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© The Rockefeller University Press,
0021-9525/2000//423 $5.00
The Journal of Cell Biology, Volume 149, Number 2,
, 2000 423-430
Original Article |
Suppression of Pyk2 Kinase and Cellular Activities by Fip200
jg19{at}cornell.edu
Proline-rich tyrosine kinase 2 (Pyk2) is a cytoplasmic tyrosine kinase implicated to play a role in several intracellular signaling pathways. We report the identification of a novel Pyk2-interacting protein designated FIP200 (FAK family kinase–interacting protein of 200 kD) by using a yeast two-hybrid screen. In vitro binding assays and coimmunoprecipitation confirmed association of FIP200 with Pyk2, and similar assays also showed FIP200 binding to FAK. However, immunofluorescent staining indicated that FIP200 was predominantly localized in the cytoplasm. FIP200 bound to the kinase domain of Pyk2 and inhibited its kinase activity in in vitro kinase assays. FIP200 also inhibited the kinase activity of the Pyk2 isolated from SYF cells (deficient in Src, Yes, and Fyn expression) and the Pyk2 mutant lacking binding site for Src, suggesting that it regulated Pyk2 kinase directly rather than affecting the associated Src family kinases. Consistent with its inhibitory effect in vitro, FIP200 inhibited activation of Pyk2 and Pyk2-induced apoptosis in intact cells, which correlated with its binding to Pyk2. Finally, activation of Pyk2 by several biological stimuli correlated with the dissociation of endogenous FIP200–Pyk2 complex, which provided further support for inhibition of Pyk2 by FIP200 in intact cells. Together, these results suggest that FIP200 functions as an inhibitor of Pyk2 via binding to its kinase domain.
Key Words: phosphorylation • FAK • tyrosine kinase • inhibitor • signal transduction
© 2000 The Rockefeller University Press
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45% amino acid identity and they both lack the Src homology 2 or 3 domains that are present in many other cytoplasmic tyrosine kinases. Both Pyk2 and FAK have large NH2- and COOH-terminal noncatalytic domains that flank a central kinase domain. The major autophosphorylation site of these kinases is also conserved. This site has been demonstrated to serve as a binding site for Src family kinases in both Pyk2 and FAK (Dikic et al. 1996). Finally, several other FAK-interacting proteins have been shown to bind Pyk2, although the biological significance of their association with Pyk2 has not been fully illustrated (Dikic et al. 1996; Salgia et al. 1996; Astier et al. 1997; Hiregowdara et al. 1997; Li and Earp 1997). Despite its structural similarity to FAK, Pyk2 appears to have different cellular roles than those of FAK. While FAK has been demonstrated to play an important role in integrin-mediated cell migration (Ilic et al. 1995; Cary et al. 1996, Cary et al. 1998; Gilmore and Romer 1996), the expression of Pyk2 did not promote cell migration in fibroblasts (Sieg et al. 1998). Recent studies also suggested that integrin signaling through FAK protected cells from apoptosis (Frisch et al. 1996) and stimulated cell cycle progression (Zhao et al. 1998). In contrast, overexpression of Pyk2 has been shown to induce apoptosis in a number of cell lines (Xiong and Parsons 1997). While its potential function in integrin signaling is not clear, Pyk2 has been suggested to play a role in a variety of other cellular processes including calcium-induced regulation of the ion channel and MAP kinase activation (Lev et al. 1995), stress-induced c-Jun NH2-terminal kinase activation (Tokiwa et al. 1996; Yu et al. 1996), and Src-mediated activation of MAP kinase signaling pathway in PC12 cells (Dikic et al. 1996).
Pyk2 is activated in response to a variety of extracellular stimuli that elevate the intracellular calcium concentration (Lev et al. 1995; Dikic et al. 1996; Tokiwa et al. 1996; Yu et al. 1996). These include activation of the nicotinic acetylcholine receptor or a voltage-gated calcium channel that induces calcium influxes, agonists for G-protein–coupled receptors such as bradykinin, lysophophatidic acid, and angiotensin II, and other agents that promote calcium release from intracellular stores (Dikic et al. 1996; Yu et al. 1996; Li and Earp 1997; Brinson et al. 1998). Therefore, it will be interesting to identify cellular proteins that interact with Pyk2 and/or regulate its activity. In this study, we identified a novel Pyk2-interacting protein using the yeast two-hybrid screen and showed that it functioned to inhibit Pyk2 kinase and cellular activity via binding to the kinase domain of Pyk2.
