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J. Biol. Chem., Vol. 282, Issue 24, 17450-17459, June 15, 2007

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Tumor Necrosis Factor- Stimulates Focal Adhesion Kinase Activity Required for Mitogen-activated Kinase-associated Interleukin 6 Expression^*

From the Department of Immunology, The Scripps Research Institute, La Jolla, California 92037

Received for publication, November 17, 2006 , and in revised form, March 30, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Focal adhesion kinase (FAK) is a cytoplasmic protein-tyrosine kinase that promotes cell migration, survival, and gene expression. Here we show that FAK signaling is important for tumor necrosis factor- (TNF)-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 knockdown and by comparisons of FAK-null (FAK^–/–) and FAK^+/+ mouse embryo fibroblasts. FAK promoted TNF-stimulated MAPK activation needed for maximal IL-6 production. FAK was not required for TNF-mediated nuclear factor-B or c-Jun N-terminal kinase activation. TNF-stimulated FAK catalytic activation and IL-6 production were inhibited by FAK N-terminal but not FAK C-terminal domain overexpression. Analysis of FAK^–/– fibroblasts stably reconstituted with wild type or various FAK point mutants showed that FAK catalytic activity, Tyr-397 phosphorylation, and the Pro-712/713 proline-rich region of FAK were required for TNF-stimulated MAPK activation and IL-6 production. Constitutively activated MAPK kinase-1 (MEK1) expression in FAK^–/– and A549 FAK short hairpin RNA-expressing cells rescued TNF-stimulated IL-6 production. Inhibition of Src protein-tyrosine kinase activity or mutation of Src phosphorylation sites on FAK (Tyr-861 or Tyr-925) did not affect TNF-stimulated IL-6 expression. Moreover, analyses of Src^–/–, Yes^–/–, and Fyn^–/– fibroblasts showed that Src expression was inhibitory to TNF-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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Focal adhesion kinase (FAK)² is best known for its role as an integrin-stimulated protein-tyrosine kinase (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 N-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 Tyr-397 (Tyr(P)-397), promotes Src family protein-tyrosine kinase binding to the FAK Tyr(P)-397 site, and facilitates the formation of the FAK-Src signaling complex that results in the secondary phosphorylation of FAK at Tyr-861 and Tyr-925 (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 overexpression 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 transcription, and the tumor necrosis factor- (TNF) cytokine is a potent stimulator of IL-6 expression (12). TNF binds to two distinct cell surface receptors (TNFR) 1 and TNFR2, which can activate a variety of intracellular signaling cascades (12). TNF can activate FAK, and this is linked to enhanced cell motility (13), gene expression (14, 15), and survival (16, 17). TNF-stimulated genes include IL-1, IL-1, IL-6, IL-8, IL-18, cycloxygenase-2, matrix metalloproteinase-9 (MMP-9), and vascular endothelial growth factor (VEGF) (18). FAK promotes matrix metalloproteinase-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-B (NF-B) is an important mediator of TNF signaling, and NF-B activation is a major regulator of TNF-stimulated IL-6 expression (21). As FAK has been linked to both TNF-stimulated NF-B activation (22) and TNF-stimulated IL-6 expression (14), we have assessed the molecular mechanism(s) of how FAK promotes TNF-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 TNF-stimulated IL-6 expression. Surprisingly, TNF-stimulated NF-B and JNK activation were normal in FAK^–/– cells, yet TNF did not function to promote IL-6 expression in the absence of FAK or in FAK shRNA knockdown cells. We found that TNF-stimulated FAK catalytic activation, FAK Tyr-397 phosphorylation, and the Pro-712/713 proline-rich region of FAK were required for TNF-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 Tyr-861 or Tyr-925 did not affect TNF-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 TNF stimulation. As TNF-stimulated FAK activation and IL-6 expression were inhibited by overexpression 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

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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 (University of California, 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 containing 10% fetal bovine serum (FBS).

