TUMOR NECROSIS FACTOR-α STIMULATES FAK ACTIVITY REQUIRED FOR MITOGEN-ACTIVATED KINASE-ASSOCIATED INTERLEUKIN 6 EXPRESSION

David D. Schlaepfer, Shihe Hou, Ssang-Taek Lim, Alok Tomar, Honggang Yu, Yangmi Lim, Dan A. Hanson, Sean A. Uryu, John Molina, and Satyajit K. Mitra

From the Department of Immunology, The Scripps Research Institute, La Jolla, CA, 92037 Running Title: FAK Regulates IL-6 Expression

Address correspondence to: David D. Schlaepfer, The Scripps Research Institute, Department of Immunology, IMM 21, 10550 N. Torrey Pines Road, La Jolla, CA 92037, Tel: 858-784-8207;

Fax: 858-784-8229; Email: dschlaep@scripps.edu

Focal adhesion kinase (FAK) is a cytoplasmic protein-tyrosine kinase (PTK) that promotes cell migration, survival, and gene expression. Here, we show that FAK signaling is important for tumor necrosis factor-α (TNFα) -induced interleukin 6 (IL-6) mRNA and protein expression in breast (4T1), lung (A549), prostate (PC-3), and neural (NB-8) tumor cells by FAK short-hairpin RNA (shRNA) knockdown and by comparisons of FAK-null (FAK-/-) and FAK+/+ mouse embryo fibroblasts. FAK promoted TNFα-stimulated mitogen-activated protein kinase (MAPK) activation needed for maximal IL-6 production. FAK was not required for TNFα-mediated Nuclear Factor kappa B or c-Jun N-terminal kinase activation. TNFα-stimulated FAK catalytic activation and IL-6 production were inhibited by FAK N-terminal but not FAK C-terminal domain over-expression. Analysis of FAK-/- fibroblasts stably reconstituted with wild type or various FAK point mutants showed that FAK catalytic activity, Y397 phosphorylation, and the P712/713 proline-rich region of FAK were required for TNFα-stimulated MAPK activation and IL-6 production. Constitutively-activated MAPK kinase-1 (MEK1) expression in FAK-/- and A549 FAK shRNA-expressing cells rescued TNFα-stimulated IL-6 production. Inhibition of Src PTK activity or mutation of Src phosphorylation sites on FAK (Y861 or Y925) did not affect TNFα-stimulated IL-6 expression. Moreover, analyses of Src-/-, Yes-/-, and Fyn-/- fibroblasts showed that Src expression was inhibitory to TNFα-stimulated IL-6 production. These studies provide evidence for a novel Src-independent FAK to MAPK signaling pathway regulating IL-6 expression with potential importance to inflammation and tumor progression.

Focal adhesion kinase (FAK) is best known for its role as an integrin-stimulated protein-tyrosine kinase (PTK) (1). FAK is recruited to sites of integrin clustering via interactions of its C-terminal domain with integrin-associated proteins such as talin and paxillin. FAK contains a central kinase domain, C-terminal proline-rich regions that serve as binding sites for Src-homology 3 (SH3) domain-containing proteins, and an amino-terminal band 4.1, ezrin, radixin, moesin homology (FERM) domain that acts to regulate FAK kinase activity through an auto-inhibitory mechanism (2,3). Integrin clustering promotes FAK activation, results in FAK phosphorylation at Y397 (pY397), promotes Src-family PTK binding to the FAK pY397 site, and facilitates the formation of FAK-Src signaling complex that results in the secondary phosphorylation of FAK at Y861 and Y925 (4,5). The FAK-Src complex can promote the tyrosine phosphorylation of various targets linked to either growth, survival, or motility-associated signaling pathways (6,7). Canonical FAK-Src integrin signaling can be blocked by over-expression of the FAK C-terminal domain termed FRNK (1). FAK can also be activated by growth factor receptors, G-protein-linked, and cytokine stimulation of cells (5), but how FAK gets activated by non-integrin stimuli remains incompletely defined. FAK also participates in immune and inflammatory response signaling by regulating cytokine production (8). FAK is important for lipopolysaccharide-induced interleukin-6 (IL-6) secretion by human and murine fibroblasts (9). IL-6 and IL-8 expression by human synoviocytes stimulated with oral streptococci protein I/II also requires FAK (10). However, the molecular mechanism(s) whereby FAK promotes IL-6 expression remain largely undefined. IL-6 is an important mediator of immune responses, inflammation, and tumor progression (11). IL-6 expression is largely regulated by gene

http://www.jbc.org/cgi/doi/10.1074/jbc.M610672200The latest version is at JBC Papers in Press. Published on April 16, 2007 as Manuscript M610672200

Copyright 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

by guest on March 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

2

transcription, and the tumor necrosis factor-α (TNFα) cytokine is a potent stimulator of IL-6 expression (12). TNFα binds to two distinct cell surface receptors (TNFR) 1 and TNFR2 which can activate a variety of intracellular signaling cascades (12). TNFα can activate FAK and this is linked to enhanced cell motility (13), gene expression (14,15), and survival (16,17). TNFα-stimulated genes include IL-1α, IL-1β, IL-6, IL-8, IL-18, cycloxygenase-2, matrix metalloproteinase-9 (MMP-9), and vascular endothelial growth factor (VEGF) (18). FAK promotes MMP-9 and VEGF mRNA expression by generating signals leading to the activation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK), respectively (19,20). In addition, the transcription factor Nuclear Factor kappa B (NF-κB) is an important mediator of TNFα signaling and NF-κB activation is a major regulator of TNFα-stimulated IL-6 expression (21). As FAK has been linked to both TNFα-stimulated NF-κB activation (22) and TNFα-stimulated IL-6 expression (14), we have assessed the molecular mechanism(s) of how FAK promote TNFα-stimulated IL-6 expression in human tumor cells and normal mouse fibroblasts. We employed FAK-/- and FAK-reconstituted fibroblasts as well as a FAK short-hairpin RNA (shRNA) knockdown to examine the role of FAK in TNFα-stimulated IL-6 expression. Surprisingly, TNFα-stimulated NF-κB and JNK activation were normal in FAK-/- cells, yet TNFα did not function to promote IL-6 expression in the absence of FAK or in FAK shRNA knockdown cells. We found that TNFα-stimulated FAK catalytic activation, FAK Y397 phosphorylation, and the P712/713 proline-rich region of FAK were required for TNFα-stimulated ERK2/mitogen-activated protein kinase (MAPK) activation and IL-6 production. Inhibition of Src activity or disruption of Src phosphorylation sites on FAK at Y861 or Y925 did not affect TNFα-stimulated IL-6 expression. However, constitutively-activated (CA) MAPK kinase-1 (MEK1) expression in FAK-/- or FAK shRNA cells rescued IL-6 production in response to TNFα stimulation. As TNFα-stimulated FAK activation and IL-6 expression were inhibited by over-expression of the FAK N-terminal FERM but not C-terminal integrin-association domain (termed FRNK), these studies have identified a novel Src-independent pathway whereby FAK kinase activity promotes signaling leading to ERK activation and IL-6 cytokine expression.

EXPERIMENTAL PROCEDURES Cell Culture - FAK+/+, FAK-/-, FAK reconstituted (clone DP3), SYF (Src-/-, c-Yes-/-, Fyn-/-) and SYF+c-Src murine embryonic fibroblasts were used as described (23-25). 4T1 breast carcinoma cells, A549 lung adenocarcinoma cells, and PC-3 prostate carcinoma cells were from ATCC (Manassas, VA). NB-8 neuroblastoma cells were generously provided by Dwayne Stupack (UC San Diego) (26). 4T1 FAK shRNA and scrambled shRNA control cells were generated as described (27). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS). Antibodies and Reagents - IκBα polyclonal, NF−κB p65 monoclonal, and FAK pY925 antibodies were from Santa Cruz (Santa Cruz, CA). Monoclonal antibodies (mAbs) to FAK (clone 4.47), and to phospho-tyrosine (4G10) were from Upstate-Millipore (Charlottesville, VA). Pyk2 and MEK1 mAbs were from BD Transduction (San Diego, CA). mAbs to β-actin (AC-74) were from Sigma-Aldrich (St. Louis, MO). Myc tag (9E10) and GFP tag antibodies were from Covance (Berkeley, CA). ERK1/2 pT185-pY187, ERK1/2, JNK1/2 pT183-pY185, FAK pY397, and FAK pY861 polyclonal antibodies were from Invitrogen-Biosource (Carlsbad, CA). FAK pY576/pY577 and MEK1/2 pS217/221 antibodies were from Cell Signaling Technologies (Danvers, MA). Phycoerythrin (PE)-conjugated anti-mouse TNFR1 and TNFR2 antibodies were from BioLegend (San Diego, CA). Rabbit polyclonal anti-FAK (5904) directed against N-terminal residues 8-27 was generated as described (24). Recombinant TNFα and IL-6 detection kits were from eBioscience (San Diego, CA). Pharmacological inhibitors PP2, PD98059, SB203580, SP600125, and wortmanin were from Calbiochem (San Diego, CA). Viral Expression - The pLentiLox shRNA vector was used to create stable knockdown of FAK expression in NB8, PC3, and A549 carcinoma cells as described (20). Targeting sequences were designed to nucleotides 394-415 of mouse FAK and nucleotides 1089-1107 of human FAK. A scrambled shRNA oligonucleotide forward 5’tgtctccgaacgtgtcacgtttcaagagaacgtga cacgttcggagacttttttc-3’ and reverse 5’tcgagaaaa aagtctccgaacgtgtcacgttctcttgaaacgtgacacgttcgg agaca3’ was used as a control. For murine Pyk2 shRNA, the oligonucleotides forward 5'tgaagta gttcttaaccgcattcaagagatgcggttaagaactacttcttttttc3’ and reverse 5'tcgagaaaaaagaagtagttcttaaccgcat

