Christof R. Hauck

Signaling through focal adhesion kinase.

Integrin receptor binding to extracellular matrix proteins generates intracellular signals via enhanced tyrosine phosphorylation events that are important for cell growth, survival, and migration. This review will focus on the functions of the focal adhesion kinase (FAK) protein-tyrosine kinase (PTK) and its role in linking integrin receptors to intracellular signaling pathways. FAK associates with several different signaling proteins such as Src-family PTKs, p130Cas, Shc, Grb2, PI 3-kinase, and paxillin. This enables FAK to function within a network of integrin-stimulated signaling pathways leading to the activation of targets such as the ERK and JNK/mitogen-activated protein kinase pathways. Focus will be placed on the structural domains and sites of FAK tyrosine phosphorylation important for FAK-mediated signaling events and how these sites are conserved in the FAK-related PTK, Pyk2. We will review what is known about FAK activation by integrin receptor-mediated events and also non-integrin stimuli. In addition, we discuss the emergence of a consensus FAK substrate phosphorylation sequence. Emphasis will also be placed on the role of FAK in generating cell survival signals and the cleavage of FAK during caspase-mediated apoptosis. An in-depth discussion will be presented of integrin-stimulated signaling events occurring in the FAK knockout fibroblasts (FAK-) and how these cells exhibit deficits in cell migration. FAK re-expression in the FAK- cells confirms the role of this PTK in the regulation of cell morphology and in promoting cell migration events. In addition, these results reinforce the potential role for FAK in promoting an invasive phenotype in human tumors.

FAK integrates growth-factor and integrin signals to promote cell migration

Here we show that cells lacking focal adhesion kinase (FAK) are refractory to motility signals from platelet-derived and epidermal growth factors (PDGF and EGF respectively), and that stable re-expression of FAK rescues these defects. FAK associates with activated PDGF- and EGF-receptor (PDGFR and EGFR) signalling complexes, and expression of the band-4.1-like domain at the FAK amino terminus is sufficient to mediate an interaction with activated EGFR. However, efficient EGF-stimulated cell migration also requires FAK to be targeted, by its carboxy-terminal domain, to sites of integrin-receptor clustering. Although the kinase activity of FAK is not needed to promote PDGF- or EGF-stimulated cell motility, kinase-inactive FAK is transphosphorylated at the indispensable Src-kinase-binding site, FAK Y397, after EGF stimulation of cells. Our results establish that FAK is an important receptor-proximal link between growth-factor-receptor and integrin signalling pathways.

Differential regulation of cell motility and invasion by FAK

Cell migration and invasion are fundamental components of tumor cell metastasis. Increased focal adhesion kinase (FAK) expression and tyrosine phosphorylation are connected with elevated tumorigenesis. Null mutation of FAK results in embryonic lethality, and FAK−/− fibroblasts exhibit cell migration defects in culture. Here we show that viral Src (v-Src) transformation of FAK−/− cells promotes integrin-stimulated motility equal to stable FAK reexpression. However, FAK−/− v-Src cells were not invasive, and FAK reexpression, Tyr-397 phosphorylation, and FAK kinase activity were required for the generation of an invasive cell phenotype. Cell invasion was linked to transient FAK accumulation at lamellipodia, formation of a FAK–Src-p130Cas–Dock180 signaling complex, elevated Rac and c-Jun NH2-terminal kinase activation, and increased matrix metalloproteinase expression and activity. Our studies support a dual role for FAK in promoting cell motility and invasion through the activation of distinct signaling pathways.

CEACAMs : their role in physiology and pathophysiology

Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) belong to a group of mammalian immunoglobulin-related glycoproteins. They are involved in cell–cell recognition and modulate cellular processes that range from the shaping of tissue architecture and neovascularization to the regulation of insulin homeostasis and T-cell proliferation. CEACAMs have also been identified as receptors for host-specific viruses and bacteria in mice and humans, respectively, making these proteins an interesting example of pathogen–host co-evolution. Forward and reverse genetics in the mouse now provide powerful novel models to elucidate the action of CEACAM family members in vivo.