Antibodies and Reagents—IB polyclonal, NF-B p65 monoclonal, and FAK Tyr(P)925 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal antibodies (mAbs) to FAK (clone 4.47) and to phosphotyrosine (4G10) were from Upstate (Charlottesville, VA). Pyk2 and MEK1 mAbs were from BD Transduction Laboratories (San Diego). mAbs to -actin (AC-74) were from Sigma. Myc tag (9E10) and GFP tag antibodies were from Covance (Berkeley, CA). ERK1/2 Thr(P)-185-Tyr(P)-187, ERK1/2, JNK1/2 Thr(P)-183-Tyr(P)-185, FAK Tyr(P)-397, and FAK Tyr(P)-861 polyclonal antibodies were from BIOSOURCE. FAK Tyr(P)-576/Tyr(P)-577 and MEK1/2 Ser(P)-217/221 antibodies were from Cell Signaling Technologies (Danvers, MA). Phycoerythrin-conjugated anti-mouse TNFR1 and TNFR2 antibodies were from BioLegend (San Diego). Rabbit polyclonal anti-FAK (5904) directed against N-terminal residues 8–27 was generated as described (24). Recombinant TNF and IL-6 detection kits were from eBioscience (San Diego). Pharmacological inhibitors PP2, PD98059, SB203580, SP600125, and wortmannin were from Calbiochem.

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'-tgtctccgaacgtgtcacgtttcaagagaacgtgacacgttcggagacttttttc-3' and reverse 5'-tcgagaaaaaagtctccgaacgtgtcacgttctcttgaaacgtgacacgttcggagaca-3' was used as a control. For murine Pyk2 shRNA, the oligonucleotides forward 5'-tgaagtagttcttaaccgcattcaagagatgcggttaagaactacttcttttttc-3' and reverse 5'-tcgagaaaaaagaagtagttcttaaccgcatctcttgaatgcggttaagaactacttca-3' were cloned into pLentiLox. Cells were sorted for GFP expression, and the shRNA efficacy was verified by immunoblotting. Rabbit CA MEK1 (S231E/S235E) was a kind gift from Jiahuai Han (The Scripps Research Institute). The 1.2-kb MEK1 cDNA was amplified by 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 were produced as described (20). FAK^–/– fibroblasts and A549 carcinoma cells were selected in puromycin (5 µg/ml), and CA-MEK1 expression was verified by immunoblotting.

HA-tagged FRNK and FRNK Ser-1034 were created and used as described (27). Briefly, cells were infected with five plaque-forming units/cell for Ad-TA (mock control) or with five Ad-TA plus 50 plaque-forming units/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 the EcoRV site of pCMV-Shuttle (Stratagene), and virus was produced using the AdEasy system (Stratagene). Cells were infected with 50 plaque-forming units/cell GFP or GFP-FAK FERM constructs and analyzed after 48 h.

GFP-FAK-reconstituted FAK^–/– Fibroblasts—GFP-FAK wild type (WT), kinase-inactive (Arg-454), and Phe-925 FAK in the retroviral pBabepuro vector were created as described (20). FAK mutants (Phe-397, Ala-712/Ala-713, and Phe-861) 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. 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 include 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 TNF (10 ng/ml) in Dulbecco's modified Eagle's medium 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 the manufacturer's instructions. All samples were analyzed in duplicate.

Fluorescence-activated Cell Sorting Analysis—FAK^+/+ and FAK^–/– fibroblasts were trypsinized, fixed with 3.7% formaldehyde solution for 10 min at room temperature, and incubated with phycoerythrin-conjugated anti-tumor necrosis factor receptor-1 (TNFR1) or TNFR2 antibodies. After extensive phosphate-buffered saline washes, cell surface TNFR1 and TNFR2 levels were analyzed by flow cytometry and compared with IgG control.

Total RNA Preparation and Reverse Transcription (RT)-PCR—Total RNA was isolated from cells using TRIzol reagent (Invitrogen). First strand synthesis was conducted using Superscript First Strand Synthesis kit (Invitrogen) with random primers and 5 µg of total RNA as template. PCR was performed using TaqPro Complete (Denville Scientific, Metuchen, NJ). IL-6 and -actin were co-amplified with specific primer pairs (IL-6 forward, 5'-gatgctaccaaactggatataatc; IL-6 reverse, 5'-ggtccttagccactccttctgtg; -actin forward, 5'-tgtgatggtgggaatgggtcag; -actin reverse, 5'-tttgatgtcacgcacgatttcc). Cycling conditions were 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min. IL-6 was amplified for 31 cycles, with -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, pH 7.4, 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 [-³²P]dCTP (PerkinElmer Life Sciences) and Klenow DNA polymerase. EMSA was performed as described previously (29). Wild type NF-B oligonucleotides were 5'-acgtgtgggattttccatg (forward) and 5'-tcgacatgggaaaatcccac (reverse), whereas mutant oligonucleotides contained the sequences 5'-acgtgttacattttcccatg (forward) and 5'-tcgacatgggaaaatgtaac (reverse) with mutations preventing NF-B binding in boldface type.