by guest on March 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

3

ctcttgaatgcggttaagaactacttca-3' were cloned into pLentiLox. Cells were sorted for green fluorescent protein (GFP) expression and the shRNA efficacy verified by immunoblotting. Rabbit CA MEK1 (S231E/S235E) was a kind gift from Jiahuai Han (Scripps). The 1.2 kB MEK1 cDNA was amplified by polymerase chain reaction (PCR), subcloned into pCMV6M to add an N-terminal Myc-tag, and the DNA sequence verified. Myc-tagged CA-MEK1 was subcloned into the lentiviral vector pCDH-puro (System BioSciences, Mountain View, CA) and recombinant lentiviruses produced as described (20). FAK-/- fibroblasts and A549 carcinoma cells were selected in puromycin (5 µg/ml) and CA-MEK1 expression verified by immunoblotting. HA-tagged FRNK and FRNK S1034 were created and used as described (27). Briefly, cells were infected with 5 plaque forming units (pfu)/cell for Ad-TA (Mock control) or with 5 Ad-TA plus 50 pfu/cell Ad-FRNK constructs. GFP FAK FERM (1-402) was generated by PCR with HindIII and BamHI ends and cloned into pEGFP-C1. Quickchange (Stratagene, La Jolla, CA) site-directed was used to create the double R177A/R178A mutation within pEGFP-C1 FAK FERM. For adenoviral expression, NheI-MluI fragments of pEGFP-C1 FAK FERM (1-402) were cloned into EcoRV site of pCMV-Shuttle (Stratagene) and virus was produced using the AdEasy system (Stratagene). Cells were infected with 50 pfu/cell GFP or GFP-FAK FERM constructs and analyzed after 48 h. GFP-FAK Reconstituted FAK-/- fibroblasts – GFP-FAK wild-type (WT), kinase-inactive (R454), and F925 FAK in the retroviral pBabepuro vector were created as described (20). FAK mutants (F397, A712/A713, and F861) in pCDNA3 (24) were subcloned into pEGFP-C1 as KpnI-XbaI fragments and 5’ untranslated sequences from FAK were removed by BspEI/ PvuI digestion and replaced by a 275 bp sequence from WT FAK cloned in pEGFP-C1 (28). All GFP-FAK constructs also contain a triple HA-tag at the FAK C-terminus and were subcloned into pBabepuro for retrovirus production. Early passage FAK-/- fibroblasts were infected with GFP-FAK retrovirus, selected in puromycin (2 µg/ml), and pooled populations of cells enriched by fluorescence-activated cell-sorting (FACS). Upon expansion, GFP-FAK expression was not stably maintained and therefore, clonal cell lines were obtained by further sorting, expanded, and GFP-FAK expression verified by immunoblotting. Three or more clonal cell lines for each GFP-FAK

construct were pooled together and comprise the GFP-FAK reconstituted cells used in this study. IL-6 ELISA - 2 X 106 cells were plated and allowed to spread for 4 h and then stimulated with or without TNFα (10 ng/ml) in DMEM containing 10% FBS for 24 h. IL-6 levels in conditioned media were measured using mouse or human IL-6 ELISA kits (eBioscience) according to manufacturer’s instructions. All samples were analyzed in duplicate. FACS Analysis - FAK+/+ and FAK-/- fibroblasts were trypsinized, fixed with 3.7% formaldehyde solution for 10 min at room temperature and incubated with PE-conjugated anti-tumor necrosis factor receptor-1 (TNFR1) or TNFR2 antibodies. After extensive PBS washes, cell surface TNFR1 and TNFR2 levels were analyzed by flow cytometry and compared to IgG control. Total RNA Preparation and (reverse-transcription polymerase chain reaction) RT-PCR - Total RNA was isolated from cells using Trizol Reagent (Invitrogen, Carlsbad, CA). First strand synthesis was conducted using Superscript First Strand Synthesis kit (Invitrogen) with random primers and 5 µg total RNA as template. PCR was performed using TaqPro Complete (Denville Scientific, Metuchen, NJ). IL-6 and β-actin were co-amplified with specific primer pairs (IL-6 forward: 5’-gatgctaccaaactggatataatc; IL-6 reverse: 5’-ggtccttagccactccttctgtg; β-actin forward: 5’-tgtgatggtgggaatgggtcag; β-actin reverse: 5’-tttgatgtcacgcacgatttcc). Cycling conditions were 95°C 30 sec, 55°C 30 sec and 72°C 1min. IL-6 was amplified for 31 cycles, with β-actin primers present in the last 18 cycles as an internal control. Protein Extracts - Total cell protein was prepared using RIPA lysis buffer (50 mM HEPES pH7.4, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10% glycerol, 1% sodium deoxycholate, 1% Triton X-100, 0.1% SDS, 10 mM sodium pyrophosphate, 100 mM NaF, 1 mM sodium orthovanadate, 10 µg/ml aprotinin, 10 µg/ml leupeptin). Cytoplasmic and nuclear extracts were isolated as described (29). Electrophoretic Mobility Shift Assay (EMSA) - Double-stranded oligonucleotides were labeled with α-32P dCTP (Perkin Elmer, Wellesley, MA) and Klenow DNA polymerase. EMSA was performed as described previously (29). Wild type NF−κB oligos were 5’-acgtgtgggattttccatg (forward) and 5’-tcgacatgggaaaatcccac (reverse) whereas mutant oligos contained the sequences 5’-acgtgttacattttcccatg (forward) and 5’-

by guest on March 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

4

tcgacatgggaaaatgtaac (reverse) with mutations preventing NF−κB binding in bold. Transfection and Luciferase Assay - FAK+/+ and FAK-/- fibroblasts were grown in 6 well plates and co-transfected with with 5 µg of NF−κB firefly luciferase reporter (from Qilin Pan, Scripps) and 1 µg of TK-Renilla luciferase (Promega, San Luis Obispo) using Lipofectamine 2000 (Invitrogen). After 24 hr, transfected cells were stimulated with 10 ng/ml TNFα for an additional 24 hr. Cellular proteins were extracted in passive lysis buffer and analyzed according to manufacturer’s instructions for Dual-Luciferase Reporter Assay (Promega). Immunofluorescence – FAK-/- or A549 cells growing on glass coverslips were fixed with 3.7% formaldehyde for 10 min and permeabilized with 0.5% Triton-X-100 and 0.05% Tween-20 detergents for 6 min. Cells were stained with 0.2 mg/ml Texas Red-conjugated phalloidin (Invitrogen) for 15 min. Photographs were taken using a monochrome CCD camera (Hamamatsu ORCA ER) and processed using OpenLab software (Improvision, Lexington MA). Phase pictures were taken at 20X (Olympus, N.A.=0.5). FAK In Vitro Kinase (IVK) Assay – FAK immunoprecipitates (5904 polyclonal and protein A agarose beads) were washed once in RIPA buffer, twice in HNTG buffer (20 mM Hepes pH 7.4, 150 mM NaCl, 0.1% Triton, and 10% glycerol), and once in kinase buffer (20 mM Hepes pH 7.4, 10% glycerol, 10 mM MgCl2, 10 mM MnCl2, 150 mM NaCl). FAK IVK activity was measured by addition of 25 µCi γ-32P ATP (6000 Ci/mmol, Perkin Elmer) for 15 min at 30oC. The assay was stopped by adding 2X Lamelli sample buffer, resolved by SDS-PAGE and transferred to PVDF membrane. Labeled FAK was visualized by autoradiography and quantified using a phosphoimager (Molecular Dynamics, Piscataway, NJ). Equal immunoprecipitation was verified by anti-FAK blotting.

