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Phosphorylation of focal adhesion kinase at Tyrosine 407 negatively regulates Ras transformation of fibroblasts
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Biochemical and Biophysical Research Communications 364 (2007) 1062–1066 www.elsevier.com/locate/ybbrc
Phosphorylation of focal adhesion kinase at Tyrosine 407 negatively regulates Ras transformation of fibroblasts Jihyun Jeon a, Hyangjin Lee a, Haein Park a, Jung-hyun Lee a, Sojoong Choi a, Jisun Hwang b, Inn-Oc Han b, Eok-Soo Oh a,* a
Department of Life Sciences, Division of Life and Pharmaceutical Sciences and the Center for Cell Signaling & Drug Discovery Research, Ewha Womans University, Daehyun-dong, Seodaemoon-Gu, Seoul 120-750, Republic of Korea b College of Medicine, Department of Physiology and Biophysics, Inha University, Incheon 402-751, Republic of Korea Received 16 October 2007 Available online 30 October 2007
Abstract Focal adhesion kinase (FAK) mediates signal transduction in response to multiple extracellular inputs, via tyrosine phosphorylation at specific residues. We recently reported that FAK Tyr-407 phosphorylation negatively regulates the enzymatic and biological activities of FAK, unlike phosphorylation of other tyrosine residues. In this study, we further investigated the effect of FAK Tyr-407 phosphorylation on cell transformation. We found that FAK Tyr-407 phosphorylation was lower in H-Ras transformed NIH3T3 and K-Ras transformed rat-2 fibroblasts than in the respective untransformed control cells. Consistently, FAK Tyr-407 phosphorylation was decreased in parallel with cell transformation in H-Ras-inducible NIH3T3 cells and increased during trichostatin A-induced detransformation of both K-Ras transformed rat-2 fibroblasts and H-Ras transformed NIH3T3 cells. In addition, overexpression of a phosphorylation-mimicking FAK Tyr-407 mutant inhibited morphological transformation of H-Ras-inducible NIH3T3 cells and inhibited invasion activity and anchorage-independent growth of H-Ras-transformed NIH3T3 cells. Taken together, these data strongly suggest that FAK Tyr-407 phosphorylation negatively regulates transformation of fibroblasts. 2007 Elsevier Inc. All rights reserved. Keywords: Focal adhesion kinase; Transformation; Tyrosine phosphorylation; Signal transduction
Focal adhesion kinase (FAK) is a non-receptor cytoplasmic tyrosine kinase that modulates various cell functions, including survival, proliferation, and migration [1,2]. The ability of FAK to transduce downstream signals is dependent on phosphorylation at tyrosine residues [3]. Six tyrosine phosphoacceptor sites, Tyr-397, Tyr-407, Tyr-576, Tyr-577, Tyr-861, and Tyr-925, have been identified in FAK. Phosphorylation of most of these sites appears to play positive regulatory roles. In particular, phosphorylation of Tyr-397, Tyr-576, and Tyr-577 is
Abbreviation: DMEM, Dulbecco’s modified Eagle’s medium; FAK, focal adhesion kinase; FBS, fetal bovine serum; HA, hemagglutinin; SDS– PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; SFM, serum-free medium. * Corresponding author. Fax: +82 2 3277 3760. E-mail address: [email protected] (E.-S. Oh). 0006-291X/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.10.134
known to directly regulate the catalytic activity of FAK [1,2]. Unlike the other five phosphorylatable tyrosine residues, FAK Tyr-407 phosphorylation seems to negatively regulate FAK activation. We recently reported that higher levels of FAK Tyr-407 phosphorylation were observed under conditions of basal FAK activity, where relatively low levels of FAK Tyr-397 phosphorylation were also seen. In addition, FAK Tyr-407 phosphorylation inhibited the enzymatic activities and biological functions of FAK [4]. Tyrosine phosphorylation within FAK may therefore both activate and inhibit enzyme functions. Protein tyrosine kinases such as FAK play crucial roles in signal transduction, but their constitutive activation often leads to cancer formation and progression [5,6]. Indeed, high levels of FAK have been found in a variety of carcinomas [7,8] and aberrant FAK activation is known to contribute to the process of cell transformation by
J. Jeon et al. / Biochemical and Biophysical Research Communications 364 (2007) 1062–1066
certain oncoproteins, such as Ras [9–11]. As FAK activation is regulated by coordinated phosphorylation at various tyrosine residues, and as manipulation of the FAK tyrosine phosphorylation pattern can lead to cellular transformation, it seems reasonable to surmise that normal cells have regulatory mechanism(s) responsible for maintaining FAK activity at basal levels. On the other hand, transformed cells must have a regulatory mechanism enhancing FAK activity. This may be achieved either by increasing activating tyrosine phosphorylation or by decreasing inhibitory tyrosine phosphorylation. Here, we report that FAK tyrosine phosphorylation at Tyr-407 inhibits transformation of NIH3T3 fibroblasts and that dephosphorylation of FAK at Tyr-407 is crucial for Ras transformation of fibroblasts. Materials and methods Reagents and antibodies. Monoclonal antibodies (mAbs) against HA (12CA5) were purchased from Roche (Tokyo, Japan) and mAbs against ERK2 (K-23) and H-Ras (F235) were purchased from Santa Cruz Biotechnology (S0anta Cruz, CA). The mAb against FAK and polyclonal Ab to phosphorylation-site specific Abs against FAK[pY407] and FAK[pY861] were purchased from BioSource Quality Controlled Biochemicals, Inc. (Morgan Hill, CA). Cell culture. NIH3T3, Ha-Ras-NIH3T3, rat-2, K-Ras-rat-2 fibroblasts, H-Ras-inducible NIH3T3 cells [9], and human colon adenocarcinoma cell line Caco2 were maintained in Dulbecco’s modified Eagle’s medium (DMEM; invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS), 10 U/ml penicillin, and 10 lg/ml streptomycin. Construction of mutant FAK mammalian expression vectors. The full length cDNA encoding FAK was subjected to site-directed mutagenesis using the Transformer site-directed mutagenesis kit (Clontech, Palo Alto, CA). The synthetic oligonucleotide, GGA AGA CAC ATT TAC CAT GCC CTC G (Tyr-407 fi Phe), or GGA AGA CAC AGA AAC CAT GCC CTC G (Tyr-407 fi Glu) were used to change Tyr-407 to phenylalanine (407F) or glutamic acid (407E). The cDNAs encoding wild-type FAK and mutant 407F and 407E were inserted into pRC/CMV at the NotI/XbaI cloning sites to generate in-frame fusions with a 3 0 sequence encoding the HA epitopes (YPYDVPDYA). Transfection. Transient transfections were carried out using LipofectAMINE reagent (Invitrogen) as described by the manufacturer. In brief, NIH3T3 cells were plated in 60-mm dishes and grown to 80% confluence for 24 h. Cells were then transfected with a mixture of 15 ll of LipofectAMINE and 4 lg of plasmid DNA. After 5 h, the transfection mixture was removed and replaced with medium containing 10% FBS. Synthesis and transfection of siRNA constructs. To design oligonucleotides targeting the mouse FAK mRNA for degradation, siRNA Design of Ambion was used. The chosen targeted FAK siRNA sequence (5 0 -AAT GCCCTAGAGAAGAAGTCC-3 0 ) was chemically synthesized by Ambion (Austin, TX) and the negative control siRNAs were purchased from Ambion. In vitro cotransfections were performed with 500 nM of mouse siRNAs and 4 lg of constructs encoding chicken FAK with and without introduced mutations (407Y, 407F, 407E), using LipofectAMINE 2000 (Invitrogen, Carlsbad, CA), according to the manufacturer’s protocols. Invasion assay. Each well of 24-well Transwell plates was coated with fibronectin (10 lg/ml) on the lower side and with Matrigel (30 lg/ll) on the upper side of the membrane and the membranes were allowed to dry for 1 h at 25 C. Cells transfected with the various constructs (5 · 104 cells) were added to the upper compartment of each well, and the plate was incubated for 6 h at 37 C in a 5% CO2 atmosphere. Non-migrated cells on the upper membrane were removed with a cotton swab. Migrated cells (located on the lower surface of the filters) were fixed for 5 min in methanol, stained with 0.6% hematoxylin, and 0.5% eosin, and then counted.
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Anchorage-independent growth in soft agarose. Each well of a 6-well culture plate was coated with 3 ml of bottom agar mixture (MEM/10% FBS/0.5% agar). After the bottom layer had solidified, 2 ml of top agar mixture (MEM/10% FBS/0.3% agar) containing HT1080 transfectants (1 · 105 cells) was added to each well, and the cultures were incubated at 37 C in a 5% CO2. Every 5 days, normal growth medium was gently layered over the cultures. Colony formation was monitored daily with a light microscope, and the formation of colonies was scored after 14 days. Immunoblotting. Cells were washed twice with PBS and were lysed in RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 10 mM NaF, 2 mM Na3VO4) containing a protease inhibitor cocktail (1 lg/ml aprotinin, 1 lg/ml anti-pain, 5 lg/ml leupeptin, 1 lg/ml pepstatin A, 20 lg/ml phenylmethylsulfonyl fluoride). The lysates were clarified by centrifugation at 14,000 rpm for 15 min at 4 C, denatured with SDS sample buffer, boiled, and analyzed by SDS–PAGE. Proteins were transferred onto polyvinylidene difluoride membranes (Amersham Pharmacia Biotech), incubated with the appropriate primary antibodies, detected with species-specific horseradish peroxidase-conjugated secondary antibodies (Amersham Life science), and visualized by enhanced chemiluminescence (ECL; Amersham Life science).
Results and discussion FAK Tyr-407 phosphorylation decreases during cell transformation High levels of FAK have been found in a variety of carcinomas and FAK is known to play a critical role in cancer progression and cell transformation [7,8]. We have also reported that FAK Tyr-407 phosphorylation negatively regulates FAK functions [4]. Therefore, we investigated whether negative regulation of FAK though FAK Tyr407 phosphorylation might affect cell transformation. We first examined FAK Tyr-407 phosphorylation in Ras-transformed fibroblasts. Compared with untransformed cells, FAK Tyr-407 phosphorylation was decreased in H-Ras transformed NIH3T3 cells (Fig. 1A, left panel). Similarly, FAK Tyr-407 phosphorylation was lower in K-Ras transformed rat-2 fibroblasts than in control untransformed cells (Fig. 1A, right panel), suggesting that FAK Tyr-407 phosphorylation might be decreased during cell transformation. To further examine the role of FAK Tyr-407 phosphorylation on cell transformation, NIH 3T3 cells were transfected with a tetracycline-inducible H-Ras expression vector. Consistent with previous data [9], these cells gradually expressed H-Ras with exhibited a transformed morphology (data not shown, 9). Also, FAK Tyr-861 phosphorylation increased in response to 2 lg/ml doxycyclin. Interestingly, FAK Tyr-407 phosphorylation decreased in a time-dependent manner over 96 h (Fig. 1B). Given that inhibition of histone deacetylase activity is known to revert transformed phenotypes of cells to detransformed phenotypes [12], we examined FAK Tyr407 phosphorylation during Trichostatin A (TSA)-induced detransformation of fibroblasts. As shown in Fig. 1C and D, TSA induced increased FAK Tyr-407 phosphorylation in both H-Ras-transformed NIH3T3 cells and K-Ras rat2 fibroblasts. Collectively, these data suggest that FAK Tyr-407 phosphorylation is decreased during Ras transformation of fibroblasts.
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Fig. 1. FAK Tyr-407 phosphorylation is decreased in H-Ras transformed NIH 3T3 cells. (A) Total cell lysates (30 lg) from either NIH3T3 and HRas-transformed NIH3T3 cells (left panel) or rat-2-fibroblasts and K-Rasrat-2 fibroblasts (right panel) were resolved by SDS–PAGE and subjected to immunoblotting with anti-FAK[pY407], or anti-FAK antibodies. (B) H-Ras-inducible NIH3T3 cells were treated with 2 lg/ml doxycyclin for the indicated periods. Cell lysates extracted at the indicated times were immunoblotted with antibodies against FAK[pY407], FAK[pY861], FAK, and H-Ras. (C) K-Ras transformed rat-2 fibroblasts were treated with 330 nM of TSA for the indicated periods of time. Phosphorylation of FAK was analyzed as described in (B). (D) Total cell lysate (30 lg) from NIH3T3 cells, H-Ras-transformed NIH3T3 cells or H-Ras-transformed NIH3T3 cells treated with 330 nM of TSA for 24 h were resolved by SDS– PAGE and subjected to immunoblotting with anti-FAK[pY407], or antiFAK antibodies.