Transfection and Luciferase Assay—FAK^+/+ and FAK^–/– fibroblasts were grown in 6-well plates and co-transfected with 5 µg of NF-B firefly luciferase reporter (from Qilin Pan, The Scripps Research Institute) and 1 µg of TK-Renilla luciferase (Promega, San Luis Obispo) using Lipofectamine 2000 (Invitrogen). After 24 h, transfected cells were stimulated with 10 ng/ml TNF for an additional 24 h. Cellular proteins were extracted in passive lysis buffer and analyzed according to the 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 0.1% Triton X-100 and 0.05% Tween 20 detergents for 6 min. Cells were stained with 0.2 ng/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 x20 (Olympus, NA = 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 of [-³²P]ATP (6000 Ci/mmol; PerkinElmer Life Sciences) for 15 min at 30 °C. The assay was stopped by adding 2x Laemmli sample buffer, resolved by SDS-PAGE, and transferred to polyvinylidene difluoride membrane. Labeled FAK was visualized by autoradiography and quantified using a PhosphorImager (GE Healthcare). Equal immunoprecipitation was verified by anti-FAK blotting.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
TNF-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 with scrambled (control) shRNA-expressing 4T1 cells as determined by ELISA (Fig. 1A). TNF is a strong promoter of IL-6 expression in many cell types (33). TNF 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 TNF-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 three cell lines, 70–90% reduction in FAK expression was accompanied by the inhibition of TNF-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 TNF stimulation. In wild type FAK^+/+ MEFs, TNF 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 TNF. Analysis of TNF receptors TNFR1 and TNFR2 by flow cytometry showed equivalent surface expression in FAK^+/+ and FAK^–/– MEFs (Fig. 1D), indicating that the defect in TNF-initiated IL-6 production is associated with the loss of intracellular FAK signaling. These results support the notion that FAK promotes TNF-induced IL-6 expression in both normal fibroblasts and carcinoma cells of murine and human origin.

View larger version (38K): [in this window] [in a new window]   FIGURE 1. FAK promotes TNF-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 were evaluated for basal (solid bars) and 10 ng/ml TNF-stimulated IL-6 secretion (open bars) by ELISA. FAK expression and actin levels in protein lysates were 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 TNF-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–/–MEFs were incubated with the indicated amount of TNF (1–100 ng/ml), and IL-6 expression was determined by ELISA. A–C, samples were tested in triplicate; error bars are ±S.D., and results represent at least two separate experiments. D, cell surface expression of TNFR1 (gray shade) and TNFR2 (dashed, open) were determined by antibody staining and flow cytometry. Anti-mouse IgG staining (black filled) was used as a control.

View larger version (91K): [in this window] [in a new window]   FIGURE 2. FAK promotes basal and TNF-induced IL-6 mRNA expression. A, semi-quantitative IL-6 RT-PCR was performed on RNA isolated from FAK+/+, FAK–/–, and FAK-reconstituted MEFs in 10% serum (Ctrl, open bars) or treated with 10 ng/ml TNF (solid bars) for 6 h prior to RNA extraction. -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 three independent experiments normalized to -actin mRNA levels. Values are fold change relative to untreated FAK+/+ cells, and error bars are ±S.D. B, FAK and actin expression levels in FAK+/+ fibroblasts expressing either anti-FAK or Scr shRNA as determined by immunoblotting. C, reduced basal (Control) and TNF-stimulated IL-6 mRNA levels in FAK shRNA expressing FAK+/+ fibroblasts compared with Scr shRNA controls as determined by RT-PCR as shown in A. Samples were tested in triplicate; error bars are ±S.D., and results represent three separate experiments. D, phase contrast images of growing FAK+/+, FAK+/+ Scr shRNA, and FAK+/+ FAK shRNA-expressing MEFs. Scale bar is 30 µm.