RESULTS TNFα-stimulated IL-6 Expression is Dependent on FAK - Pro- and anti-inflammatory cytokine production can influence tumor progression (30). FAK expression and activity promote 4T1 breast carcinoma tumor growth and metastasis in Balb/c mice (20,27). As 4T1 tumors also induce a leukemoid reaction (31) and metastatic 4T1 lung tumors were associated with lung edema and immune cell infiltration (27), we tested whether FAK signaling was involved in promoting IL-6 expression as this cytokine promotes inflammation in breast cancer (32). Basal levels of secreted IL-6 were reduced >10-fold in 4T1 FAK shRNA compared to scrambled (control) shRNA-expressing 4T1 cells as determined by ELISA (Fig. 1A). TNFα is a strong promoter of IL-6 expression in many cell types (33). TNFα promoted 3-fold increased IL-6 secretion from 4T1 control cells whereas in 4T1 FAK shRNA cells, IL-6 levels remained below the basal level of 4T1 controls (Fig. 1A). To test whether FAK facilitates TNFα-stimulated IL-6 expression in human tumor cells, we analyzed NB8 neuroblastoma, A549 lung carcinoma, and PC3 prostate carcinoma cells stably expressing either scrambled or FAK shRNA (Fig. 1B). In all 3 cell lines, 70 to 90% reduction in FAK expression was accompanied by the inhibition of TNFα-stimulated IL-6 production. As fibroblasts in the tumor stroma can also contribute to the production of inflammatory cytokines (30), we compared the responses of murine FAK+/+p53-/- and FAK-/-p53-/- embryonic fibroblasts (MEFs) to TNFα stimulation. In wild type FAK+/+ MEFs, TNFα stimulation resulted in a dose-dependent increase of IL-6 protein secretion (Fig. 1C). In contrast, FAK-/- MEFs produced only marginal amounts of IL-6 in response to TNFα. Analysis of TNFα receptors TNFR1 and TNFR2 by flow cytometry showed equivalent surface expression in FAK+/+ and FAK-/- MEFs (Fig. 1D) indicating that the defect in TNFα-initiated IL-6 production is associated with the loss of intracellular FAK signaling. These results support the notion that FAK promotes TNFα-induced IL-6 expression in both normal fibroblasts and carcinoma cells of murine and human origin. FAK Promotes IL-6 mRNA Expression - Since TNFα can promote IL-6 mRNA transcription, we performed semi-quantitative RT-PCR analyses on TNFα-treated FAK+/+, FAK-/-, and FAK-reconstituted FAK-/- MEFs (clone DP3) to

by guest on March 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

5

determine whether IL-6 expression in FAK-/- MEFs was blocked at the mRNA level. By normalizing the IL-6 amplified band to the internal actin control in all reactions, RT-PCR analysis revealed that basal and TNFα-stimulated IL-6 mRNA levels were reduced ~3-fold in FAK-/- compared to FAK+/+ and FAK-reconstituted MEFs (Fig. 2A). One caveat of using FAK-/- MEFs for signaling studies is that the expression of the FAK-related kinase Pyk2 is elevated in these cells (34). To confirm that the inhibition of TNFα-stimulated IL-6 expression was due to lack of FAK and not due to interference by Pyk2, FAK expression in FAK+/+ MEFs was inhibited via lentiviral anti-FAK shRNA (Fig. 2B). Stable knockdown (~85%) of FAK levels in FAK+/+ MEFs resulted in a 4-5 fold reduction in both basal and TNFα-stimulated IL-6 mRNA levels compared to scrambled shRNA-expressing FAK+/+ MEFs (Fig. 2C). Notably, stable FAK knockdown did not promote an alteration in cell morphology compared to scrambled (Scr) shRNA-expressing MEFs (Fig. 2D) and there was no compensatory change in Pyk2 levels in FAK shRNA-expressing FAK+/+ p53-/- MEFs (data not shown). These results support the conclusion that TNFα effects on IL-6 expression are dependent on FAK and occur in part through effects on IL-6 mRNA expression. FAK is not Required for TNFα-induced NF-κB Activation - A major signaling pathway promoting TNFα-induced IL-6 mRNA transcription is through NF-κB activation (12). As a previous study showed that TNFα-induced NF-κB activation may be impaired in FAK-/- fibroblasts (14), we investigated the regulation of NF-κB in FAK+/+ and FAK-/- fibroblasts (Fig. 3). In unstimulated cells, NF-κB is cytoplasmically-localized and kept inactive in part through an association with the inhibitory protein, IκB. TNFα enhances the phosphorylation of IκBα that triggers its rapid degradation, thus releasing NF-κB and allowing for NF-κB nuclear translocation. In both FAK+/+ and FAK-/- cells, TNFα induced a rapid and identical reduction of IκBα protein levels within 5 min followed by IκBα re-synthesis after 60 min (Fig. 3A). Similar results were obtained with anti-FAK shRNA-expressing FAK+/+ cells (Fig. 3B) and Pyk2 shRNA-expressing FAK-/- cells (Fig. 3C and D). To determine whether IκBα degradation corresponded to NF-κB nuclear translocation, control and TNFα-treated (30 min) cells were separated into cytoplasmic and nuclear fractions. TNFα equally stimulated the degradation of IκBα

and corresponding to the nuclear translocation of the NF-κB p65 subunit in FAK+/+, FAK shRNA-expressing FAK+/+, FAK-/-, and Pyk2 shRNA-expressing FAK-/- MEFs (Fig. 3D). These results support the conclusion that FAK or Pyk2 are not required for TNFα-stimulated IκBα degradation and NF-κB nuclear translocation. To determine whether nuclear translocated NF-κB in FAK-/- cells is impaired in its ability to bind DNA and promote transcription, we performed electrophoretic mobility shift assays (EMSA) with lysates of FAK-/- and FAK+/+ MEFs. TNFα stimulation resulted in increased NF−κB 32P-labeled DNA binding within 30 min in both FAK-/- and FAK+/+ MEFs compared to untreated controls (Fig. 3E). 100-fold excess of cold wild type (WT) oligonucleotide addition disrupted NF-κB binding whereas similar addition of NF-κB binding site-mutated oligonucleotides did not affect NF-κB-specific binding (Fig. 3E). Consistent with the nuclear translocation and EMSA results, NF-κB promoter-reporter transcriptional assays confirmed that TNFα stimulated NF-κB transactivation ability in both FAK-/- and FAK+/+ MEFs with FAK-/- MEFs exhibiting higher activity levels (Fig. 3F). These results support the conclusion that NF-κB is fully functional in FAK-/- MEFs and therefore not directly associated with the IL-6 expression defect in response to TNFα. TNFα Activates FAK but FAK-associated Signaling Promoting IL-6 Expression Does Not Require Src – Many stimuli that promote FAK activation and Y397 phosphorylation also lead to the formation of a FAK-Src signaling complex (7). As FAK has been shown to be activated by TNFα in other cell types (13,15), FAK in vitro kinase (IVK) assays were performed after TNFα-stimulation of FAK+/+ MEFs. Compared to basal levels of FAK IVK activity in serum-starved cells, maximal (>5-fold change) FAK IVK activity occurred within 5 to 15 min after TNFα addition to FAK+/+ MEFs (Fig. 4A). Src was not detected in the FAK immunoprecipitates (IPs) after TNFα-stimulation (data not shown) and FAK activation occurred with equal kinetics in the presence of the Src PP2 inhibitor (Fig. 4A). These results support the notion that Src does not function upstream or facilitate FAK activation in TNFα-stimulated FAK+/+ MEFs. Notably, TNFα-stimulated FAK activation at 15 min was reflected in ~2-fold increased FAK Y397 phosphorylation and >5-fold increased FAK Y576/Y577 phosphorylation within the kinase domain as