Fig. 2. FAK Tyr-407 phosphorylation in Ras-transformed fibroblasts does not increase during cell cycle arrest. (A) NIH3T3 cells (left, top panel), H-Ras transformed NIH 3T3 cells (left, bottom panel), rat-2 fibroblasts (right, top panel), or K-Ras transformed rat-2 fibroblasts (right, bottom panel) were cultured for the indicated periods to achieve higher density saturation at confluence. Total cell lysates were immunoblotted with anti-FAK[pY407] and anti-FAK antibodies. Exposure times for H-Ras-NIH3T3 cells were longer than for NIH3T3 cells. (B) Either NIH3T3 cells (top panle) or H-Ras-NIH3T3 cells (bottom panel) were starved for the indicated periods. Total cell lysates (20 lg) were immunoblotted with the indicated specific antibodies.
FAK Tyr-407 phosphorylation in Ras-transformed fibroblasts did not increase during cell cycle arrest We have shown that FAK Tyr-407 phosphorylation in NIH3T3 cells increases during cell cycle arrest induced by serum starvation and contact inhibition [4]. To examine the role of FAK Tyr-407 phosphorylation in cell cycle arrest, both NIH3T3 and H-Ras-transformed NIH3T3 cells were grown to high density saturations at confluence. Unlike NIH3T3 cells, which showed a gradual increase in FAK Tyr-407 phosphorylation due to contact inhibition (Fig. 2A, left top panel), H-Ras transformed cells did not show this effect (Fig. 2A, left bottom panel). Similarly, an increase of FAK Tyr-407 phosphorylation was detected in rat-2 fibroblasts (Fig. 2A, top panel), but not in K-Ras rat-2 fibroblasts cells (Fig. 2A, bottom panel). Consistently, FAK Tyr-407 phosphorylation increased in NIH3T3 cells (Fig. 2B, top panel), but not in H-Ras transformed cells, during serum starvation (Fig. 2B, bottom panel). Thus, it was hardly seen cell cycle arrest-induced FAK Tyr-407 phosphorylation in Ras-transformed fibroblasts. All these data suggest that decreased FAK Tyr407 phosphorylation may be necessary for Ras-induced cell transformation (Fig. 3). FAK phosphorylation at Tyr-407 inhibits H-Ras mediated cell transformation To investigate the level of FAK Tyr-407 phosphorylation during cell transformation, NIH3T3 cells stably
Fig. 3. FAK phosphorylation at Tyr-407 inhibits H-Ras mediated cell transformation. (A) H-Ras-inducible NIH3T3 cells transfected with 4 lg of either empty vector (vector), HA-tagged FAK (407Y), or HA-tagged Y407E mutant FAK (407E) were treated with 2 lg/ml doxycyclin for 24 h. The number of cells showing the transformed cell morphology of fibroblast was counted. (B) H-Ras-transformed NIH3T3 cells were cotransfected with 500 nM of siRNA targeting mFAK and with 4 lg of either empty vector () or the indicated chicken FAK (cFAK) mutant constructs. Exponentially growing cells were lysed, and total cell lysates were analyzed by SDS/PAGE followed by blotting with anti-FAK, antiHA, and anti-ERK antibodies. (C) Cells were loaded onto the upper compartments of Matrigel-coated Transwell chambers and incubated for 16 h. The number of invasive cells was counted, and the percent of invasive cells relative to that of the empty vector control are shown. (D) Cells (1 · 105) were seeded in soft agar and allowed to grow 21 days, and the number of viable colonies was counted. All results represent the average of at least three independent experiments.
expressing a tetracycline-inducible H-Ras expression vector were transfected with the empty vector, wild-type FAK (407Y), or phosphorylation-mimicking mutant FAK
J. Jeon et al. / Biochemical and Biophysical Research Communications 364 (2007) 1062–1066
(407E) and the morphological transformations of the cell lines were then analyzed. In response to 2 lg/ml doxycyclin, cells exhibited a spindle-like morphology with small round cell bodies, indicting morphological transformation (data not shown, 9). Compared with cells transfected with vector control (Vec), cells transfected with 407Y showed enhanced morphological transformation of NIH3T3 cells induced by H-Ras expression (Fig. 3A). Interestingly, however, cells transfected with the Y407E mutant showed the significant decrease of morphological transformation. Consistent with the morphological changes, H-Ras-transformed NIH3T3 cells transfected with the 407Y mutant FAK showed increased invasion activity. This was more prominent in H-Ras-transformed NIH3T3 cells transfected with the 407F mutant FAK than in cells transfected with the control vector. In contrast, transfection of H-Rastransformed NIH3T3 cells with the 407E mutant FAK inhibited invasion activity (data not shown, but refer to Fig. 3C and D), suggesting a negative role for FAK phosphorylation at Tyr-407 during Ras transformation of fibroblasts. To further investigate the role of phosphorylation of FAK residue Tyr-407 in cell transformation, we designed a unique 21-bp small interfering RNA (siRNA) sequence targeted against the mouse FAK (mFAK) mRNA, and used this siRNA to knockdown mFAK expression in HRas-NIH3T3 cells (Fig. 3B). As expected, H-Ras-NIH3T3 cells transfected with mFAK siRNA showed decreased expression of FAK (Fig. 3B, compare lanes 1 and 2). We then re-expressed recombinant HA-tagged chicken FAK (cFAK) in the knockdown NIH3T3 cells and compared attributes of the knockdown cells with those of cells expressing recombinant wild-type or mutant FAK proteins
Fig. 4. FAK phosphorylation at Tyr-407 inhibits the invasion activity of Caco2 cells. Caco2 cells were transfected with 4 lg of either empty vector (vector), HA-tagged FAK (407Y), HA-tagged Y407F mutant FAK (407F), or HA-tagged Y407E mutant FAK (407E). Total cell lysates (30 lg) were resolved by SDS–PAGE and subjected to immunoblotting with anti-HA and anti-FAK antibodies. Erk2 was used as a loading control (top panel). Cells were loaded onto the upper compartments of Matrigel-coated Transwell chambers and incubated for 16 h. The number of invasive cells was counted, and the percent of invasive cells relative to that of the empty vector control are shown (bottom panel).