View larger version (67K): [in this window] [in a new window]   FIGURE 3. TNF-induced NF-B activation is not impaired in the absence of FAK. A, FAK+/+ and FAK–/–fibroblasts were incubated with 10 ng/ml TNF for the indicated times, proteins extracted, followed by IB and -actin immunoblotting. B, FAK shRNA expressing FAK+/+ cells were stimulated with or without TNF for 30 min followed by immunoblotting for IB and -actin levels. C, anti-Pyk2 or Scr shRNA expression in FAK–/–fibroblasts was used to inhibit Pyk2 expression as verified by anti-Pyk2 and actin immunoblotting. D, TNF-induced IB degradation and nuclear translocation of the p65 NF-B 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 TNF for 30 min. Nuclear extracts were isolated and tested for NF-B binding to 32P-labeled double-stranded NF-B oligonucleotides by electrophoretic mobility shift assay. 100-Fold excess of cold wild type (WT) or mutated (mut) NF-B oligonucleotides was added to verify binding specificity. N.S., nonspecific. F, FAK+/+ and FAK–/–cells were co-transfected with NF-B promoter-luciferase reporter and treated with 10 ng/ml TNF for 24 h. Luciferase activity is presented as fold increase relative to NF-B promoter activity in untreated FAK+/+ cells (control). Samples were tested in triplicate, error bars are ±S.D., and results represent two separate experiments.

TNF Activates FAK but FAK-associated Signaling Promoting IL-6 Expression Does Not Require Src—Many stimuli that promote FAK activation and Tyr-397 phosphorylation also lead to the formation of a FAK-Src signaling complex (7). As FAK has been shown to be activated by TNF in other cell types (13, 15), FAK IVK assays were performed after TNF stimulation of FAK^+/+ MEFs. Compared with basal levels of FAK IVK activity in serum-starved cells, maximal (>5-fold change) FAK IVK activity occurred within 5–15 min after TNF addition to FAK^+/+ MEFs (Fig. 4A). Src was not detected in the FAK immunoprecipitates (IPs) after TNF 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 TNF-stimulated FAK^+/+ MEFs. Notably, TNF-stimulated FAK activation at 15 min was reflected in 2-fold increased FAK Tyr-397 phosphorylation and >5-fold increased FAK Tyr-576/Tyr-577 phosphorylation within the kinase domain as detected by phosphospecific blotting of FAK IPs (Fig. 4B). TNF addition did not promote changes in FAK Tyr-861 phosphorylation (Fig. 4B), and analyses using phosphospecific antibodies to FAK Tyr-925 were not specific (data not shown).

To determine whether Src activity was required for TNF-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 TNF-induced IL-6 mRNA expression. Instead, PP2 addition resulted in 4-fold increased IL-6 mRNA levels over that stimulated by TNF in PP3-treated control MEFs (Fig. 4C). This result supports the notion that Src may act as an inhibitor to TNF-induced IL-6 expression. To test this possibility, triple null SYF fibroblasts as well as SYF MEFs re-expressing c-Src were analyzed for TNF-stimulated IL-6 production by ELISA (Fig. 4D). Src re-expression resulted in a 6-fold reduction in both basal and TNF-induced IL-6 expression in SYF MEFs. Together, these results support the conclusion that FAK is rapidly activated in response to TNF in an Src-independent manner. Additionally, our results support the notion that FAK and Src may have opposing functions in TNF-stimulated signaling.