The Helicobacter pylori CagA protein induces cortactin dephosphorylation and actin rearrangement by c‐Src inactivation

The gastric pathogen Helicobacter pylori translocates the CagA protein into epithelial cells by a type IV secretion process. Translocated CagA is tyrosine phosphorylated (CagAP‐Tyr) on specific EPIYA sequence repeats by Src family tyrosine kinases. Phos phorylation of CagA induces the dephosphorylation of as yet unidentified cellular proteins, rearrangements of the host cell actin cytoskeleton and cell scattering. We show here that CagAP‐Tyr inhibits the catalytic activity of c‐Src in vivo and in vitro. c‐Src inactivation leads to tyrosine dephosphorylation of the actin binding protein cortactin. Concomitantly, cortactin is specifically redistributed to actin‐rich cellular protrusions. c‐Src inactivation and cortactin dephosphorylation are required for rearrangements of the actin cytoskeleton. Moreover, CagAP‐Tyr‐mediated c‐Src inhibition downregulates further CagA phosphorylation through a negative feedback loop. This is the first report of a bacterial virulence factor that inhibits signalling of a eukaryotic tyrosine kinase and on a role of c‐Src inactivation in host cell cytoskeletal rearrangements.

FRNK blocks v-Src-stimulated invasion and experimental metastases without effects on cell motility or growth.

Focal adhesion kinase (FAK) was first identified as a viral Src (v‐Src) substrate, but the role of FAK in Src transformation events remains undefined. We show that stable expression of the FAK C‐terminal domain (termed FRNK) in v‐Src‐transformed NIH 3T3 fibroblasts inhibited cell invasion through Matrigel and blocked experimental metastases in nude mice without effects on cell motility. FRNK inhibitory activity was dependent upon its focal contact localization. FRNK expression disrupted the formation of a v‐Src‐FAK signaling complex, inhibited p130Cas tyrosine phosphorylation, and attenuated v‐Src‐stimulated ERK and JNK kinase activation. However, FRNK did not affect v‐Src‐stimulated Akt activation, cell growth in soft agar, or subcutaneous tumor formation in nude mice. FRNK‐expressing cells exhibited decreased matrix metalloproteinase‐2 (MMP‐2) mRNA levels and MMP‐2 secretion. Transient FRNK expression in human 293 cells inhibited exogenous MMP‐2 promoter activity and overexpression of wild‐type but not catalytically‐inactive (Ala‐404) MMP‐2 rescued v‐Src‐stimulated Matrigel invasion in the presence of FRNK. Our findings show the importance of FAK in Src‐stimulated cell invasion and support a role for Src‐FAK signaling associated with elevated tumor cell metastases.

The focal adhesion kinase : A regulator of cell migration and invasion

Cell migration plays an important role in embryonic development, wound healing, immune responses, and in pathological phenomena such as tissue invasion and metastasis formation. In this review, we summarize recent reports that connect the focal adhesion kinase (FAK) to cell migration and invasion. FAK is a nonreceptor protein tyrosine kinase involved in signal transduction from integrin‐enriched focal adhesion sites that mediate cell contact with the extracellular matrix. Multiple protein‐protein interaction sites allow FAK to associate with adapter and structural proteins allowing for the modulation of mitogen‐activated protein (MAP) kinases, stress‐activated protein (SAP) kinases, and small GTPase activity. FAK‐enhanced signals have been shown to mediate the survival of anchorage‐dependent cells and are critical for efficient cell migration in response to growth factor receptor and integrin stimulation. Elevated expression of FAK in human tumors has been correlated with increased malignancy and invasiveness. Because recent findings show that FAK contributes to the secretion of matrix‐metalloproteinases, FAK may represent an important checkpoint in coordinating the dynamic processes of cell motility and extracellular matrix remodeling during tumor cell invasion.

Migration of human melanoma cells depends on extracellular pH and Na+/H+ exchange.