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(compare lanes 3, 4, and 5). Consistently, H-Ras-transformed NIH3T3 cells transfected with the 407E mutant showed decreased invasion compared with cells transfected with 407Y (Fig. 3C). In addition, the 407E mutant expression caused decrease anchorage-independent growth in soft agar of H-Ras-transformed NIH3T3 cells (Fig. 3D), indicating that dephosphorylation of FAK Tyr-407 is critical for H-Ras transformation of NIH3T3 cells. Collectively, all these data further support the idea that FAK Tyr-407 phosphorylation negatively regulates cell transformation. FAK phosphorylation at Tyr-407 inhibits the invasion activity of colon cancer cells FAK is known to play a critical role in the regulation of tumor activity in various cancer cells including carcinomas as well as sarcomas [13–16]. We next investigated whether FAK Tyr-407 phosphorylation might be involved in the regulation of tumor activity in human colon adenocarcinoma cells. Caco2 cells were transfected with FAK or FAK mutants, and the invasion activities of the resulting cell lines were investigated (Fig. 4). Compared to vectortransfected cells, cell invasion was increased in cells transfected with either 407Y or 407F, but was markedly inhibited in cells transfected with 407E. It is thus likely that FAK Tyr-407 phosphorylation contributes to the regulation of colon cancer activity, in a manner similar to the Tyr-407 phosphorylation effect on cell transformation. In non-transformed cells, it is likely that FAK activity is maintained at basal levels through FAK Tyr-407 phosphorylation [9], which in turn negatively regulates cell transformation. To become transformed, non-transformed cells may acquire a regulatory mechanism enhancing FAK activity, probably by removal of inhibitory tyrosine phosphorylation (FAK Tyr-407 phosphorylation). The removal of FAK Tyr-407 phosphorylation results in FAK Tyr-397 phosphorylation followed by FAK interaction with Src and other adaptor molecules contributing to the transformation of fibroblasts. FAK Tyr-407 phosphorylation therefore plays a crucial role in maintaining basal FAK activity and in preventing fibroblasts from spontaneous transformation. This study implies that a full understanding of the role of FAK Tyr-407 phosphorylation may be essential for molecular dissection of cell transformation. At this point, however, it is unclear how altered FAK Tyr-407 phosphorylation is associated with transformation of fibroblasts. The identification of tyrosine kinases and tyrosine phosphatases that specifically regulate FAK Tyr-407 phosphorylation and dephosphorylation will illuminate the relationship between FAK Tyr-407 phosphorylation and cell transformation. Acknowledgments This study was supported by a grant of the Molecular and Cellular BioDiscovery Research Program (CBM-
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01B-2-1 to E.S.O.), and in part by a grant of the National R&D Program for Cancer Control, Ministry of Health & Welfare, Republic of Korea (0420070-1 to E.S.O.) and Grant No. R15-2006-020 from the NCRC program of the MOST and the KOSEF through the Center for Cell Signaling & Drug Discovery Research at Ewha Womans University. References [1] J.L. Guan, Focal adhesion kinase in integrin signaling, Matrix Biol. 16 (1997) 195–200. [2] S.K. Hanks, T.R. Polte, Signaling through focal adhesion kinase, Bioessays 19 (1997) 137–145. [3] D.D. Schlaepfer, S.K. Mitra, Multiple connections link FAK to cell motility and invasion, Curr. Opin. Genet. Dev. 14 (2004) 92–101. [4] Y. Lim, H. Park, J. Jeon, I. Han, J. Kim, E.H. Jho, E.S. Oh, Focal adhesion kinase is negatively regulated by phosphorylation at tyrosine 407, J. Biol. Chem. 282 (2007) 10398–10404. [5] G.W. McLean, N.O. Carragher, E. Avizienyte, J. Evans, V.G. Brunton, M.C. Frame, The role of focal-adhesion kinase in cancer—a new therapeutic opportunity, Nat. Rev. Cancer 5 (2005) 505–515. [6] D.D. Schlaepfer, S.K. Mitra, D. Ilic, Control of motile and invasive cell phenotypes by focal adhesion kinase, Biochim. Biophys. Acta 1692 (2004) 77–102. [7] T.P. Hecker, C.L. Gladson, Focal adhesion kinase in cancer, Front Biosci. 8 (2003) s705–s714. [8] L.J. Kornberg, Focal adhesion kinase and its potential involvement in tumor invasion and metastasis, Head Neck 20 (1998) 745–752.