View larger version (47K): [in this window] [in a new window]   FIGURE 4. TNF activates FAK independent of Src and signaling is blocked by FAK FERM domain overexpression. A, FAK+/+ fibroblasts were stimulated by TNF in the presence or absence of the Src inhibitor PP2 (20 µM) for the indicated times. FAK immunoprecipitates (IPs) were analyzed for associated IVK activity in a [-32P]ATP autophosphorylation assay (top), and FAK levels in the IPs were verified by immunoblotting (bottom). WB, Western blot. B, FAK+/+ fibroblasts were stimulated by TNF for the indicated time and FAK IPs analyzed by immunoblotting with Tyr(P)-397, Tyr(P)-576/Tyr(P)-577, Tyr(P)-861 (pY397, pY576/pY577, pY861), and FAK phosphospecific or anti-FAK antibodies. C, FAK+/+ fibroblasts were stimulated with 10 ng/ml TNF 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 were evaluated by RT-PCR with internal actin controls. Shown is an inverted image of ethidium bromide-stained gel and results ±S.D. represent two separate 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 TNF-stimulated (open bars) IL-6 protein expression by ELISA. Samples were tested in triplicate, error bars are ±S.D., and results represent three separate experiments. E, FAK+/+ fibroblasts were mock-treated or transduced with adenovirus for HA-tagged FRNK or FRNK Ser-1034 and after 48 h were stimulated with 10 ng/ml TNF 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 TNF 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 ±S.D. from two experiments. G, cell lysates were made (with or without TNF 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 phosphospecific Tyr(P)-576/Tyr(P)-577 and anti-FAK immunoblotting.

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 TNF-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 overexpressed 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). Overexpression of WT but not R177A/178A FAK FERM resulted in 2-fold inhibition of TNF-stimulated IL-6 expression (Fig. 4F). Notably, WT FAK FERM overexpression blocked TNF-stimulated FAK activation as determined by anti-Tyr(P)-576/Tyr(P)-577 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 TNF-stimulated FAK activation occurs through a novel FERM domain mechanism.

View larger version (44K): [in this window] [in a new window]   FIGURE 5. FAK kinase activity, Tyr-397 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 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 with autofluorescence levels of parental FAK–/– cells (filled peaks). 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 TNF-stimulated (open bars) IL-6 protein expression by ELISA. Samples were tested in triplicate, error bars are ±S.D., and results represent two separate experiments.

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 TNF was evaluated by ELISA (Fig. 5D). Wild type (WT) and two phosphorylation site GFP-FAK mutants (Phe-861 and Phe-925) restored both low level basal and TNF-stimulated IL-6 expression compared with GFP-expressing FAK^–/– controls. In contrast, phosphorylation site mutant Phe-397 FAK, kinase-inactive Arg-454 FAK, and SH3-binding site mutant Ala-712/713 FAK did not mediate IL-6 induction after TNF 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 TNF-stimulated IL-6 expression.

FAK Facilitates TNF-stimulated MAPK Activation—Since FAK mutants Phe-397, Arg-454, and Ala-712/713 failed to promote TNF-stimulated IL-6 production, we investigated the potential signaling defects associated with these mutations. Phosphospecific blotting after TNF stimulation was performed, and comparisons were made between WT GFP-FAK and mutant-reconstituted FAK^–/– MEFs (Fig. 6). Anti-phosphotyrosine (Tyr(P)) blotting showed that FAK mutants Phe-397, Arg-454, and Ala-712/713 exhibited reduced phosphorylation under basal and after TNF stimulation (Fig. 6A). Analysis of IB degradation after TNF 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 TNF addition as detected by phosphospecific immunoblotting (Fig. 6C).

Analysis of ERK/MAPK activation by phosphospecific antibodies directed to the MEK1 (Thr-185/Tyr-187) phosphorylation sites on ERK1/2 showed weak to no reactivity in GFP-expressing FAK^–/– MEFs after TNF addition (Fig. 6D). In contrast, ERK1/2 were rapidly phosphorylated within 5 min with a peak activation occurring at 15 min in TNF-stimulated GFP-FAK WT-reconstituted MEFs. FAK^–/– fibroblasts re-expressing FAK mutants Phe-397, Arg-454, or Ala-712/713 that were defective for TNF-stimulated IL-6 production showed no activation of ERK1/2 at 5 min and lower levels of ERK1/2 phosphorylation at 15 min compared with FAK WT-reconstituted cells (Fig. 6D). As both Phe-861 and Phe-925 FAK-reconstituted cells showed equivalent levels of TNF-stimulated ERK/MAPK activation compared with WT FAK reconstituted MEFs (data not shown), these studies support the conclusion that FAK facilitates signaling leading to ERK/MAPK activation needed for TNF-stimulated IL-6 expression. For FAK, this involves FAK kinase activity, Tyr-397 phosphorylation, and FAK association with SH3 domain-containing proteins but not FAK phosphorylation at Tyr-861 or Tyr-925.