by guest on March 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

6

detected by phospho-specific blotting of FAK IPs (Fig. 4B). TNFα addition did not promote changes in FAK Y861 phosphorylation (Fig. 4B) and analyses using phospho-specific antibodies to FAK Y925 were not specific (data not shown). To determine if Src activity was required for TNFα-stimulated increased IL-6 mRNA levels, FAK+/+ cells were treated with the PP2 Src inhibitor (20 µM) or inactive control PP3 compound (20 µM) and evaluated for IL-6 mRNA changes by RT-PCR (Fig. 4C). PP2 did not block TNFα-induced IL-6 mRNA expression. Instead, PP2 addition resulted in ~4-fold increased IL-6 mRNA levels over that stimulated by TNFα in PP3-treated control MEFs (Fig. 4C). This result supports the notion that Src may act as an inhibitor to TNFα-induced IL-6 expression. To test this possibility, triple null Src-/-, c-Yes-/- and Fyn-/- (SYF) fibroblasts as well as SYF MEFs re-expressing c-Src were analyzed for TNFα-stimulated IL-6 production by ELISA (Fig. 4D). Src re-expression resulted in 6-fold reduction in both basal and TNFα-induced IL-6 expression in SYF MEFs. Together, these results support the conclusion that FAK is rapidly activated in response to TNFα in a Src-independent manner. Additionally, our results support the notion that FAK and Src may have opposing functions in TNFα-stimulated signaling. TNFα-stimulated FAK catalytic activation and IL-6 production are inhibited by FAK N-terminal domain over-expression – Integrin-stimulated FAK activation can be blocked by over-expression of the FAK C-terminal domain termed FRNK which acts as a competitive inhibitor of the linkage of FAK to integrins (see Fig. 5a). A point mutant (Ala-1034 to Ser, S1034) disrupts paxillin binding to FRNK and serves as a control (24). Surprisingly, transient adenoviral (Ad) FRNK or FRNK S1034 over-expression had no significant effect on TNFα-stimulated IL-6 expression in FAK+/+ MEFs as determined by ELISA (Fig. 4E). This result further supports the notion that TNFα-stimulated FAK activation is distinct from integrin activation of FAK. FAK contains a central kinase domain, the C-terminal region that directs FAK to integrins, and a N-terminal FERM domain. The FAK FERM domain consists of a 3-lobed structure (2) that acts as both a regulatory region controlling FAK activity (3,35) and a region that connects FAK to growth factor receptors (36). To determine whether the FAK FERM domain is involved in TNFα-stimulated signaling, green fluorescent

fusion proteins (GFP) of FAK FERM 1-402 (wild type, WT) and a FERM double point-mutant R177A/R178A were transiently over-expressed by Ad transduction of FAK+/+ MEFs (Fig. 4F). The FERM R177A/R178A mutations are located within the FERM F2 lobe and may disrupt the folding integrity of this region (2). Over-expression of WT but not R177A/178A FAK FERM resulted in ~2-fold inhibition of TNFα-stimulated IL-6 expression (Fig. 4F). Notably, WT FAK FERM over-expression blocked TNFα-stimulated FAK activation as determined by anti-pY576/pY577 immunoblotting of FAK IPs whereas GFP or GFP-FERM R177A/178A had no effect on endogenous FAK activity (Fig. 4G). Together, these results support the notion that TNFα-stimulated FAK activation occurs through a novel FERM domain mechanism. Reconstitution of FAK-/- Fibroblasts Shows that FAK Signaling Promotes IL-6 Expression - Few studies to date have reported the stable re-expression of various FAK mutants in FAK-/- MEFs. To identify the required signaling features of FAK that facilitate TNFα-induced IL-6 expression, GFP-fusion proteins of WT FAK and various FAK point mutants were created (Fig. 5A) and stable pooled populations of GFP-FAK reconstituted MEFs were established (Fig. 5B). The panel of FAK mutants include: F397, a mutation of Y397 to phenylalanine (F) that eliminates the major FAK phosphorylation site and abolishes Src homology 2 (SH2) domain-containing protein binding to this site; R454, a mutation of K454 to arginine (R) which inhibits ATP binding to create a kinase-defective mutant; A712/713, a double mutation of P712 and P713 into alanines (A) that disrupts the first of two SH3 domain binding sites in the FAK C-terminal domain; F861, a mutation of Y861 to F that prevents phosphorylation; and F925, a mutation of Y925 to F that prevents phosphorylation and SH2 domain-containing proteins such as Grb2 binding to this site. Immunoblotting confirmed that the different pooled populations of FAK-/- MEFs expressed comparable levels of GFP-FAK and Pyk2 (Fig. 5C). The ability of wild type FAK and various FAK mutants to rescue FAK-/- defects in IL-6 production in response to TNFα was evaluated by ELISA (Fig. 5D). Wild type (WT) and two phosphorylation site GFP-FAK mutants (F861 and F925) restored both low level basal and TNFα-stimulated IL-6 expression compared to GFP-expressing FAK-/- controls. In contrast,

by guest on March 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

7

phosphorylation site mutant F397 FAK, kinase-inactive R454 FAK, and SH3 binding site mutant A712/713 FAK did not mediate IL-6 induction after TNFα stimulation (Fig. 5D). These results emphasize the importance of FAK kinase activity and its ability to associate with proteins containing SH2 and SH3 domains in mediating TNFα stimulated IL-6 expression. FAK Facilitates TNFα-stimulated MAPK Activation - Since FAK mutants F397, R454, and A712/713 failed to promote TNFα-stimulated IL-6 production, we investigated the potential signaling defects associated with these mutations. Phospho-specific blotting after TNFα stimulation was performed and comparisons made between WT GFP-FAK and mutant-reconstituted FAK-/- MEFs (Fig. 6). Anti-phosphotyrosine (pY) blotting showed that FAK mutants F397, R454, and A712/713 exhibited reduced phosphorylation under basal and after TNFα-stimulation (Fig. 6A). Analysis of IκBα degradation after TNFα addition showed that this occurred equally in FAK-/-, FAK WT, and in FAK mutant-expressing MEFs (Fig. 6B). Additionally, the kinetics of JNK activation occurred similarly in WT and mutant FAK expressing MEFs with maximal activation occurring at 15 min after TNFα addition as detected by phosphospecific immunoblotting (Fig. 6C). Analysis of ERK/MAPK activation by phospho-specific antibodies directed to the MEK1 (T185/Y187) phosphorylation sites on ERK1/2 showed weak to no reactivity in GFP-expressing FAK-/- MEFs after TNFα addition (Fig. 6D). In contrast, ERK1/2 were rapidly phosphorylated within 5 min with a peak activation occurring at 15 min in TNFα-stimulated GFP-FAK WT reconstituted MEFs. FAK-/- fibroblasts re-expressing FAK mutants F397, R454, or A712/713 that were defective for TNFα-stimulated IL-6 production showed no activation of ERK1/2 at 5 min and lower levels of ERK1/2 phosphorylation at 15 min compared to FAK WT reconstituted cells (Fig. 6D). As both F861 and F925 FAK reconstituted cells showed equivalent levels of TNFα-stimulated ERK/MAPK activation compared to WT FAK reconstituted MEFs (data not shown), these studies support the conclusion that FAK facilitates signaling leading to ERK/MAPK activation needed for TNFα-stimulated IL-6 expression. For FAK, this involves FAK kinase activity, Y397 phosphorylation, and FAK association with SH3

domain-containing proteins but not FAK phosphorylation at Y861 or Y925. Constitutively-active MEK1 Expression Rescues TNFα-stimulated IL-6 Production in FAK-/- fibroblasts and FAK shRNA-expressing Carcinoma Cells – The regulation of TNFα-stimulated IL-6 mRNA transcription occurs through the activation of multiple signaling cascades (12). Comparisons of parental FAK-/- and FAK+/+ MEFs showed that loss of FAK was associated with the inhibition of TNFα-stimulated ERK/MAPK phosphorylation (Fig. 7A). Using FAK+/+ MEFs, pharmacological inhibition of MAPK kinase-1 (MEK1) (PD98059) or JNK (SP600125), resulted in partial blockage (~60-80%) of TNFα-induced IL-6 secretion whereas treatment of FAK+/+ cells with wortmannin to inhibit phosphatidylinositol 3-kinase had no effect (Fig. 7B). To determine if the loss of TNFα-stimulated ERK/MAPK activation in the absence of FAK is directly connected to the loss of IL-6 production, Myc-tagged constitutively-active (CA) MEK1 was stably expressed in FAK-/- MEFs (Fig. 7C). CA-MEK1 expression, 2-3 fold above endogenous MEK1, resulted in high levels of MEK1 and ERK/MAPK phosphorylation (Fig. 7C). This was accompanied by subtle changes in FAK-/- cell morphology with the enhancement of filopodia-like actin protrusions in FAK-/- CA-MEK1 MEFs (Fig. 7D). Importantly, CA-MEK1 expression in FAK-/- MEFs rescued both basal and TNFα-stimulated IL-6 production equal to FAK-reconstituted MEFs (Fig. 7E). To determine whether this response is cell type independent, comparisons were made between A549 lung carcinoma cells expressing FAK or scrambled (Scr) shRNAs (Fig. 8A). Loss of FAK in A549 cells results in the inhibition of TNFα-stimulated IL-6 expression (Fig. 1B) and is associated with lower levels of TNFα-stimulated MEK1 and ERK/MAPK activation as determined by phosphospecific immunoblotting (Fig. 8A). CA-MEK1 was stably-expressed in A549 FAK shRNA cells and this resulted in higher levels of ERK/MAPK phosphorylation (Fig. 8B), but no significant changes in A549 cell morphology compared to parental and FAK shRNA-expressing cells (Fig. 8C). Importantly, CA-MEK1 rescued TNFα-stimulated IL-6 expression in A549 FAK shRNA cells without effects on basal IL-6 production (Fig. 8D). Together, these results support the conclusion that TNFα-stimulated FAK activity promotes signals leading to ERK/MAPK

by guest on March 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

8

activation necessary for efficient IL-6 production in fibroblasts and carcinoma cells.