[9] Y. Lim, I. Han, J. Jeon, H. Park, Y.Y. Bahk, E.S. Oh, Phosphorylation of focal adhesion kinase at tyrosine 861 is crucial for Ras transformation of fibroblasts, J. Biol. Chem. 279 (2004) 29060–29065. [10] N. Benlimame, Q. He, S. Jie, D. Xiao, Y.J. Xu, M. Loignon, D.D. Schlaepfer, FAK signaling is critical for ErbB-2/ErbB-3 receptor cooperation for oncogenic transformation and invasion, J. Cell Biol. 171 (2005) 505–516. [11] H.R. Reiske, J.H. Zhao, D.C. Han, L.A. Cooper, J.L. Guan, Analysis of FAK-associated signaling pathways in the regulation of cell cycle progression, FEBS Lett. 486 (2000) 275–280. [12] Y. Lim, I. Han, H.J. Kwon, E.S. Oh, Trichostatin A-induced detransformation correlates with decreased focal adhesion kinase phosphorylation at tyrosine 861 in Ras-transformed fibroblasts, J. Biol. Chem. 277 (2002) 12735–12740. [13] H.G. Yu, S.L. Tong, Y.M. Ding, J. Ding, X.M. Fang, X.F. Fang, X.F. Zhang, Z.J. Liu, Y.H. Zhou, Q.S. Liu, H.S. Luo, J.P. Yu, Enhanced expression of cholecystokinin-2 receptor promotes the progression of colon cancer through activation of focal adhesion kinase, Int. J. Cancer 119 (2006) 2724–2732. [14] R.S. Sawhney, M.M. Cookson, Y. Omar, J. Hauser, M.G. Brattain, Integrin alpha2-mediated ERK and calpain activation play a critical role in cell adhesion and motility via focal adhesion kinase signaling: identification of a novel signaling pathway, J. Biol. Chem. 281 (2006) 8497–8510. [15] H. Park, I. Han, H.J. Kwon, E.S. Oh, Focal adhesion kinase regulates syndecan-2-mediated tumorigenic activity of HT1080 fibrosarcoma cells, Cancer Res. 65 (2005) 9899–9905. [16] M. Hanada, K. Tanaka, Y. Matsumoto, F. Nakatani, R. Sakimura, T. Matsunobu, X. Li, T. Okada, T. Nakamura, M. Takasaki, Y. Iwamoto, Focal adhesion kinase is activated in invading fibrosarcoma cells and regulates metastasis, Clin. Exp. Metastasis 22 (2005) 485–494.
Biochemical and Biophysical Research Communications 364 (2007) 1062–1066 www.elsevier.com/locate/ybbrc
Phosphorylation of focal adhesion kinase at Tyrosine 407 negatively regulates Ras transformation of fibroblasts Jihyun Jeon a, Hyangjin Lee a, Haein Park a, Jung-hyun Lee a, Sojoong Choi a, Jisun Hwang b, Inn-Oc Han b, Eok-Soo Oh a,* a
Department of Life Sciences, Division of Life and Pharmaceutical Sciences and the Center for Cell Signaling & Drug Discovery Research, Ewha Womans University, Daehyun-dong, Seodaemoon-Gu, Seoul 120-750, Republic of Korea b College of Medicine, Department of Physiology and Biophysics, Inha University, Incheon 402-751, Republic of Korea Received 16 October 2007 Available online 30 October 2007
Abstract Focal adhesion kinase (FAK) mediates signal transduction in response to multiple extracellular inputs, via tyrosine phosphorylation at specific residues. We recently reported that FAK Tyr-407 phosphorylation negatively regulates the enzymatic and biological activities of FAK, unlike phosphorylation of other tyrosine residues. In this study, we further investigated the effect of FAK Tyr-407 phosphorylation on cell transformation. We found that FAK Tyr-407 phosphorylation was lower in H-Ras transformed NIH3T3 and K-Ras transformed rat-2 fibroblasts than in the respective untransformed control cells. Consistently, FAK Tyr-407 phosphorylation was decreased in parallel with cell transformation in H-Ras-inducible NIH3T3 cells and increased during trichostatin A-induced detransformation of both K-Ras transformed rat-2 fibroblasts and H-Ras transformed NIH3T3 cells. In addition, overexpression of a phosphorylation-mimicking FAK Tyr-407 mutant inhibited morphological transformation of H-Ras-inducible NIH3T3 cells and inhibited invasion activity and anchorage-independent growth of H-Ras-transformed NIH3T3 cells. Taken together, these data strongly suggest that FAK Tyr-407 phosphorylation negatively regulates transformation of fibroblasts. 2007 Elsevier Inc. All rights reserved. Keywords: Focal adhesion kinase; Transformation; Tyrosine phosphorylation; Signal transduction
Focal adhesion kinase (FAK) is a non-receptor cytoplasmic tyrosine kinase that modulates various cell functions, including survival, proliferation, and migration [1,2]. The ability of FAK to transduce downstream signals is dependent on phosphorylation at tyrosine residues [3]. Six tyrosine phosphoacceptor sites, Tyr-397, Tyr-407, Tyr-576, Tyr-577, Tyr-861, and Tyr-925, have been identified in FAK. Phosphorylation of most of these sites appears to play positive regulatory roles. In particular, phosphorylation of Tyr-397, Tyr-576, and Tyr-577 is
Abbreviation: DMEM, Dulbecco’s modified Eagle’s medium; FAK, focal adhesion kinase; FBS, fetal bovine serum; HA, hemagglutinin; SDS– PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; SFM, serum-free medium. * Corresponding author. Fax: +82 2 3277 3760. E-mail address: [email protected] (E.-S. Oh). 0006-291X/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.10.134
known to directly regulate the catalytic activity of FAK [1,2]. Unlike the other five phosphorylatable tyrosine residues, FAK Tyr-407 phosphorylation seems to negatively regulate FAK activation. We recently reported that higher levels of FAK Tyr-407 phosphorylation were observed under conditions of basal FAK activity, where relatively low levels of FAK Tyr-397 phosphorylation were also seen. In addition, FAK Tyr-407 phosphorylation inhibited the enzymatic activities and biological functions of FAK [4]. Tyrosine phosphorylation within FAK may therefore both activate and inhibit enzyme functions. Protein tyrosine kinases such as FAK play crucial roles in signal transduction, but their constitutive activation often leads to cancer formation and progression [5,6]. Indeed, high levels of FAK have been found in a variety of carcinomas [7,8] and aberrant FAK activation is known to contribute to the process of cell transformation by
J. Jeon et al. / Biochemical and Biophysical Research Communications 364 (2007) 1062–1066