Their glycolytic metabolism imposes an increased acid load upon tumour cells. The surplus protons are extruded by the Na+/H+ exchanger (NHE) which causes an extracellular acidification. It is not yet known by what mechanism extracellular pH (pHe) and NHE activity affect tumour cell migration and thus metastasis. We studied the impact of pHe and NHE activity on the motility of human melanoma (MV3) cells. Cells were seeded on/in collagen I matrices. Migration was monitored employing time lapse video microscopy and then quantified as the movement of the cell centre. Intracellular pH (pHi) was measured fluorometrically. Cell–matrix interactions were tested in cell adhesion assays and by the displacement of microbeads inside a collagen matrix. Migration depended on the integrin α2β1. Cells reached their maximum motility at pHe∼7.0. They hardly migrated at pHe 6.6 or 7.5, when NHE was inhibited, or when NHE activity was stimulated by loading cells with propionic acid. These procedures also caused characteristic changes in cell morphology and pHi. The changes in pHi, however, did not account for the changes in morphology and migratory behaviour. Migration and morphology more likely correlate with the strength of cell–matrix interactions. Adhesion was the strongest at pHe 6.6. It weakened at basic pHe, upon NHE inhibition, or upon blockage of the integrin α2β1. We propose that pHe and NHE activity affect migration of human melanoma cells by modulating cell–matrix interactions. Migration is hindered when the interaction is too strong (acidic pHe) or too weak (alkaline pHe or NHE inhibition).

CD66-mediated phagocytosis of Opa52 Neisseria gonorrhoeae requires a Src-like tyrosine kinase- and Rac1-dependent signalling pathway

The interaction of Neisseria gonorrhoeae with human phagocytes is a hallmark of gonococcal infections. Recently, CD66 molecules have been characterized as receptors for Opa52‐expressing gonococci on human neutrophils. Here we show that Opa52‐expressing gonococci or Escherichia coli or F(ab) fragments directed against CD66, respectively, activate a signalling cascade from CD66 via Src‐like protein tyrosine kinases, Rac1 and PAK to Jun‐N‐terminal kinase. The induced signal is distinct from Fcγ‐receptor‐mediated signalling and is specific for Opa52, since piliated Opa− gonococci, commensal Neisseria cinerea or E.coli do not stimulate this signalling pathway. Inhibition of Src‐like kinases or Rac1 prevents the uptake of Opa52 bacteria, demonstrating the crucial role of this signalling cascade for the opsonin‐independent, Opa52/CD66‐mediated phagocytosis of pathogenic Neisseria.

Global Regulatory Impact of ClpP Protease of Staphylococcus aureus on Regulons Involved in Virulence, Oxidative Stress Response, Autolysis, and DNA Repair

Staphylococcus aureus is an important pathogen, causing a wide range of infections including sepsis, wound infections, pneumonia, and catheter-related infections. In several pathogens ClpP proteases were identified by in vivo expression technologies to be important for virulence. Clp proteolytic complexes are responsible for adaptation to multiple stresses by degrading accumulated and misfolded proteins. In this report clpP, encoding the proteolytic subunit of the ATP-dependent Clp protease, was deleted, and gene expression of DeltaclpP was determined by global transcriptional analysis using DNA-microarray technology. The transcriptional profile reveals a strong regulatory impact of ClpP on the expression of genes encoding proteins that are involved in the pathogenicity of S. aureus and adaptation of the pathogen to several stresses. Expression of the agr system and agr-dependent extracellular virulence factors was diminished. Moreover, the loss of clpP leads to a complete transcriptional derepression of genes of the CtsR- and HrcA-controlled heat shock regulon and a partial derepression of genes involved in oxidative stress response, metal homeostasis, and SOS DNA repair controlled by PerR, Fur, MntR, and LexA. The levels of transcription of genes encoding proteins involved in adaptation to anaerobic conditions potentially regulated by an Fnr-like regulator were decreased. Furthermore, the expression of genes whose products are involved in autolysis was deregulated, leading to enhanced autolysis in the mutant. Our results indicate a strong impact of ClpP proteolytic activity on virulence, stress response, and physiology in S. aureus.