DISCUSSION In this study, we confirm that FAK is required for TNFα-stimulated IL-6 mRNA production and protein secretion by comparisons of FAK-/- and FAK+/+ fibroblasts as was first shown by Funakoshi-Tago et. al. (14). However, our findings directly contradict the previous conclusions that only NF−κB activation by TNFα is defective in FAK-/- cells. In contrast, we found that all aspects of TNFα induced NF−κB activation including IκBα degradation, NF−κB nuclear translocation, NF−κB DNA binding, and stimulated NF−κB transactivation activity are equivalent in FAK+/+, FAK-/-, FAK+/+ FAK shRNA-, and FAK-/- Pyk2 shRNA-expressing fibroblasts. The activation of JNK by TNFα is also not affected by the loss of FAK. However, we find that MEK-ERK/MAPK activation is impaired by the loss of FAK and this was not directly evaluated by Funakoshi-Tago et. al. Moreover, it remains undetermined how such opposite results could have been generated from comparisons of the same cell lines. To this end, we stably-expressed FAK shRNA in various carcinoma cell lines to verify whether FAK has a general and cell type-independent role in regulating IL-6 expression. We found that loss of FAK was also associated with the inhibition of TNFα-stimulated IL-6 production. In both FAK-/- and FAK shRNA-expressing A549 lung carcinoma cells, constitutively-active MEK1 expression rescued the stimulated production of IL-6 in response to TNFα. Together, these results support the conclusion that FAK expression is required for efficient ERK/MAPK activation after TNFα stimulation and that a FAK-ERK/MAPK signaling connection is necessary for maximal IL-6 production in fibroblasts and carcinoma cells. To obtain insights in the mechanism through which FAK promotes TNFα-stimulated IL-6 production, we stably reconstituted FAK-/- fibroblasts with wildtype and various GFP-FAK point mutants that disrupt particular aspects of FAK signaling connections. No studies to date have created such a panel of FAK mutants stably re-expressed within FAK-/- cells. The GFP-FAK mutants include: F397, disrupting the major FAK phosphorylation site; R454, creating a kinase-defective mutant; A712/713, disrupting the first of two SH3 domain binding sites in the FAK C-

terminal domain; F861 and F925, mutations that prevent phosphorylation of these FAK C-terminal domain sites. Compared to GFP-expressing FAK-/- cells, WT GFP-FAK re-expression rescued the defects in both TNFα-stimulated ERK activation and IL-6 production. Additionally, FAK F861 or F925 mutations equally promoted TNFα-stimulated IL-6 expression as did WT FAK. Although Y861 FAK phosphorylation is implicated in regulating p130Cas binding (37), the signaling connections of this FAK phosphorylation site remain undefined. However, integrin-stimulated FAK phosphorylation at Y925 and Grb2 binding to FAK is one of several pathways through which FAK can enhance integrin-stimulated ERK activation (4). The fact that F925 FAK promoted TNFα-stimulated IL-6 expression is interesting as F925 FAK did not promote Src-mediated ERK/MAPK activation regulating vascular endothelial growth factor (VEGF) expression (20) and F925 FAK expression in FAK-/- cells did not promote matrix metalloproteinase-9 expression after TNFα stimulation as did WT FAK (15). Together, these results suggest that FAK can promote ERK activation through multiple pathways and that distinct multi-protein FAK signaling complexes may have unique biological consequences. The reconstitution studies of FAK-/- cells showed that FAK mutants defective in catalytic activity, Y397 phosphorylation, and the P712/713 region did not promote TNFα-stimulated ERK activation or IL-6 expression as did WT FAK. The importance of FAK catalytic activity and Y397 phosphorylation in TNFα signaling to ERK is similar to integrin-stimulated FAK activation where these FAK mutations block the formation of a FAK-Src signaling complex (4,7). However, pharmacological inhibition of Src did not block TNFα-stimulated FAK catalytic activation. Instead, inhibition of Src expression or activity led to increased IL-6 production after TNFα stimulation of cells. Additionally, Src phosphorylation sites Y861 and Y925 on FAK were not required for TNFα-stimulated signaling promoting IL-6 expression. Moreover, integrin activation of FAK is blocked by C-terminal FRNK domain expression whereas FRNK did not affect TNFα-stimulated IL-6 production. Thus, despite the shared requirements of FAK catalytic activity and Y397 phosphorylation, integrin and TNFα stimulation of cells are likely to promote FAK activation and signaling to ERK through distinct mechanisms.

by guest on March 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

9

Insights into the potential molecular mechanism(s) of TNFα-mediated FAK activation came from results showing that FAK FERM domain over-expression could inhibit both TNFα-stimulated FAK activation and IL-6 production. This occurred without alterations in the basal level of FAK activity. This is important as previous studies have shown that exogenous FAK FERM can bind to the FAK kinase region and act as an trans-inhibitor of FAK activity (3). Whereas regulatory FERM interactions with the FAK kinase domain are disrupted by mutations in the F1 lobe of the FERM domain (35), we found that FERM F2 lobe mutations (R177A/R178A)

resulted in an exogenous FERM domain that was highly expressed but did not block TNFα-stimulated FAK activation. We interpret this result as supportive of a role for FERM F2 domain in binding to a potential TNFα-stimulated activator of FAK. As FAK forms a complex with the receptor interacting protein (RIP)1 after TNFα-stimulation (14), that FAK FERM domain can form a complex with RIP (38), and that RIP expression is required for TNFα-stimulated ERK activation (39), future studies will be focused on testing this potential signaling linkage in mediating TNFα-stimulated FAK activation.

REFERENCES 1. Parsons, J. T. (2003) J. Cell Sci. 116, 1409-1416 2. Ceccarelli, D. F., Song, H. K., Poy, F., Schaller, M. D., and Eck, M. J. (2006) J. Biol. Chem. 281,

252-259 3. Cooper, L. A., Shen, T. L., and Guan, J. L. (2003) Mol. Cell. Biol. 23(22), 8030-8041 4. Schlaepfer, D. D., Hauck, C. R., and Sieg, D. J. (1999) Prog. Biophys. Mol. Biol. 71, 435-478 5. Schlaepfer, D. D., Mitra, S. K., and Ilic, D. (2004) Biochim. Biophys. Acta 1692, 77-102 6. Cox, B. D., Natarajan, M., Stettner, M. R., and Gladson, C. L. (2006) J. Cell. Biochem. 99, 35-52 7. Mitra, S. K., and Schlaepfer, D. D. (2006) Curr. Opin. Cell Biol. 18, 516-523 8. Watanabe, Y., Tamura, M., Osajima, A., Anai, H., Kabashima, N., Serino, R., and Nakashima,

Y. (2003) Kidney Int. 64, 431-440 9. Zeisel, M. B., Druet, V. A., Sibilia, J., Klein, J. P., Quesniaux, V., and Wachsmann, D. (2005) J.

Immunol. 174, 7393-7397 10. Neff, L., Zeisel, M., Druet, V., Takeda, K., Klein, J. P., Sibilia, J., and Wachsmann, D. (2003) J.

Biol. Chem. 278, 27721-27728 11. Hodge, D. R., Hurt, E. M., and Farrar, W. L. (2005) Eur. J. Cancer 41, 2502-2512 12. MacEwan, D. J. (2002) Cell Signal 14, 477-492 13. Corredor, J., Yan, F., Shen, C. C., Tong, W., John, S. K., Wilson, G., Whitehead, R., and Polk,

D. B. (2003) Am. J. Physiol. Cell Physiol. 284, C953-961 14. Funakoshi-Tago, M., Sonoda, Y., Tanaka, S., Hashimoto, K., Tago, K., Tominaga, S., and

Kasahara, T. (2003) J. Biol. Chem. 278, 29359-29365 15. Mon, N. N., Hasegawa, H., Thant, A. A., Huang, P., Tanimura, Y., Senga, T., and Hamaguchi,

M. (2006) Cancer Res. 66, 6778-6784 16. Huang, D., Khoe, M., Befekadu, M., Chung, S., Takata, Y., Ilic, D., and Bryer-Ash, M. (2006)

Am. J. Physio.l Cell Physiol. (PMID: 17135301) 17. Takahashi, R., Sonoda, Y., Ichikawa, D., Yoshida, N., Eriko, A. Y., and Tadashi, K. (2006)

Biochim. Biophys. Acta. (PMID: 17197095) 18. Aggarwal, B. B., Shishodia, S., Sandur, S. K., Pandey, M. K., and Sethi, G. (2006) Biochem.