certain oncoproteins, such as Ras [9–11]. As FAK activation is regulated by coordinated phosphorylation at various tyrosine residues, and as manipulation of the FAK tyrosine phosphorylation pattern can lead to cellular transformation, it seems reasonable to surmise that normal cells have regulatory mechanism(s) responsible for maintaining FAK activity at basal levels. On the other hand, transformed cells must have a regulatory mechanism enhancing FAK activity. This may be achieved either by increasing activating tyrosine phosphorylation or by decreasing inhibitory tyrosine phosphorylation. Here, we report that FAK tyrosine phosphorylation at Tyr-407 inhibits transformation of NIH3T3 fibroblasts and that dephosphorylation of FAK at Tyr-407 is crucial for Ras transformation of fibroblasts. Materials and methods Reagents and antibodies. Monoclonal antibodies (mAbs) against HA (12CA5) were purchased from Roche (Tokyo, Japan) and mAbs against ERK2 (K-23) and H-Ras (F235) were purchased from Santa Cruz Biotechnology (S0anta Cruz, CA). The mAb against FAK and polyclonal Ab to phosphorylation-site specific Abs against FAK[pY407] and FAK[pY861] were purchased from BioSource Quality Controlled Biochemicals, Inc. (Morgan Hill, CA). Cell culture. NIH3T3, Ha-Ras-NIH3T3, rat-2, K-Ras-rat-2 fibroblasts, H-Ras-inducible NIH3T3 cells [9], and human colon adenocarcinoma cell line Caco2 were maintained in Dulbecco’s modified Eagle’s medium (DMEM; invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS), 10 U/ml penicillin, and 10 lg/ml streptomycin. Construction of mutant FAK mammalian expression vectors. The full length cDNA encoding FAK was subjected to site-directed mutagenesis using the Transformer site-directed mutagenesis kit (Clontech, Palo Alto, CA). The synthetic oligonucleotide, GGA AGA CAC ATT TAC CAT GCC CTC G (Tyr-407 fi Phe), or GGA AGA CAC AGA AAC CAT GCC CTC G (Tyr-407 fi Glu) were used to change Tyr-407 to phenylalanine (407F) or glutamic acid (407E). The cDNAs encoding wild-type FAK and mutant 407F and 407E were inserted into pRC/CMV at the NotI/XbaI cloning sites to generate in-frame fusions with a 3 0 sequence encoding the HA epitopes (YPYDVPDYA). Transfection. Transient transfections were carried out using LipofectAMINE reagent (Invitrogen) as described by the manufacturer. In brief, NIH3T3 cells were plated in 60-mm dishes and grown to 80% confluence for 24 h. Cells were then transfected with a mixture of 15 ll of LipofectAMINE and 4 lg of plasmid DNA. After 5 h, the transfection mixture was removed and replaced with medium containing 10% FBS. Synthesis and transfection of siRNA constructs. To design oligonucleotides targeting the mouse FAK mRNA for degradation, siRNA Design of Ambion was used. The chosen targeted FAK siRNA sequence (5 0 -AAT GCCCTAGAGAAGAAGTCC-3 0 ) was chemically synthesized by Ambion (Austin, TX) and the negative control siRNAs were purchased from Ambion. In vitro cotransfections were performed with 500 nM of mouse siRNAs and 4 lg of constructs encoding chicken FAK with and without introduced mutations (407Y, 407F, 407E), using LipofectAMINE 2000 (Invitrogen, Carlsbad, CA), according to the manufacturer’s protocols. Invasion assay. Each well of 24-well Transwell plates was coated with fibronectin (10 lg/ml) on the lower side and with Matrigel (30 lg/ll) on the upper side of the membrane and the membranes were allowed to dry for 1 h at 25 C. Cells transfected with the various constructs (5 · 104 cells) were added to the upper compartment of each well, and the plate was incubated for 6 h at 37 C in a 5% CO2 atmosphere. Non-migrated cells on the upper membrane were removed with a cotton swab. Migrated cells (located on the lower surface of the filters) were fixed for 5 min in methanol, stained with 0.6% hematoxylin, and 0.5% eosin, and then counted.
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Anchorage-independent growth in soft agarose. Each well of a 6-well culture plate was coated with 3 ml of bottom agar mixture (MEM/10% FBS/0.5% agar). After the bottom layer had solidified, 2 ml of top agar mixture (MEM/10% FBS/0.3% agar) containing HT1080 transfectants (1 · 105 cells) was added to each well, and the cultures were incubated at 37 C in a 5% CO2. Every 5 days, normal growth medium was gently layered over the cultures. Colony formation was monitored daily with a light microscope, and the formation of colonies was scored after 14 days. Immunoblotting. Cells were washed twice with PBS and were lysed in RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 10 mM NaF, 2 mM Na3VO4) containing a protease inhibitor cocktail (1 lg/ml aprotinin, 1 lg/ml anti-pain, 5 lg/ml leupeptin, 1 lg/ml pepstatin A, 20 lg/ml phenylmethylsulfonyl fluoride). The lysates were clarified by centrifugation at 14,000 rpm for 15 min at 4 C, denatured with SDS sample buffer, boiled, and analyzed by SDS–PAGE. Proteins were transferred onto polyvinylidene difluoride membranes (Amersham Pharmacia Biotech), incubated with the appropriate primary antibodies, detected with species-specific horseradish peroxidase-conjugated secondary antibodies (Amersham Life science), and visualized by enhanced chemiluminescence (ECL; Amersham Life science).
Results and discussion FAK Tyr-407 phosphorylation decreases during cell transformation High levels of FAK have been found in a variety of carcinomas and FAK is known to play a critical role in cancer progression and cell transformation [7,8]. We have also reported that FAK Tyr-407 phosphorylation negatively regulates FAK functions [4]. Therefore, we investigated whether negative regulation of FAK though FAK Tyr407 phosphorylation might affect cell transformation. We first examined FAK Tyr-407 phosphorylation in Ras-transformed fibroblasts. Compared with untransformed cells, FAK Tyr-407 phosphorylation was decreased in H-Ras transformed NIH3T3 cells (Fig. 1A, left panel). Similarly, FAK Tyr-407 phosphorylation was lower in K-Ras transformed rat-2 fibroblasts than in control untransformed cells (Fig. 1A, right panel), suggesting that FAK Tyr-407 phosphorylation might be decreased during cell transformation. To further examine the role of FAK Tyr-407 phosphorylation on cell transformation, NIH 3T3 cells were transfected with a tetracycline-inducible H-Ras expression vector. Consistent with previous data [9], these cells gradually expressed H-Ras with exhibited a transformed morphology (data not shown, 9). Also, FAK Tyr-861 phosphorylation increased in response to 2 lg/ml doxycyclin. Interestingly, FAK Tyr-407 phosphorylation decreased in a time-dependent manner over 96 h (Fig. 1B). Given that inhibition of histone deacetylase activity is known to revert transformed phenotypes of cells to detransformed phenotypes [12], we examined FAK Tyr407 phosphorylation during Trichostatin A (TSA)-induced detransformation of fibroblasts. As shown in Fig. 1C and D, TSA induced increased FAK Tyr-407 phosphorylation in both H-Ras-transformed NIH3T3 cells and K-Ras rat2 fibroblasts. Collectively, these data suggest that FAK Tyr-407 phosphorylation is decreased during Ras transformation of fibroblasts.