View larger version (85K): [in this window] [in a new window]   FIGURE 6. FAK–/– or FAK mutant reconstituted fibroblasts that are defective for TNF-stimulated IL-6 expression exhibit reduced TNF-mediated ERK activation. A–D, the indicated GFP-only or GFP-FAK-reconstituted fibroblasts were grown to confluence, serum-starved for 16 h, and treated with TNF (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 TNF-stimulated IB degradation as determined by anti-IB blotting. C, kinetics of TNF-stimulated JNK activation as determined by blotting with phosphospecific antibodies to Thr(P)-183-Tyr(P)-185 JNK (pJNK1/2). D, kinetics of TNF-stimulated ERK-MAPK kinase activation as determined by blotting with phosphospecific antibodies to Thr(P)-185-Tyr(P)-187 ERK1/2 (pERK) or to total ERK1/2 protein.

View larger version (48K): [in this window] [in a new window]   FIGURE 7. MEK-ERK activation is sufficient to promote TNF-induced IL-6 expression in the absence of FAK. A, FAK+/+ or FAK–/– fibroblasts were serum-starved and treated with TNF (10 ng/ml) for the indicated times prior to protein extraction and blotting with phosphospecific antibodies to Thr(P)-185-Tyr(P)-187 ERK1/2 (pERK) or to total ERK1/2 protein. B, FAK+/+ fibroblasts were pretreated for 30 min with various kinase inhibitors (40 µM PD98059, 20 µM SP600125, or 250 nM wortmannin), incubated with/without 10 ng/ml TNF for 24 h in the continued presence of inhibitors. IL-6 production was measured by ELISA. Samples were tested in triplicate, error bars are ±S.D., and results represent two separate experiments. C, stable expression of CA Myc-tagged MEK1 in FAK–/– fibroblasts compared with vector control is verified by blotting protein lysates with anti-Myc tag, MEK1, or actin antibodies. Phosphospecific antibody blotting was used to visualize activated MEK1 (pMEK1, Ser(P)-217-Ser(P)-221) or activated ERK1/2 (pERK, Thr(P)-187-Tyr(P)-187). 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 TNF-stimulated (open bars) IL-6 protein expression by ELISA. Samples were tested in triplicate, error bars are ±S.D., and results represent three separate experiments.

View larger version (83K): [in this window] [in a new window]   FIGURE 8. CA-MEK1 expression rescues TNF IL-6 expression in A549 FAK shRNA carcinoma cells. A, TNF-stimulated MEK1 (pMEK1, Ser(P)-217-Ser(P)-221) and ERK1/2 activation (pERK, Thr(P)-187-Tyr(P)-187) as determined by phosphospecific antibody blotting of 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, Thr(P)-185-Tyr(P)-187) 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 TNF-stimulated (open bars) IL-6 protein expression by ELISA. Samples were tested in triplicate, error bars are ±S.D., and results represent three separate experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
In this study, we confirm that FAK is required for TNF-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-B activation by TNF is defective in FAK^–/– cells. In contrast, we found that all aspects of TNF induced NF-B activation, including IB degradation, NF-B nuclear translocation, NF-B DNA binding, and stimulated NF-B transactivation activity, are equivalent in FAK^+/+-, FAK^–/–-, FAK^+/+-, FAK shRNA-, and FAK^–/– Pyk2 shRNA-expressing fibroblasts. The activation of JNK by TNF 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. (14). 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 TNF-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 TNF. Together, these results support the conclusion that FAK expression is required for efficient ERK/MAPK activation after TNF 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 TNF-stimulated IL-6 production, we stably reconstituted FAK^–/– fibroblasts with wild type 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 the following: Phe-397, disrupting the major FAK phosphorylation site; Arg-454, creating a kinase-defective mutant; Ala-712/713, disrupting the first of two SH3 domain binding sites in the FAK C-terminal domain; and Phe-861 and Phe-925, mutations that prevent phosphorylation of these FAK C-terminal domain sites. Compared with GFP-expressing FAK^–/– cells, WT GFP-FAK re-expression rescued the defects in both TNF-stimulated ERK activation and IL-6 production. Additionally, FAK Phe-861 or Phe-925 mutations equally promoted TNF-stimulated IL-6 expression as did WT FAK. Although Tyr-861 FAK phosphorylation is implicated in regulating p130^Cas binding (37), the signaling connections of this FAK phosphorylation site remain undefined. However, integrin-stimulated FAK phosphorylation at Tyr-925 and Grb2 binding to FAK is one of several pathways through which FAK can enhance integrin-stimulated ERK activation (4). The fact that Phe-925 FAK promoted TNF-stimulated IL-6 expression is interesting as Phe-925 FAK did not promote Src-mediated ERK/MAPK activation regulating VEGF expression (20), and Phe-925 FAK expression in FAK^–/– cells did not promote matrix metalloproteinase-9 expression after TNF stimulation as did WT FAK (15). Together, these results suggest that FAK can promote ERK activation through multiple pathways and that distinct multiprotein FAK signaling complexes may have unique biological consequences.