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Construction of cDNA Expression Vectors
pKH3-Pyk2 and pKH3-FAK have been described previously (Zheng et al. 1998). Pyk2 mutant Y402F was created by the PCR overlap extension method as described previously (Cary et al. 1996). The COOH-terminal FIP200 (residues 1,374–1,951) was excised from the prey plasmid pGAD33 and inserted into pGEX-KG to generate pGEX-CT-FIP. The insert is also cloned into the mammalian expression vector pSG5-Flag (gift of Dr. G. Mosialos, Harvard Medical School, Boston, MA) to generate pSG5-CT-FIP with in-frame fusion of the Flag epitope at the NH2 terminus. The full-length cDNA encoding FIP200 was provided by Dr. T. Nagase (Kazusa DNA Research Institute, Japan). Using this cDNA as a template, PCR was performed to generate a 1.9-kb NH2-terminal fragment of FIP200 (NT-FIP; residues 1–641) with an addition of an EcoRV linker at the 5' end (GATATC) before the ATG start codon and an internal BglII site at the 3' end. This fragment was digested with EcoRV and BglII, and was inserted into the corresponding cloning site of pSG5-Flag to generate plasmid pSG5-NT-FIP. A 3.9-kb fragment encoding residues 642–1,591 of FIP200 was isolated from the full-length clone by BglII digestion, and was inserted into the corresponding site in pSG5-NT-FIP to generate the plasmid pSG5-FIP200 encoding the full-length FIP200. The Flag epitope is fused in-frame to the ATG start codon in both pSG5-NT-FIP and pSG5-FIP200. All constructs were verified by DNA sequencing.
Cell Culture
293T, RASM, and Rat1 cells were obtained from ATCC and maintained in DME supplemented with 10% FBS (Life Technologies, Inc.). NIH3T3 cells were maintained in DME plus 10% FCS (Life Technologies, Inc.). CHO cells were maintained in F12 medium plus 10% fetal bovine serum. SYF cells (Klinghoffer et al. 1999) were gifts from Drs. L. Cary, R. Klinghoffer, and P. Soriano (Fred Hutchinson Cancer Research Center) and were maintained in DME supplemented with 10% FCS. Transient transfections of 293T, NIH3T3, CHO, and Rat1 cells were performed using Lipofectamine (Life Technologies, Inc.) according to the manufacturer's guidelines.
Preparation of GST Fusion Proteins and In Vitro Binding Assays
GST fusion proteins were produced and purified as described previously (Chen et al. 1995), except that a protease-defective Escherichia coli strain, BL21-Dex, was used. GST fusion proteins (5 µg) were immobilized on glutathione-agarose beads, and then incubated for 90 min at 4°C with lysates (200 µg) prepared from 293T cells that had been transfected with pKH3-Pyk2 or pKH3-FAK. After washing, the bound proteins were analyzed by Western blotting with anti-HA (1:600) as described below.
Immunoprecipitation and Western Blot
Cells were lysed with modified RIPA lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.3% sodium deoxycholate, 0.1% NP-40, 10% glycerol, 1.5 mM MgCl2, 1 mM EDTA, 0.2 mM EGTA, 20 mM NaF, 25 µM ZnCl2, 1 mM NaVO4, 1 mM PMSF, 10 µg/ml aprotinin, and 2 µg/ml leupeptin) as described previously (Zhao et al. 1998). Immunoprecipitation was carried out at 4°C by incubating cell lysates for 2 h with indicated antibodies, followed by an incubation for 1 h with protein A–Sepharose or protein G–Plus. Immunoprecipitates were washed three times in lysis buffer without protease inhibitors. The beads were resuspended in SDS-PAGE sample buffer, boiled for 5 min, and resolved by SDS-PAGE. Western blotting was performed with appropriate antibodies as indicated, using the Amersham ECL system as described previously (Chen et al. 1995; Zheng et al. 1998). In some experiments, whole cell lysates were analyzed directly by Western blotting.
In Vitro Kinase Assay
Cells were treated with 400 mM sorbitol for 5 min and lysed in 1% NP-40 lysis buffer as described previously (Zheng et al. 1998). The lysates were immunoprecipitated with anti-Pyk2 antibodies. They were washed three times with NP-40 buffer and once with 50 mM Tris, pH 7.4. Aliquots of the samples were subjected to in vitro kinase assays in kinase buffer (50 mM Tris, pH 7.4, 10 mM MnCl2, 20 µCi 
-[32P]ATP, and 10 µg E4Y1) for 20 min at room temperature in the presence of various amounts (0–5 µg) of GST or GST-CT-FIP. The kinase reactions were stopped by the addition of SDS sample buffer, boiled for 5 min, and resolved on SDS-PAGE. The gel was dried and subjected to autoradiography. The phosphorylated E4Y1 was also subjected to phosphoimage quantitative analysis by using the scanner model Storm 840 and ImageQuant IQMac v1.2 (Molecular Dynamics). The in vitro kinase assays for FAK were performed as described previously (Zhao et al. 1998).