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Fig. 1. FAK Tyr-407 phosphorylation is decreased in H-Ras transformed NIH 3T3 cells. (A) Total cell lysates (30 lg) from either NIH3T3 and HRas-transformed NIH3T3 cells (left panel) or rat-2-fibroblasts and K-Rasrat-2 fibroblasts (right panel) were resolved by SDS–PAGE and subjected to immunoblotting with anti-FAK[pY407], or anti-FAK antibodies. (B) H-Ras-inducible NIH3T3 cells were treated with 2 lg/ml doxycyclin for the indicated periods. Cell lysates extracted at the indicated times were immunoblotted with antibodies against FAK[pY407], FAK[pY861], FAK, and H-Ras. (C) K-Ras transformed rat-2 fibroblasts were treated with 330 nM of TSA for the indicated periods of time. Phosphorylation of FAK was analyzed as described in (B). (D) Total cell lysate (30 lg) from NIH3T3 cells, H-Ras-transformed NIH3T3 cells or H-Ras-transformed NIH3T3 cells treated with 330 nM of TSA for 24 h were resolved by SDS– PAGE and subjected to immunoblotting with anti-FAK[pY407], or antiFAK antibodies.
Fig. 2. FAK Tyr-407 phosphorylation in Ras-transformed fibroblasts does not increase during cell cycle arrest. (A) NIH3T3 cells (left, top panel), H-Ras transformed NIH 3T3 cells (left, bottom panel), rat-2 fibroblasts (right, top panel), or K-Ras transformed rat-2 fibroblasts (right, bottom panel) were cultured for the indicated periods to achieve higher density saturation at confluence. Total cell lysates were immunoblotted with anti-FAK[pY407] and anti-FAK antibodies. Exposure times for H-Ras-NIH3T3 cells were longer than for NIH3T3 cells. (B) Either NIH3T3 cells (top panle) or H-Ras-NIH3T3 cells (bottom panel) were starved for the indicated periods. Total cell lysates (20 lg) were immunoblotted with the indicated specific antibodies.
FAK Tyr-407 phosphorylation in Ras-transformed fibroblasts did not increase during cell cycle arrest We have shown that FAK Tyr-407 phosphorylation in NIH3T3 cells increases during cell cycle arrest induced by serum starvation and contact inhibition [4]. To examine the role of FAK Tyr-407 phosphorylation in cell cycle arrest, both NIH3T3 and H-Ras-transformed NIH3T3 cells were grown to high density saturations at confluence. Unlike NIH3T3 cells, which showed a gradual increase in FAK Tyr-407 phosphorylation due to contact inhibition (Fig. 2A, left top panel), H-Ras transformed cells did not show this effect (Fig. 2A, left bottom panel). Similarly, an increase of FAK Tyr-407 phosphorylation was detected in rat-2 fibroblasts (Fig. 2A, top panel), but not in K-Ras rat-2 fibroblasts cells (Fig. 2A, bottom panel). Consistently, FAK Tyr-407 phosphorylation increased in NIH3T3 cells (Fig. 2B, top panel), but not in H-Ras transformed cells, during serum starvation (Fig. 2B, bottom panel). Thus, it was hardly seen cell cycle arrest-induced FAK Tyr-407 phosphorylation in Ras-transformed fibroblasts. All these data suggest that decreased FAK Tyr407 phosphorylation may be necessary for Ras-induced cell transformation (Fig. 3). FAK phosphorylation at Tyr-407 inhibits H-Ras mediated cell transformation To investigate the level of FAK Tyr-407 phosphorylation during cell transformation, NIH3T3 cells stably
Fig. 3. FAK phosphorylation at Tyr-407 inhibits H-Ras mediated cell transformation. (A) H-Ras-inducible NIH3T3 cells transfected with 4 lg of either empty vector (vector), HA-tagged FAK (407Y), or HA-tagged Y407E mutant FAK (407E) were treated with 2 lg/ml doxycyclin for 24 h. The number of cells showing the transformed cell morphology of fibroblast was counted. (B) H-Ras-transformed NIH3T3 cells were cotransfected with 500 nM of siRNA targeting mFAK and with 4 lg of either empty vector () or the indicated chicken FAK (cFAK) mutant constructs. Exponentially growing cells were lysed, and total cell lysates were analyzed by SDS/PAGE followed by blotting with anti-FAK, antiHA, and anti-ERK antibodies. (C) Cells were loaded onto the upper compartments of Matrigel-coated Transwell chambers and incubated for 16 h. The number of invasive cells was counted, and the percent of invasive cells relative to that of the empty vector control are shown. (D) Cells (1 · 105) were seeded in soft agar and allowed to grow 21 days, and the number of viable colonies was counted. All results represent the average of at least three independent experiments.
