Integrin-mediated signal transduction in cells lacking focal adhesion kinase p125FAK

ABSTRACT The hepatitis B virus (HBV) X protein (HBx) is a multifunctional regulator of cellular signal transduction and transcription pathways and has a critical role in HBV replication. Much of the cytoplasmic signal transduction activity associated with HBx expression and its stimulation of viral replication is attributable to HBx-induced activation of calcium signaling pathways involving Pyk2 and Src tyrosine kinases. To further characterize upstream signal transduction pathways that are required for HBx activity, including activation of Src and mitogen-activated protein kinase (MAPK) cascades, we determined whether focal adhesion kinase (FAK), a known regulator of Src family kinases and the other member of the Pyk2/FAK kinase family, is activated by HBx. We report that HBx activates FAK and that FAK activation is important for multiple HBx functions. Dominant inhibiting forms of FAK blocked HBx activation of Src kinases and downstream signal transduction, HBx stimulation of NF-κB and AP-1-dependent transcription, and HBV DNA replication. We also demonstrate that HBx-induced activation of FAK is dependent on cellular calcium signaling, which is modulated by HBx. Moreover, prolonged expression of HBx increases both FAK activity and its level of expression. FAK activation may play a role in cellular transformation and cancer progression. HBx stimulation of FAK activity and abundance may also be relevant as a potential cofactor in HBV-associated hepatocellular carcinoma.

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Interleukin 1-induced calcium signalling in chondrocytes requires focal adhesions

Biochemical Journal ◽

10.1042/bj3240653 ◽

1997 ◽

Vol 324 (2) ◽

pp. 653-658 ◽

Cited By ~ 44

Author(s):

Laura LUO ◽

Tony CRUZ ◽

Christopher McCULLOCH

Keyword(s):

Signal Transduction ◽

Focal Adhesion ◽

Cell Attachment ◽

Interleukin 1 ◽

Primary Cultures ◽

Adhesion Formation ◽

Focal Adhesions ◽

Ion Concentration ◽

Significant Difference ◽

In Cells

The cytokine interleukin 1 (IL-1) is an important mediator of connective-tissue destruction in arthritic joints but the mechanisms by which IL-1 mediates signal transduction in chondrocytes is poorly understood. Previous results have indicated that IL-1 receptors co-localize with focal adhesions [Qwarnstrom, Page, Gillis and Dower (1988) J. Biol. Chem. 263, 8261–8269], discrete adhesive domains of cells that function in cell attachment and possibly in signal transduction. We have determined whether focal adhesions restrict IL-1-induced Ca2+ signalling in primary cultures of bovine chondrocytes. In cells grown for 24 h on fibronectin, the basal intracellular Ca2+ ion concentration ([Ca2+]i) was 100±3 nM. Optimal increases of [Ca2+]i above baseline were induced by 10 nM IL-1 (183±30 nM above baseline). There was no significant difference between cells plated on fibronectin or type II collagen (P > 0.2; 233±90 nM above baseline). Ca2+ transients were significantly decreased by the inclusion of 0.5 mM EGTA in the bathing buffer (74±11 nM above baseline), and 1 μM thapsigargin completely blocked Ca2+ transients. Cells plated on poly-(l-lysine) or suspended cells showed no Ca2+ increases, whereas cells grown on fibronectin exhibited IL-1-induced Ca2+ responses that corresponded temporally to the time-dependent cell spreading after plating on fibronectin. Cells plated on poly-(l-lysine) and incubated with fibronectin-coated beads exhibited vinculin staining in association with the beads. In identical cell preparations, IL-1 induced a 136±39 nM increase of [Ca2+]i above baseline in response to 10 nM IL-1β. There were no IL-1-induced Ca2+ increases when cells on poly-(l-lysine) were incubated with fibronectin-coated beads for only 15 min at 37 °C, in cells maintained for 3 h at 4 °C, in cells incubated with BSA beads for 3 h at 37 °C, or in cells pretreated with cytochalasin D. Labelling of IL-1 receptors with 125I-IL-1β showed 3-fold more specific labelling of focal adhesion complexes in cells incubated with fibronectin-coated beads compared with cells incubated with BSA-coated beads, indicating that IL-1 receptor binding or the number of IL-1 receptors was increased in focal adhesions. These results indicate that, in chondrocytes, IL-1-induced Ca2+ signalling is dependent on focal adhesion formation and that focal adhesions recruit IL-1 receptors by redistribution in the cell membrane.

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Signal transduction through integrins: A central role for focal adhesion kinase?

BioEssays ◽

10.1002/bies.950170309 ◽

1995 ◽

Vol 17 (3) ◽

pp. 229-236 ◽

Cited By ~ 197

Author(s):

Alan Richardson ◽

J. Thomas Parsons

Keyword(s):

Signal Transduction ◽

Focal Adhesion Kinase ◽

Focal Adhesion

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Interleukin-2 induces 2-integrin-dependent signal transduction involving the focal adhesion kinase-related protein B (fakB)

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.95.12.6959 ◽

1998 ◽

Vol 95 (12) ◽

pp. 6959-6964 ◽

Cited By ~ 6

Author(s):

J. Brockdorff ◽

S. B. Kanner ◽

M. Nielsen ◽

N. Borregaard ◽

C. Geisler ◽

...