The reconstitution studies of FAK^–/– cells showed that FAK mutants defective in catalytic activity, Tyr-397 phosphorylation, and the Pro-712/713 region did not promote TNF-stimulated ERK activation or IL-6 expression as did WT FAK. The importance of FAK catalytic activity and Tyr-397 phosphorylation in TNF 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 TNF-stimulated FAK catalytic activation. Instead, inhibition of Src expression or activity led to increased IL-6 production after TNF stimulation of cells. Additionally, Src phosphorylation sites Tyr-861 and Tyr-925 on FAK were not required for TNF-stimulated signaling promoting IL-6 expression. Moreover, integrin activation of FAK is blocked by C-terminal FRNK domain expression, whereas FRNK did not affect TNF-stimulated IL-6 production. Thus, despite the shared requirements of FAK catalytic activity and Tyr-397 phosphorylation, integrin and TNF stimulation of cells are likely to promote FAK activation and signaling to ERK through distinct mechanisms.

Insights into the potential molecular mechanism(s) of TNF-mediated FAK activation came from results showing that FAK FERM domain overexpression could inhibit both TNF-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). Although 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 TNF-stimulated FAK activation. We interpret this result as supportive of a role for FERM F2 domain in binding to a potential TNF-stimulated activator of FAK. As FAK forms a complex with the receptor interacting protein (RIP) 1 after TNF-stimulation (14), that FAK FERM domain can form a complex with RIP (38), and that RIP expression is required for TNF-stimulated ERK activation (39), future studies will be focused on testing this potential signaling linkage in mediating TNF-stimulated FAK activation.

	FOOTNOTES

 
^* This work was supported in part by CA102310 [GenBank] and CA87038 from the National Institutes of Health. This is manuscript 18575-IMM from The Scripps Research Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported in part by National Institutes of Health Grant CA75240 and American Heart Association Established Investigator Award 0540115N. To whom correspondence should be addressed: The Scripps Research Institute, Dept. of Immunology, IMM 21, 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-784-8207; Fax: 858-784-8289; E-mail: dschlaep{at}scripps.edu.

² The abbreviations used are: FAK, focal adhesion kinase; CA, constitutively activated; EMSA, electrophoretic mobility shift assay; ERK, extracellular signal-regulated kinase; FAK^–/–, FAK-null; FRNK, FAK-related non-kinase; GFP, green fluorescent protein; JNK, c-Jun N-terminal kinase; IL-6, interleukin 6; MEK1, MAPK kinase-1; MAPK, mitogen-activated protein kinase; mAbs, monoclonal antibodies; NF-B, nuclear factor-B; RT, reverse transcription; Scr, scrambled; shRNA, short-hairpin RNA; TNFR1, tumor necrosis factor receptor-1; TNF, tumor necrosis factor-; VEGF, vascular endothelial growth factor; and WT, wild type; ELISA, enzyme-linked immunosorbent assay; HA, hemagglutinin; IVK, in vitro kinase; IP, immunoprecipitate; RIP, receptor interacting protein; Ad, adenoviral; SH, Src homology; SYF, Src-Yes-Fyn null.

	ACKNOWLEDGMENTS

 
We thank Alan Saluk and the Scripps Flow Cytometry facility for outstanding technical assistance in creating GFP-FAK reconstituted cell lines.

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