Pharmacol. 72, 1605-1621 19. Hsia, D. A., Mitra, S. K., Hauck, C. R., Streblow, D. N., Nelson, J. A., Ilic, D., Huang, S., Li, E.,

Nemerow, G. R., Leng, J., Spencer, K. S., Cheresh, D. A., and Schlaepfer, D. D. (2003) J. Cell Biol. 160, 753-767

20. Mitra, S. K., Mikolon, D., Molina, J. E., Hsia, D. A., Hanson, D. A., Chi, A., Lim, S. T., Bernard-Trifilo, J. A., Ilic, D., Stupack, D. G., Cheresh, D. A., and Schlaepfer, D. D. (2006) Oncogene 25, 5969-5984

21. Vanden Berghe, W., Vermeulen, L., De Wilde, G., De Bosscher, K., Boone, E., and Haegeman, G. (2000) Biochem. Pharmacol. 60, 1185-1195

by guest on March 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

10

22. Zhang, H. M., Keledjian, K. M., Rao, J. N., Zou, T., Liu, L., Marasa, B. S., Wang, S. R., Ru, L., Strauch, E. D., and Wang, J. Y. (2006) Am. J. Physiol. Cell Physiol. 290, C1310-1320

23. Ilic, D., Furuta, Y., Kanazawa, S., Takeda, N., Sobue, K., Nakatsuji, N., Nomura, S., Fujimoto, J., Okada, M., Yamamoto, T., and Aizawa, S. (1995) Nature 377, 539-544

24. Sieg, D. J., Hauck, C. R., and Schlaepfer, D. D. (1999) J. Cell Sci. 112, 2677-2691 25. Hsia, D. A., Lim, S. T., Bernard-Trifilo, J. A., Mitra, S. K., Tanaka, S., den Hertog, J., Streblow,

D. N., Ilic, D., Ginsberg, M. H., and Schlaepfer, D. D. (2005) Mol. Cell. Biol. 25, 9700-9712 26. Stupack, D. G., Teitz, T., Potter, M. D., Mikolon, D., Houghton, P. J., Kidd, V. J., Lahti, J. M.,

and Cheresh, D. A. (2006) Nature 439, 95-99 27. Mitra, S. K., Lim, S. T., Chi, A., and Schlaepfer, D. D. (2006) Oncogene 25, 4429-4440 28. Ilic, D., Almeida, E. A., Schlaepfer, D. D., Dazin, P., Aizawa, S., and Damsky, C. H. (1998) J.

Cell. Biol. 143, 547-560 29. Hou, S., Guan, H., and Ricciardi, R. P. (2003) J. Biol. Chem. 278, 45994-45998 30. Coussens, L. M., and Werb, Z. (2002) Nature 420, 860-867 31. Dupre, S. A., and Hunter, K. W., Jr. (2006) Exp. Mol. Pathol. 82, 12-24 32. Bachelot, T., Ray-Coquard, I., Menetrier-Caux, C., Rastkha, M., Duc, A., and Blay, J. Y. (2003)

Br. J. Cancer 88, 1721-1726 33. Philip, M., Rowley, D. A., and Schreiber, H. (2004) Semin. Cancer Biol. 14, 433-439 34. Sieg, D. J., Ilic, D., Jones, K. C., Damsky, C. H., Hunter, T., and Schlaepfer, D. D. (1998) EMBO

J. 17, 5933-5947 35. Cohen, L. A., and Guan, J. L. (2005) J. Biol. Chem. 280, 8197-8207 36. Chen, S. Y., and Chen, H. C. (2006) Mol. Cell Biol. 26, 5155-5167 37. Lim, Y., Han, I., Jeon, J., Park, H., Bahk, Y. Y., and Oh, E. S. (2004) J. Biol. Chem. 279, 29060-

29065 38. Kurenova, E., Xu, L. H., Yang, X., Baldwin, A. S., Jr., Craven, R. J., Hanks, S. K., Liu, Z. G., and

Cance, W. G. (2004) Mol. Cell. Biol. 24, 4361-4371 39. Devin, A., Lin, Y., and Liu, Z. G. (2003) EMBO Rep. 4, 623-627 ACKNOWLEDGMENTS We thank Alan Saluk and the Scripps Flow Cytometry facility for outstanding technical assistance in creating GFP-FAK reconstituted cell lines. This work was supported by CA102310 and CA87038 from the NIH. David Schlaepfer is supported in part by CA75240 and an American Heart Association Established Investigator award (0540115N). This is manuscript 18575-IMM from The Scripps Research Institute. The abbreviations used are: CA, constitutively-activated; DMEM, Dulbecco’s modified Eagle’s medium; EMSA, Electrophoretic Mobility Shift Assay; ERK, extracellular signal-regulated kinase; FAK-/-, FAK-null; FACS, fluorescence-activated cell-sorting; FAK, focal adhesion kinase; FERM, band 4.1, ezrin, raxidin, moesin; FRNK, FAK-related non-kinase; GFP, green fluorescent protein; JNK, c-Jun N-terminal kinase; IL-6, interleukin 6; MEK1, MAPK kinase-1; MMP-9, matrix metalloproteinase-9; MAPK, mitogen-activated protein kinase; mABs, monoclonal antibodies; NF-κB, Nuclear Factor kappa B; PE, phycoerythrin; PCR, polymerase chain reaction; PTK, protein-tyrosine kinase; RT, reverse-transcription; Scr, scrambled; shRNA, short-hairpin RNA; TNFR1, tumor necrosis factor receptor-1; TNFα, tumor necrosis factor-α; VEGF, vascular endothelial growth factor; and WT, wild-type.