Immunofluorescence
Cells were processed for immunofluorescence staining as described previously (Zhao et al. 1998; Zheng et al. 1998). The primary antibodies used were polyclonal anti-Flag (1:300), monoclonal anti-HA (1:200), and monoclonal antivinculin (1:50). The secondary antibodies used were fluorescein-conjugated goat anti–rabbit IgG (1:300), and rhodamine-conjugated goat anti–mouse IgG (1:300). The cells were mounted on Slowfade (Molecular Probes), examined, and photographed using an Olympus fluorescent microscope (100x).
Apoptosis Assay
Rat1 cells were cotransfected with a plasmid encoding GFP and expression vectors as indicated. After 24 h, the cells were washed with PBS, fixed in 4% paraformaldehyde in PBS for 15 min at room temperature, and permeabilized in 0.5% Triton X-100 in PBS for 15 min at room temperature. The nuclei were stained with 0.5 µg/ml Hoechst in PBS at 37°C for 10 min. Normal nuclei, apoptotic nuclei with fragmented nuclei, and condensed chromatin were counted under an Olympus fluorescent microscope. Approximately 40 GFP+ cells were counted in several random fields. At least four independent experiments were performed for each transfection. Expression of transfected genes was verified by Western blotting with respective antibodies. Statistical analyses were performed by Minitab Release 10.5Xtra (Minitab Inc.).
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Based on its interaction with both Pyk2 and FAK as well as its apparent molecular mass (see below), we designated the full-length protein as FIP200 and the longest insert (encoded by pGAD33) of the three clones as CT-FIP (COOH-terminal FIP200). Analysis of the FIP200 protein sequence suggested it to be a cytoplasmic protein (e.g., no signal peptide for secretion, no transmembrane domain, and no nuclear localization signals). Residues 1,085–1,225 showed a high sequence homology (84% identity) with the mouse coiled-coil protein 1 and, therefore, was designated as a CC1-like region. In addition, a leucine zipper segment was found between residues 1,371 and 1,391 of FIP200 (Fig. 1 A).
To determine if FIP200 could bind directly to Pyk2 in vitro, a GST fusion protein containing CT-FIP (GST-CT-FIP) was prepared and tested for its binding to recombinant Pyk2 expressed in mammalian cells. Fig. 1 B shows that Pyk2 bound GST-CT-FIP (middle lane), but not GST alone (left lane). To detect FIP200 association with Pyk2 in intact cells, 293T cells were cotransfected with pKH3-Pyk2 encoding HA-tagged Pyk2 and expression vectors encoding Flag-tagged FIP200, CT-FIP, and NH2-terminal FIP200 (NT-FIP; residues 1–641), or the vector alone as a control. Coimmunoprecipitation of the lysates showed that Pyk2 was associated with CT-FIP and FIP200, but not NT-FIP, in intact cells (Fig. 1 C). Western blotting of the immunoprecipitates with anti-Flag showed expression levels of FIP200 and its fragments (Fig. 1 D). Direct analysis of the lysates by Western blotting with anti-Pyk2 confirmed comparable expression of Pyk2 in all samples (Fig. 1 E). Consistent with the in vitro binding and transfection studies, association of endogenous Pyk2 and FIP200 was also detected by coimmunoprecipitation (Fig. 1 F). Together, these results demonstrated Pyk2 interaction with FIP200, and indicated that this interaction is mediated by the FIP200 COOH-terminal region.
Pyk2 and FAK are closely related tyrosine kinases that share similar structural organization and significant sequence homology (Avraham et al. 1995; Lev et al. 1995; Sasaki et al. 1995). Therefore, we also examined the possibility of FIP200 association with FAK by both in vitro binding and in vivo coimmunoprecipitation experiments. Fig. 2 A shows that FAK bound to GST-CT-FIP200 (middle lane), but not GST alone (left lane). To detect in vivo association, 293T cells were cotransfected with pKH3-FAK and pSG5-FIP200, pSG5-CT-FIP, or pSG5-Flag vector alone as a control. 2 d after transfection, cell lysates were prepared and immunoprecipitated with anti-Flag. Fig. 2 B shows that FAK was associated with both the full-length (left lane) and COOH-terminal fragment of FIP200 (middle lane), but was absent in the immune complex from cells cotransfected with the control vector alone (right lane). Together, these results suggested that FIP200 may also associate with FAK via its COOH-terminal region.
