellular matrix (ECM) signaling pathways. Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that plays key roles in transmitting ECM signals by integrins. In this study, we delineated patterns of FAK expression and tyrosine phosphorylation (Y397) in the developing lens and investigated its regulation by FGF2. We also examined FAK expression and activation during disrupted fiber differentiation in mice expressing a dominant-negative TGFbeta receptor.MethodsFAK expression and activation (phosphorylation on Y397) was studied in embryonic and postnatal rodent lenses by in situ hybridization, immunofluorescence, and western blotting. Rat lens explants were used to investigate the effects of FGF2 on FAK expression and activation. Immunofluorescence and western blotting were used to examine FAK expression and phosphorylation in transgenic mice that express a dominant-negative TGFbeta receptor.ResultsFAK is widely expressed and phosphorylated during embryonic stages of lens morphogenesis and differentiation. However, in postnatal lenses its expression and activation becomes restricted to the posterior germinative zone and the transitional zone at the lens equator. While both NH2- and COOH-terminal antibodies revealed cytoplasmic and membrane-associated staining in lens cells, the NH2-terminal antibody also showed FAK was present in fiber cell nuclei. In vitro, FAK expression and phosphorylation on Y397 were increased by concentrations of FGF2 that initiate lens epithelial cell migration (10 ng/ml) and differentiation (50 ng/ml) but not proliferation (5 ng/ml). Moreover, reactivity for Y397 phosphorylated FAK is prominent in the nuclei of differentiating fibers both in vivo and in vitro. Disruption of TGFbeta-like signals by ectopic expression of a dominant-negative TGFbeta receptor (TbetaRII(D/N)) results in abnormal lens fiber differentiation in transgenic mice. While FAK expression is initiated normally in the posterior germinative zone of TbetaRII(D/N) transgenic lenses, as fiber differentiation proceeds, FAK becomes localized to a perinuclear compartment, decreases its association with the cytoskeleton and is poorly phosphorylated on Y(397).ConclusionsFAK is widely expressed and activated during early lens morphogenesis. During secondary lens fiber differentiation, FAK is expressed and phosphorylated on Y397 as epithelial cells exit the cell cycle, initiate migration at the equator, and undergo differentiation in the transitional zone. During terminal fiber differentiation an NH2-terminal fragment of FAK, including Y397, is translocated to the nucleus. The expression, activation, and nuclear localization of FAK are regulated, at least partly, by FGF2. FAK activity and subcellular localization are also modulated by TGFbeta-like signals. In fiber cells of TbetaRII(D/N) transgenic lenses, FAK is abnormally retained in a perinuclear compartment, loses its association with the cytoskeleton, and is poorly phosphorylated. These data suggest that integrin signaling via FAK plays important roles during lens differentiation, mediated by FGFs and TGFbeta-superfamily signals.

Project description:BackgroundMetastasis is the leading cause of death among patients with colorectal cancer (CRC). Therefore, it is important to explore the molecular mechanisms of metastasis to develop effective therapeutic targets for CRC. In the present study, ribosomal protein L21 (RPL21) was considered as being involved in promoting CRC metastasis, yet the underlying mechanism requires further investigation.MethodsImmunohistochemistry, western blotting, and quantitative reverse transcription polymerase chain reaction were performed to measure the expression of RPL21 and lysosome-associated membrane protein 3 (LAMP3) in CRC tissues and cells. Wound healing, transwell migration, and invasion assays were performed to study the migration and invasion of cultured CRC cells. An orthotopic CRC mouse model was developed to investigate the metastatic ability of CRC. Transcriptome sequencing was conducted to identify the genes related to RPL21. The dual-luciferase reporter gene assay was performed to determine the transcriptional activity of transcription factor EB (TFEB). The GST/His pull-down assay was performed to investigate the specific binding sites of RPL21 and LAMP3. The cell adhesion assay was performed to determine the adhesion ability of CRC cells. Immunofluorescence staining was performed to observe focal adhesions (FAs).ResultsRPL21 was highly expressed in CRC, contributing to tumor invasiveness and poor patient prognosis. Functionally, RPL21 promoted the migration and invasion of CRC cells in vitro and tumor metastasis in vivo. Moreover, LAMP3 was identified as being highly related to RPL21 and was essential in promoting the migration and invasion of CRC cells. Mechanistically, RPL21 activated the transcriptional function of TFEB to upregulate LAMP3 expression. RPL21 directly bound to the aa 341-416 domain of LAMP3 via its aa 1-40 and aa 111-160 segments. The combination of RPL21 and LAMP3 enhanced the stability of the RPL21 protein by suppressing the degradation of the ubiquitin-proteasome system. Furthermore, RPL21 and LAMP3 promoted the formation of immature FAs by activating the FAK/paxillin/ERK signaling pathway.ConclusionsRPL21 promoted invasion and metastasis by regulating FA formation in a LAMP3-dependent manner during CRC progression. The interaction between RPL21 and LAMP3 may function as a potential therapeutic target against CRC.

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Direct interaction of focal adhesion kinase (FAK) with Met is required for FAK to promote hepatocyte growth factor-induced cell invasion.

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Direct interaction of focal adhesion kinase (FAK) with Met is required for FAK to promote hepatocyte growth factor-induced cell invasion.

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