by guest on March 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

11

FIGURE LEGENDS FIGURE 1. FAK promotes TNFα-stimulated IL-6 secretion in normal and tumor cells. A, Conditioned media from murine 4T1 breast carcinoma cells stably expressing anti-FAK or scrambled shRNA was evaluated for basal (solid bars) and 10 ng/ml TNFα-stimulated IL-6 secretion (open bars) by ELISA. FAK expression and actin levels in protein lysates from 4T1 FAK shRNA or control cells as determined by immunoblotting. B, NB8 neuroblastoma, A549 lung carcinoma, or PC-3 prostate carcinoma expressing either anti-human FAK or scrambled shRNA were evaluated for TNFα-stimulated (10 ng/ml) IL-6 expression by ELISA. FAK or actin expression in the indicated cells was determined by immunoblotting. C, FAK+/+ and FAK-/- mouse embryo fibroblasts (MEFs) were incubated with the indicated amount of TNFα (1 to 100 ng/ml) and IL-6 expression determined by ELISA. Panels A-C, Samples were tested in triplcate, error bars are +/- SD, and results represent at least two separate experiments. D, Cell surface expression of tumor necrosis factor receptor-1 (TNFR1) (grey shade) and TNFR2 (dashed, open) were determined by antibody staining and flow cytometry. Anti-mouse IgG staining (black filled) was used as a control. FIGURE 2. FAK promotes basal and TNFα-induced IL-6 mRNA expression. A, Semi-quantitative IL-6 RT-PCR was performed on RNA isolated from FAK+/+, FAK-/- and FAK-reconstituted mouse embryo fibroblasts (MEFs) in 10% serum (Control, open bars) or treated with 10 ng/ml TNFα (solid bars) for 6 h prior to RNA extraction. β-actin was co-amplified with IL-6 as an internal control. Representative ethidium bromide stained gels are shown as inverted images. Values represent densitometric quantitation of IL-6 bands from 3 independent experiments normalized to β-actin mRNA levels. Values are fold change relative to untreated FAK+/+ cells and error bars are +/- SD. B, FAK and actin expression levels in FAK+/+ fibroblasts expressing either anti-FAK or scrambled (Scr) shRNA as determined by immunoblotting. C, Reduced basal (Control) and TNFα-stimulated IL-6 mRNA levels in FAK shRNA expressing FAK+/+ fibroblasts compared to Scr shRNA controls as determined by RT-PCR as shown in panel A. Samples were tested in triplicate, error bars are +/- SD, and results represent three separate experiments. D, Phase contrast images of growing FAK+/+, FAK+/+ Scr shRNA, and FAK+/+ FAK shRNA-expressing mouse embryo fibroblasts (MEFs). Scale bar is 30 µm. FIGURE 3. TNFα-induced NF-κB activation is not impaired in the absence of FAK. A, FAK+/+ and FAK-/- fibroblasts were incubated with 10 ng/ml TNFα for the indicated times, proteins extracted, followed by IκBα and β-actin immunoblotting. B, FAK shRNA expressing FAK+/+ cells were stimulated with or without TNFα for 30 min followed by immunoblotting for IκBα and β-actin levels. C, Anti-Pyk2 or scrambled (Scr) shRNA expression in FAK-/- fibroblasts was used to inhibit Pyk2 expression as verified by anti-Pyk2 and actin immunoblotting. D, TNFα-induced IκBα degradation and nuclear translocation of the p65 NF-κB subunit as determined by immunoblotting of nuclear and cytoplasmic cell fractions were similar in FAK+/+, FAK-/-, and FAK-/-Pyk2 shRNA-expressing fibroblasts. E, FAK+/+ and FAK-/- fibroblasts were incubated with or without TNFα for 30 min. Nuclear extracts were isolated and tested for NF−κB binding to 32P-labeled double-stranded NF−κB oligonucleotides by electrophoretic mobility shift assay. 100-fold excess of cold wild type (WT) or mutated (mut) NF−κB oligonucleotides were added to verify binding specificity. F, FAK+/+ and FAK-/- cells were co-transfected with NF−κB promoter-luciferase reporter and treated with 10 ng/ml TNFα for 24hr. Luciferase activity is presented as fold increase relative to NF−κB promoter activity in untreated FAK+/+ cells (Control). Samples were tested in triplcate, error bars are +/- SD, and results represent two separate experiments. FIGURE 4. TNFα activates FAK independent of Src and signaling is blocked by FAK FERM domain over-expression. A, FAK+/+ fibroblasts were stimulated by TNFα in the presence or absence of the Src inhibitor PP2 (20 µM) for the indicated times. FAK immunoprecipitates (IPs) were analyzed for associated in vitro kinase (IVK) activity in a γ-32P ATP autophosphorylation assay (top) and FAK levels in the IPs were verified by immunoblotting (bottom). B, FAK+/+ fibroblasts were stimulated by TNFα for the indicated time and FAK IPs analyzed by immunoblotting with pY397, pY576/pY577, pY861 FAK phospho-specific or anti-FAK antibodies. C, FAK+/+ fibroblasts were stimulated with 10 ng/ml TNFα in the presence of 20 µM PP2 Src inhibitor or 20 µM PP3 control for 6 h and the effects of Src inhibition on IL-6 mRNA expression was evaluated by RT-PCR with internal actin controls. Shown is an inverted image of ethidium bromide stained gel and results +/- SD represent two separate

by guest on March 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

12

experiments. D, Src-Yes-Fyn null (SYF) fibroblasts and SYF cells stably re-expressing c-Src were analyzed for basal (solid bars) and 10 ng/ml TNFα-stimulated (open bars) IL-6 protein expression by ELISA. Samples were tested in triplcate, error bars are +/- SD, and results represent three separate experiments. E, FAK+/+ fibroblasts were mock-treated or transduced with adenovirus for HA-tagged FRNK or FRNK S1034 and after 48 h, stimulated with 10 ng/ml TNFα for 24 h. FRNK expression was determined by HA-tag immunoblotting. F, FAK+/+ fibroblasts were transduced with adenovirus for GFP, GFP-FAK FERM (1-402), or GFP-FAK FERM R177A/R178A and after 48 h, stimulated with 10 ng/ml TNFα for 24 h. GFP and GFP-FAK FERM expression were determined by anti-GFP immunoblotting. E and F, Secreted IL-6 levels were determined by ELISA and values represent +/- SD from two experiments. G, Cell lysates were made (with or without TNFα addition for 15 min) from the indicated FAK+/+ fibroblasts transduced with GFP, GFP-FAK FERM, or GFP-FAK FERM R177A/R178A adenovirus for 48 h. FAK IPs (using C-terminal domain targeted antibodies) were sequentially analyzed by phospho-specific pY576/pY577 and anti-FAK immunoblotting. FIGURE 5. FAK kinase activity, Y397 phosphorylation, and C-terminal proline-rich motifs are needed for FAK-mediated IL-6 expression. A, Schematic representation of mutations within GFP-FAK constructs used to reconstitute FAK-/- fibroblasts. Indicated is the band 4.1, ezrin, radixin, moesin (FERM), central kinase, and C-terminal focal adhesion targeting (FAT) domains. Filled regions indicate proline-rich motifs and all constructs contain HA-tags at the C-terminus. FRNK encompasses the FAK C-terminal proline-rich and FAK domains. B, Pooled populations of control GFP-expressing FAK-/- or the indicated GFP-FAK reconstituted fibroblasts were analyzed for GFP expression by flow cytometry (open peaks) and compared to autofluorescence levels of parental FAK-/- cells (filled peak). C, Protein extracts from the indicated GFP-control or GFP-FAK reconstituted FAK-/- cells were immunoblotted with antibodies to GFP, Pyk2, and actin. D, WT and mutant FAK reconstituted fibroblasts were analyzed for basal (solid bars) and 10 ng/ml TNFα-stimulated (open bars) IL-6 protein expression by ELISA. Samples were tested in triplcate, error bars are +/- SD, and results represent two separate experiments. FIGURE 6. FAK-/- or FAK mutant reconstituted fibroblasts that are defective for TNFα-stimulated IL-6 expression exhibit reduced TNFα-mediated ERK activation. Panels A-D, The indicated GFP-only or GFP-FAK reconstituted fibroblasts were grown to confluence, serum starved for 16 h, and treated with TNFα (10 ng/ml) for the indicated times prior to protein extraction and blotting. A, FAK phosphorylation was evaluated by anti-phosphotyrosine (pY) and anti-GFP blotting. B, Kinetics of TNFα-stimulated IκB degradation as determined by anti-IκBα blotting. C, Kinetics of TNFα-stimulated JNK activation as determined by blotting with phospho-specific antibodies to pT183-pY185 JNK (pJNK1/2). D, Kinetics of TNFα-stimulated ERK-MAPK kinase activation as determined by blotting with phospho-specific antibodies to pT185-pY187 ERK1/2 (pERK) or to total ERK1/2 protein. FIGURE 7. MEK-ERK activation is sufficient to promote TNFα-induced IL-6 expression in the absence of FAK. A, FAK+/+ or FAK-/- fibroblasts were serum starved and treated with TNFα (10 ng/ml) for the indicated times prior to protein extraction and blotting with phospho-specific antibodies to pT185-pY187 ERK1/2 (pERK) or to total ERK1/2 protein. B, FAK+/+ fibroblasts were pre-treated for 30 min with various kinase inhibitors (40 µM PD98059, 20 µM SP600125, or 250 nM wortmanin), incubated with/ without 10ng/ml TNFα for 24 hr in the continued presence of inhibitors. IL-6 production was measured by ELISA. Samples were tested in triplcate, error bars are +/- SD, and results represent two separate experiments. C, Stable expression of constitutively active (CA) Myc-tagged MEK1 in FAK-/- fibroblasts compared to vector control is verified by blotting protein lysates with anti-Myc tag, MEK1, or actin antibodies. Phospho-specific antibody blotting was used to visualize activated MEK1 (pMEK1, pS217pS221) or activated ERK1/2 (pERK, pT185-pY187). D, Phase contrast images and phalloidin-stained actin of FAK-/- and FAK-/- (CA-MEK1) fibroblasts. Scale bar is 30 µm. E, FAK-/-, FAK-reconstituted, and FAK-/- CA-MEK1 fibroblasts were analyzed for basal (solid bars) and 10 ng/ml TNFα-stimulated (open bars) IL-6 protein expression by ELISA. Samples were tested in triplcate, error bars are +/- SD, and results represent three separate experiments.

by guest on March 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

13

FIGURE 8. CA-MEK1 expression rescues TNFα IL-6 expression in A549 FAK shRNA carcinoma cells. A, TNFα-stimulated MEK1 (pMEK1, pS217pS221) and ERK1/2 activation (pERK, pT185-pY187) as determined by phospho-specific antibody blotting of scrambled (Scr) and FAK shRNA-expressing A549 cells. Total FAK and MEK1 expression were verified by blotting. B, Stable CA MEK1 expression in FAK shRNA A549 cells as detected by anti-Myc blotting. Activated ERK1/2 (pERK, pT185-pY187) was determined by phosphospecific blotting. Total ERK and actin were verified by blotting. C, Phase contrast images of growing A549 Scr shRNA, A549 FAK shRNA, and A549 FAK shRNA+ CA-MEK1 cells. Scale bar is 30 µm. D, A549 Scr shRNA, A549 FAK shRNA, and A549 FAK shRNA+ CA-MEK1 cells were analyzed for basal (solid bars) and 10 ng/ml TNFα-stimulated (open bars) IL-6 protein expression by ELISA. Samples were tested in triplcate, error bars are +/- SD, and results represent three separate experiments.