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80% at high doses of GST-CT-FIP. These results suggested that FIP200 might function as an inhibitor of Pyk2 by binding to its kinase domain directly.
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To determine whether binding of FIP200 to Pyk2 inhibited its kinase activity in intact cells, expression vectors encoding Pyk2 and FIP200 or its fragments were cotransfected into CHO cells. 2 d after transfection, the cells were treated with sorbitol and the lysates were prepared from these cells. HA-tagged Pyk2 was immunoprecipitated with anti-Pyk2 and analyzed for its tyrosine phosphorylation by Western blotting with antiphosphotyrosine mAb PY20. Fig. 5 A shows that tyrosine phosphorylation of Pyk2 was significantly increased by sorbitol treatment, as observed previously (top; Zheng et al. 1998). Cotransfection with CT-FIP reduced the increase in Pyk2 phosphorylation, whereas coexpression of the pSG5 vector had no effect. Western blotting of the immunoprecipitates with anti-Pyk2 verified similar levels of Pyk2 expression in these samples (middle). Western blotting with anti-Flag confirmed the expression of CT-FIP in the cotransfection samples (bottom). Similar studies indicated that cotransfection of the FIP200 (Fig. 5 B) but not the NT-FIP fragment (Fig. 5 C), also inhibited Pyk2 phosphorylation in response to sorbitol. Together, these results indicated that FIP200 could function as an inhibitor of Pyk2 in intact cells, and the inhibitory activity correlated with FIP200 binding to Pyk2 via its COOH-terminal fragment.
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Discussion |
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Like FAK, Pyk2 has large NH2- and COOH-terminal domains flanking the central kinase domain, which have been shown to interact with several signaling molecules and cytoskeletal proteins (Dikic et al. 1996; Salgia et al. 1996; Astier et al. 1997; Hiregowdara et al. 1997). Interestingly, we found that FIP200 bound to Pyk2 at the kinase domain itself, which is the first such case for Pyk2 or FAK. Although FIP200 contains two potential tyrosine phosphorylation sites (Fig. 1 A), cotransfection of Pyk2 with FIP200 did not induce FIP200 phosphorylation, suggesting that FIP200 was unlikely to be a substrate for Pyk2 (data not shown). In contrast, several lines of evidence indicated that FIP200 may function as an inhibitor of Pyk2. A GST fusion protein containing CT-FIP200 (binding segment for Pyk2) inhibited Pyk2 kinase activity in a dose-dependent manner in vitro. Coexpression of FIP200 or CT-FIP with Pyk2 in CHO cells reduced sorbitol-induced phosphorylation of Pyk2 in vivo. Finally, expression of FIP200 or CT-FIP also blocked Pyk2-triggered apoptosis in Rat1 cells. In contrast, the NT-FIP lacking Pyk2 binding activity did not affect Pyk2 activation by sorbitol or induction of apoptosis by Pyk2 in the same assays. Together, these data suggested strongly that FIP200 functioned as an inhibitor of Pyk2 by binding to its kinase domain.
The mechanism of inhibition of Pyk2 by FIP200 is not clear at the present. It is possible that binding of FIP200 to the kinase domain of Pyk2 inhibits its kinase activity directly. Alternatively, association of FIP200 to Pyk2 may prevent binding of Src family kinases to the major autophosphorylation site Y402 near the kinase domain, thereby reducing the kinase activity of the Pyk2/Src complex. However, several lines of data suggested that this latter possibility is less likely. First, FIP200 lacks the Src homology 2 domain for binding any phosphotyrosines, and it bound to the Y402F Pyk2 mutant both in vitro and in vivo (data not shown). Therefore, FIP200 does not compete with Src for binding Y402 of Pyk2. Second, a GST fusion protein containing the COOH-terminal FIP200 inhibited the kinase activity of the 402F mutant as effectively as that of wild-type Pyk2 (Fig. 4 C). Finally, it also inhibited the kinase activity of the Pyk2 isolated from SYF cells, which are deficient in the expression of Src family kinases Src, Yes, and Fyn (Klinghoffer et al. 1999; Fig. 4 B). Therefore, it is likely that FIP200 inhibited Pyk2 kinase activity directly by binding to its kinase domain rather than by interfering with binding or the activity of associated Src family kinases. In any case, our findings of FIP200 as an inhibitor of Pyk2 activity suggested the possibility that some of the activators of Pyk2 may activate the kinase by decreasing association of FIP200 with Pyk2 (Fig. 6). It is also likely that other upstream regulators of FIP200 may enhance FIP200 binding to Pyk2, thus, leading to inactivation of Pyk2.