expressing a tetracycline-inducible H-Ras expression vector were transfected with the empty vector, wild-type FAK (407Y), or phosphorylation-mimicking mutant FAK
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(407E) and the morphological transformations of the cell lines were then analyzed. In response to 2 lg/ml doxycyclin, cells exhibited a spindle-like morphology with small round cell bodies, indicting morphological transformation (data not shown, 9). Compared with cells transfected with vector control (Vec), cells transfected with 407Y showed enhanced morphological transformation of NIH3T3 cells induced by H-Ras expression (Fig. 3A). Interestingly, however, cells transfected with the Y407E mutant showed the significant decrease of morphological transformation. Consistent with the morphological changes, H-Ras-transformed NIH3T3 cells transfected with the 407Y mutant FAK showed increased invasion activity. This was more prominent in H-Ras-transformed NIH3T3 cells transfected with the 407F mutant FAK than in cells transfected with the control vector. In contrast, transfection of H-Rastransformed NIH3T3 cells with the 407E mutant FAK inhibited invasion activity (data not shown, but refer to Fig. 3C and D), suggesting a negative role for FAK phosphorylation at Tyr-407 during Ras transformation of fibroblasts. To further investigate the role of phosphorylation of FAK residue Tyr-407 in cell transformation, we designed a unique 21-bp small interfering RNA (siRNA) sequence targeted against the mouse FAK (mFAK) mRNA, and used this siRNA to knockdown mFAK expression in HRas-NIH3T3 cells (Fig. 3B). As expected, H-Ras-NIH3T3 cells transfected with mFAK siRNA showed decreased expression of FAK (Fig. 3B, compare lanes 1 and 2). We then re-expressed recombinant HA-tagged chicken FAK (cFAK) in the knockdown NIH3T3 cells and compared attributes of the knockdown cells with those of cells expressing recombinant wild-type or mutant FAK proteins
Fig. 4. FAK phosphorylation at Tyr-407 inhibits the invasion activity of Caco2 cells. Caco2 cells were transfected with 4 lg of either empty vector (vector), HA-tagged FAK (407Y), HA-tagged Y407F mutant FAK (407F), or HA-tagged Y407E mutant FAK (407E). Total cell lysates (30 lg) were resolved by SDS–PAGE and subjected to immunoblotting with anti-HA and anti-FAK antibodies. Erk2 was used as a loading control (top panel). Cells were loaded onto the upper compartments of Matrigel-coated Transwell chambers and incubated for 16 h. The number of invasive cells was counted, and the percent of invasive cells relative to that of the empty vector control are shown (bottom panel).
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(compare lanes 3, 4, and 5). Consistently, H-Ras-transformed NIH3T3 cells transfected with the 407E mutant showed decreased invasion compared with cells transfected with 407Y (Fig. 3C). In addition, the 407E mutant expression caused decrease anchorage-independent growth in soft agar of H-Ras-transformed NIH3T3 cells (Fig. 3D), indicating that dephosphorylation of FAK Tyr-407 is critical for H-Ras transformation of NIH3T3 cells. Collectively, all these data further support the idea that FAK Tyr-407 phosphorylation negatively regulates cell transformation. FAK phosphorylation at Tyr-407 inhibits the invasion activity of colon cancer cells FAK is known to play a critical role in the regulation of tumor activity in various cancer cells including carcinomas as well as sarcomas [13–16]. We next investigated whether FAK Tyr-407 phosphorylation might be involved in the regulation of tumor activity in human colon adenocarcinoma cells. Caco2 cells were transfected with FAK or FAK mutants, and the invasion activities of the resulting cell lines were investigated (Fig. 4). Compared to vectortransfected cells, cell invasion was increased in cells transfected with either 407Y or 407F, but was markedly inhibited in cells transfected with 407E. It is thus likely that FAK Tyr-407 phosphorylation contributes to the regulation of colon cancer activity, in a manner similar to the Tyr-407 phosphorylation effect on cell transformation. In non-transformed cells, it is likely that FAK activity is maintained at basal levels through FAK Tyr-407 phosphorylation [9], which in turn negatively regulates cell transformation. To become transformed, non-transformed cells may acquire a regulatory mechanism enhancing FAK activity, probably by removal of inhibitory tyrosine phosphorylation (FAK Tyr-407 phosphorylation). The removal of FAK Tyr-407 phosphorylation results in FAK Tyr-397 phosphorylation followed by FAK interaction with Src and other adaptor molecules contributing to the transformation of fibroblasts. FAK Tyr-407 phosphorylation therefore plays a crucial role in maintaining basal FAK activity and in preventing fibroblasts from spontaneous transformation. This study implies that a full understanding of the role of FAK Tyr-407 phosphorylation may be essential for molecular dissection of cell transformation. At this point, however, it is unclear how altered FAK Tyr-407 phosphorylation is associated with transformation of fibroblasts. The identification of tyrosine kinases and tyrosine phosphatases that specifically regulate FAK Tyr-407 phosphorylation and dephosphorylation will illuminate the relationship between FAK Tyr-407 phosphorylation and cell transformation. Acknowledgments This study was supported by a grant of the Molecular and Cellular BioDiscovery Research Program (CBM-
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01B-2-1 to E.S.O.), and in part by a grant of the National R&D Program for Cancer Control, Ministry of Health & Welfare, Republic of Korea (0420070-1 to E.S.O.) and Grant No. R15-2006-020 from the NCRC program of the MOST and the KOSEF through the Center for Cell Signaling & Drug Discovery Research at Ewha Womans University. References [1] J.L. Guan, Focal adhesion kinase in integrin signaling, Matrix Biol. 16 (1997) 195–200. [2] S.K. Hanks, T.R. Polte, Signaling through focal adhesion kinase, Bioessays 19 (1997) 137–145. [3] D.D. Schlaepfer, S.K. Mitra, Multiple connections link FAK to cell motility and invasion, Curr. Opin. Genet. Dev. 14 (2004) 92–101. [4] Y. Lim, H. Park, J. Jeon, I. Han, J. Kim, E.H. Jho, E.S. Oh, Focal adhesion kinase is negatively regulated by phosphorylation at tyrosine 407, J. Biol. Chem. 282 (2007) 10398–10404. [5] G.W. McLean, N.O. Carragher, E. Avizienyte, J. Evans, V.G. Brunton, M.C. Frame, The role of focal-adhesion kinase in cancer—a new therapeutic opportunity, Nat. Rev. Cancer 5 (2005) 505–515. [6] D.D. Schlaepfer, S.K. Mitra, D. Ilic, Control of motile and invasive cell phenotypes by focal adhesion kinase, Biochim. Biophys. Acta 1692 (2004) 77–102. [7] T.P. Hecker, C.L. Gladson, Focal adhesion kinase in cancer, Front Biosci. 8 (2003) s705–s714. [8] L.J. Kornberg, Focal adhesion kinase and its potential involvement in tumor invasion and metastasis, Head Neck 20 (1998) 745–752.
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