by guest on March 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

IL-6 ELISA

FAKshRNA -- ++-- ++ -- ++

IL-6

(pg/

ml)

NB-8 A549 PC-3

50

100

150

200

250

FAK

actin

TNFα

B

0

50

100

150

200IL-6 ELISA

IL-6

(pg/

ml)

4T1 Breastcarcinoma cells

A

C

TNFαα(ng/ml)

IL-6

(pg/

ml)

FAK+/+ MEFs FAK-/- MEFs

1400

1200

800

400

00 1 10 100 0 1 10 100

IL-6 ELISA D

FAK-/-TNFR1

TNFR2

Cel

lNum

ber

Log fluorescent intensity

FAK+/+TNFR1

TNFR2

Fig. 1

FAK

actin

--++FAKshRNA

TNFαBasal

TNFαBasal

by guest on March 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

FAK

FAK

shR

NA

Scr

shR

NA

actin

0

0.5

1

1.5

2

2.5

TNFαControl

FAK+/+ MEFs

FAKshRNA

ScrshRNA

FAK+/+ FAK-/- (+FAK)FAK-/-

actin

IL-6

4

3

2

1

0

(Fol

d-F

AK

+/+

Ctrl

)

FAK+/+ FAK-/- FAK-/- (+FAK)

RT-PCR

Ctrl TNF TNF TNF

269-

514-Ctrl Ctrl

Fig. 2

FAK +/+ MEFs

A

TNFαControl

B

C

D FAK+/+ MEFs

FAK+/+ MEFs Scr shRNA

FAK+/+ MEFs FAK shRNA

IL-6

mR

NA

Lev

els

(Fol

d-F

AK

+/+

Ctrl

)IL

-6m

RN

AL

evel

s

by guest on March 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

IκBα

p65

Cytoplasm

Nuclear

Scr Pyk2shRNAD FAK +/+ -/- -/- -/-

- -C TNF C TNF C TNF C TNF

IκBα

0 5 10 20 30 60 0 5 10 20 30 60A

actin

FAK+/+ FAK-/-

TNFα +/+ -/- -/-FAKshRNA Scr Pyk2

C

Pyk2

actin

IκBα

Ctrl TNF Ctrl TNF

B

FAK+/+

ScrambledFAK

shRNA

actin

Fig. 3

TNF - + - + + +Cold oligo - - - - mut WT

NF-κB

N.S.

E FAK+/+ FAK-/- FAK-/-

NF-κB DNA Binding

0

1

2

3

4

5

(Fol

d-F

AK

+/+

Ctrl

)

F

FAK+/+ FAK-/-

NF-κB Promoter Activity

(min)

TNFαControl

(free)

(shifted)

Luci

fera

seA

ctiv

ity

by guest on March 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Fig. 4

actin

IL-6

TNF700

600

500

400

300

200

1000

SYF SYF+ c-Src

IL-6

(pg

/ml)

0

1

2

3

4

5

(Fol

d-F

AK

+/+

TNF)

0 5 15 30 0 5 15 30

IVK (32P)

WB: FAK

FAK IPs : IVK Assay

PP2 (20 µM) - - - - + + + +

TNF (10 ng/ml)

TNF+PP2

IL-6 ELISA

(min)

-FAK

A

C D

-FAK

TNFαControl

FAK+/+ MEFs

FAK+/+ MEFs

pY397

pY861

FAK

FAK IPs

0 5 15 30 (min)

TNFα (10 ng/ml)

pY576/pY577

B

25

20

15

10

5

0Control FRNK

IL-6

(ng

/ml)

IL-6 ELISAETNFα (10 ng/ml)

FRNKS1034

FAK+/+ MEFs

PP3 PP2

FAK+/+ MEFs

AdConstruct

25

20

15

10

5

0GFP GFP-FAK

FERM

IL-6

(ng

/ml)

IL-6 ELISAFTNFα (10 ng/ml)

FAK+/+ MEFs

AdConstruct

GFP-FAKFERM

R177/178A

FRNK S1034

HABlot

FERMGFP R177/178

GFPBlot

50- 75-

30-

G

0 15 0 15 0 15

pY576/pY577

FAK

(min)

TNFα (10 ng/ml)

GFPGFP-FAK

FERM

GFP-FAKFERM

R177/178A

FAK IPs

IL-6

mR

NA

Lev

els

by guest on March 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

neg GFP

WT F397

R454 A712/713

F861 F925

KinaseFERM FAT

F397

A712

/713

R45

4

F861

F925

F

GFP

1600

1400

1200

1000

800

600

400

200

0

IL-6

(pg/

ml)

GFP FAK F397 R454 A712/3 F861 F925

actin

GFP-FAK

Pyk2

FAK-/- Fibroblasts

B

D

Fig. 5

GFP

FAK

WT

F397

R454

A712

/713

F861

F925

-150

-100

-45

FAK-/- Fibroblasts

IL-6 ELISA

A

TNFαControl

CFACS Profiles

Log Fluorescent Intensity101 102 103 104

Cel

lNum

ber

HA

FAT HAFRNK

by guest on March 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

GFP-FAK

pERK

ERK1/2

pY

IκBα

pJNK1/2

Fig. 6

GFP FAK WT F397 R454 A712/713TNF

(10ng/ml) 0 5 15 30 0 5 15 30 0 5 15 30 0 5 15 30 0 5 15 (min)

GFP FAK WT F397 R454 A712/713TNF

(10ng/ml) 0 5 15 30 0 5 15 30 0 5 15 30 0 5 15 30 0 5 15 (min)

C

B

A GFP FAK WT F397 R454 A712/713TNF

(10ng/ml) 0 5 15 30 0 5 15 30 0 5 15 30 0 5 15 30 0 5 15 (min)

-150

-150

D

GFP FAK WT F397 R454 A712/713TNF

(10ng/ml) 0 5 15 30 0 5 15 30 0 5 15 30 0 5 15 30 0 5 15 (min)

GFP or GFP-FAK Reconstituted FAK-/- MEFs

by guest on March 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

CAMEK1

Control

Myc-MEK1

actin

FAK-/- MEFsC

pERK

A

Fig. 7

FAK+/+ MEFs

FAK-/- MEFs

pERK

pERK

-ERK2

-ERK2

1600

1400

1200

1000

800

600

400

200

0IL

-6(p

g/m

l)

PD98059

SP600125

Wortmanin

IL-6 ELISA - FAK+/+ MEFsBTNFαControl

DMSO

TNFα (10 ng/ml)0 5 15 30 (min)

TNFα (10 ng/ml)0 5 15 30 (min)

FAK-/- (CA-MEK1) MEFs

ph

ase

acti

n

FAK-/- MEFs

pMEK1

MEK1

D

IL-6 ELISA

TNFα (10 ng/ml)Basal

FAK-/-

2

4

(Fol

dov

erco

ntro

l)

6

8

10

FAK-/-(+FAK)

FAK-/-+

CA-MEK1Control

0

E

IL-6

Lev

els

by guest on March 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

IL-6 ELISA

TNFα (10 ng/ml)Basal

A549Scr

shRNA

A549 FAKshRNA

100

IL-6

(pg/

ml)

200

300

400

D

A549 FAKshRNA

+CA-MEK1

0

Fig. 8

+CA

MEK1

FAKsh

RNA

A549 FAK shRNA

FAKsh

RNA

Scrsh

RNA

pMEK1

MEK1

A

FAK

B

pERK

A549

pERK

actin

Myc-MEK1

ERK1/2

A549 Scr shRNA

C

A549 FAK shRNA

A549 FAK shRNA + CA-MEK1

TNFα (10 ng/ml)15 min

by guest on March 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Lim, Dan A. Hanson, Sean A. Uryu, John Molina and Satyajit K. MitraDavid D. Schlaepfer, Shihe Hou, Ssang-Taek Lim, Alok Tomar, Honggang Yu, Yangmi

kinase-associated interleukin 6 expression stimulates FAK activity required for mitogen-activatedαTumor necrosis factor-

published online April 16, 2007J. Biol. Chem. 

  10.1074/jbc.M610672200Access the most updated version of this article at doi:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

by guest on March 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from