Keyword(s):

Signal Transduction ◽

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Interleukin 2 ◽

Related Protein ◽

Dependent Signal ◽

Protein B

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Integrin-mediated signal transduction linked to Ras pathway by GRB2 binding to focal adhesion kinase

Nature ◽

10.1038/372786a0 ◽

1994 ◽

Vol 372 (6508) ◽

pp. 786-791 ◽

Cited By ~ 1064

Author(s):

David D. Schlaepfer ◽

Steven K. Hanks ◽

Tony Hunter ◽

Peter van der Geer

Keyword(s):

Signal Transduction ◽

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Ras Pathway

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Optogenetic model reveals cell shape regulation through FAK and Fascin

Journal of Cell Science ◽

10.1242/jcs.258321 ◽

2021 ◽

Author(s):

Jean A. Castillo-Badillo ◽

N. Gautam

Keyword(s):

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Cell Shape ◽

Spherical Shape ◽

Cell Types ◽

Experimental Models ◽

Molecular Processes ◽

Different Cell Types ◽

Shape Changes ◽

In Cells

Cell shape regulation is important but the mechanisms that govern shape are not fully understood, in part due to limited experimental models where cell shape changes and underlying molecular processes can be rapidly and non-invasively monitored in real time. Here, we use an optogenetic tool to activate RhoA in the middle of mononucleated macrophages to induce contraction, resulting in a side with the nucleus that retains its shape and a non-nucleated side which was unable to maintain its shape and collapsed. In cells overexpressing focal adhesion kinase (FAK), the non-nucleated side exhibited a wide flat morphology and was similar in adhesion area to the nucleated side. In cells overexpressing fascin, an actin bundling protein, the non-nucleated side assumed a spherical shape and was similar in height to the nucleated side. This effect of fascin was also observed in fibroblasts even without inducing furrow formation. Based on these results, we conclude that FAK and fascin work together to maintain cell shape by regulating adhesion area and height, respectively, in different cell types.

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Direct interaction of v-Src with the focal adhesion kinase mediated by the Src SH2 domain.

Molecular Biology of the Cell ◽

10.1091/mbc.5.4.413 ◽

1994 ◽

Vol 5 (4) ◽

pp. 413-421 ◽

Cited By ~ 223

Author(s):

Z Xing ◽

H C Chen ◽

J K Nowlen ◽

S J Taylor ◽

D Shalloway ◽

...

Keyword(s):

Signal Transduction ◽

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Intracellular Signaling ◽

Direct Interaction ◽

Sh2 Domain ◽

Sh2 Domains ◽

Intracellular Signaling Molecules

The recently described focal adhesion kinase (FAK) has been implicated in signal transduction pathways initiated by cell adhesion receptor integrins and by neuropeptide growth factors. To examine the mechanisms by which FAK relays signals from the membrane to the cell interior, we carried out a series of experiments to detect potential FAK interactions with proteins containing Src homology 2 (SH2) domains that are important intracellular signaling molecules. Using v-Src-transformed NIH3T3 cells, we showed that FAK was present in the immune-complex precipitated by anti-Src antibody, suggesting potential interaction of FAK with v-Src in vivo. We also showed potentially direct interaction of FAK with v-Src in vivo using the yeast two-hybrid system. Using recombinant FAK expressed in insect cells and bacterial fusion proteins containing Src SH2 domains, we showed direct binding of FAK to the Src SH2 domain but not to the SH3 domain in vitro. A kinase-defective mutant of FAK, which is not autophosphorylated, did not interact with the Src SH2 domain under the same conditions, suggesting the involvement of the FAK autophosphorylation sites. Treatment of FAK with a protein-tyrosine phosphatase decreased its binding to the Src SH2 domain, whereas autophosphorylation in vitro increased its binding. These results confirm the importance of FAK autophosphorylation sites in its interaction with SH2 domain-containing proteins. Taken together, these results suggest that FAK may mediate signal transduction events initiated on the cell surface by kinase activation and autophosphorylation that result in its binding to other key intracellular signaling molecules.

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Focal adhesion kinase: at the crossroads of signal transduction

Journal of Cell Science ◽

10.1242/jcs.110.4.401 ◽

1997 ◽

Vol 110 (4) ◽

pp. 401-407 ◽

Cited By ~ 16

Author(s):

D. Ilic ◽

C.H. Damsky ◽

T. Yamamoto

Keyword(s):

Signal Transduction ◽

Extracellular Matrix ◽

Cell Migration ◽

Tyrosine Kinase ◽

Focal Adhesion Kinase ◽

Signaling Pathways ◽

Focal Adhesion ◽

Nonreceptor Tyrosine Kinase ◽

Definitive Evidence ◽

Multiple Signaling

Morphogenetic processes during development, including cell migration, depend on signals from both the extracellular matrix (ECM) and soluble signaling factors. Extensive evidence has shown that the nonreceptor tyrosine kinase, focal adhesion kinase (FAK), is activated in response to both kind of signal. The most definitive evidence that FAK is directly downstream of signals initiated by the ECM comes from comparing the phenotypes of mice deficient for FAK and the ECM molecule, fibronectin: in both cases embryos die at about E8.5 and display almost identical severe vascular and other mesodermal defects. It is now clear that there are additional FAK-like proteins, indicating the existence of a FAK family. Furthermore, FAK is not located at adhesive sites in all cells where it is expressed. This, plus extensive data indicating that FAK becomes activated in response to several soluble signaling factors, suggests that the FAK family may be at the crossroads of multiple signaling pathways that affect cell and developmental processes.

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High Levels of Heat Shock Proteins 90 and 27 in CD34-Positive Cells from Myelodysplastic Syndromes (MDS) Are Associated with Higher Expression and Activation of Focal Adhesion Kinase (FAK) and with Disease Progression.

Blood ◽

10.1182/blood.v114.22.289.289 ◽

2009 ◽

Vol 114 (22) ◽

pp. 289-289

Author(s):

Françoise Solly ◽

Pascale Flandrin-Gresta ◽

Carmen Aanei ◽

Jérôme Cornillon ◽

Emmanuelle Tavernier ◽

...

Keyword(s):

Signal Transduction ◽

High Risk ◽

Heat Shock ◽

Heat Shock Protein ◽

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Conflicts Of Interest ◽

Focal Adhesions ◽

Cd34 Cells ◽

Phosphorylated Akt

Abstract Abstract 289 MDS are characterized by a high risk of evolution into acute myeloid leukemia (AML). The pathogenesis of this evolution is still unclear. Some studies indicate that aberrant activation of survival signaling pathways is involved. The 90-kDa heat shock protein (HSP90) is implicated in the conformational maturation and stabilization of protein kinases and has key roles in signal transduction, protein folding, and protein degradation. HSP90 levels are increased in AML cells, and associated with resistance to chemotherapy induced apoptosis. Moreover, HSP90 is involved in the formation of focal adhesions. Focal Adhesion Kinase (FAK), a non-receptor tyrosine kinase, and a client of HSP90, is a member of the integrin-mediated signal transduction pathway. FAK was found over-expressed and constitutively activated in solid tumors. In AML, FAK expression is associated with enhanced blast migration and poor prognosis. FAK also exerts a potent antiapoptotic effect through adhesion to extracellular matrix and stromal cells. Finally 27-kDa heat shock protein (HSP27), a small HSP, prevents apoptosis by interfering with the mitochondrial pathway of apoptosis. In a cancer cell line, HSP27 has been shown to regulate cell adhesion via modulation of FAK. The aim of our study was to investigate the role of HSP90 in high-risk MDS and its potential role on focal adhesion. The expression of HSP90, HSP27, phosphorylated FAK (pFAK), and phosphorylated Akt (pAkt) was assessed by multicolor flow cytometry in bone marrow (BM) mononuclear (MNC) and CD34+ cells from 177 MDS samples at diagnosis: 96 refractory anemias with excess of blasts (RAEB), 58 refractory anemias (RA) and 23 chronic myelomonocytic leukemias (CMML). The levels of HSP90, HSP27, FAK, pFAK, and pAkt in MNC from RAEB patients, were significantly higher than in those measured in RA or CMML patients (p<10−5) (Table). The same difference was observed in CD34+ cells, showing that the increased levels observed in RAEB MNC were not a consequence of increased percentage of blasts. In control marrows (n=5), the percentages of positive cells in MNC and CD34+ populations were similar to that in RA. The percentages of HSP90, HSP27, FAK, pFAK and pAKT were significantly correlated with those of blasts and CD34+ cells, and tended to be higher in cases with high-risk cytogenetics. Consequently the risk of transformation into AML was significantly higher when these proteins were highly expressed. The effects of inhibition of HSP90 were evaluated in 30 RAEB samples by incubating MNC with 17-Alyl-amino-gelgenamycin (17-AAG) at concentrations of 1 and 5μM for 24 hours in liquid culture. A downregulation of HSP90, pFAK and pAKT was observed in CD34+ cells at 12 hours, followed by increased apoptosis at 24h as assessed by activated caspase 3 and annexin V staining. Our results suggest that FAK, HSP90 and Akt activation are associated with cell survival and may contribute to the progression of MDS to leukemia. Moreover this signaling network could be a therapeutic target through HSP90 inhibition by 17-AAG. Disclosures: No relevant conflicts of interest to declare.

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