Two novel Pyk2-interacting proteins, Nirs and Pap, have been identified recently through the use of the yeast two-hybrid screen or another yeast-based cloning strategy (Andreev et al. 1999; Lev et al. 1999). Nirs are transmembrane proteins related to the Drosophila retinal degeneration B protein (Lev et al. 1999). Pap (
and β isoforms) is a cytoplasmic protein with Arf-GAP activity, which is involved in regulation of vesicular transport (Andreev et al. 1999). Both proteins appear to interact with Pyk2 but not FAK. However, consistent with the similar structures of Pyk2 and FAK, several FAK-binding proteins have been shown to associate with Pyk2 (Salgia et al. 1996; Dikic et al. 1996; Astier et al. 1997; Hiregowdara et al. 1997). Interestingly, FIP200 could also bind to FAK via its COOH-terminal region as shown by both in vitro binding and coimmunoprecipitation (Fig. 2). The COOH-terminal FIP200 inhibited FAK kinase activity in vitro (Fig. 4 D). We have previously observed association of FAK with a tyrosine-phosphorylated cellular protein of 200 kD upon PDGF stimulation (Chen and Guan 1996). However, we did not observe any significant stimulation of tyrosine phosphorylation of FIP200 by PDGF (data not shown), although we could not completely exclude the possibility of FIP200 being the previously described pp200 associated with FAK (Chen and Guan 1996). Further experiments will be necessary to determine regulation and potential cellular functions of FIP200 interaction with FAK.
Previous Northern blot analysis showed expression of FIP200 mRNA in a wide variety of tissues (Nagase et al. 1996). Consistent with this, we have found that FIP200 is expressed in many cell lines as detected by Western blotting with anti-FIP200 (data not shown). Furthermore, immunofluorescent staining of transfected FIP200 with epitope tag antibodies (Fig. 3) as well as staining of endogenous FIP200 with anti-FIP200 (data not shown) showed a predominantly diffuse cytoplasmic distribution of FIP200. This distribution is consistent with its interaction with the mainly cytoplasmic tyrosine kinase Pyk2 (Zheng et al. 1998). However, the cytoplasmic localization of FIP200 is not in conflict with its potential interactions with proteins in other subcellular compartments but facing the cytoplasm (e.g., FAK in focal contacts).
In contrast to the wide distribution of FIP200, Pyk2 is expressed most abundantly in restricted tissues and cells, such as the brain and hematopoietic cells (Avraham et al. 1995; Lev et al. 1995; Sasaki et al. 1995). This suggests that FIP200 may have other functions in cells lacking Pyk2 expression, perhaps by its interactions with other cellular proteins such as FAK. In addition, residues 1,085–1,225 in FIP200 have a significant sequence homology with the protein CC1, which has been shown to interact with the cellular protein stathmin (Maucuer et al. 1995). Stathmin is a ubiquitous, cytosolic 19-kD protein, possibly involved in integrating diverse intracellular regulatory pathways (Sobel 1991). Potential interactions of FIP200 with stathmin or other proteins and their cellular functions remain to be investigated.
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Acknowledgments |
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This research was supported by the National Institutes of Health grants GM48050 and GM52890 to J.-L. Guan. J.-L. Guan is an Established Investigator of the American Heart Association.
Submitted: 24 September 1999
Revised: 6 February 2000
Accepted: 2 March 2000
Abbreviations used in this paper: CT-FIP, COOH-terminal FIP200; FAK, focal adhesion kinase; FIP200, FAK family kinase–interacting protein of 200 kD; GST, glutathione-S-transferase; HA, hemagglutinin; NT-FIP, NH2-terminal FIP200; Pyk2, proline-rich tyrosine kinase 2.
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0.15 in comparison to the value from cells transfected with the control vector (Minitab Release 10.5 Xtra). (B) Rat-1 cells were cotransfected with a plasmid encoding GFP, pKH3-Pyk2 (P), and expression vectors encoding FIP200, NT-FIP, CT-FIP, or vector alone (C), as indicated. They were analyzed as in A, and the results show the mean and SD of a percentage of apoptotic/GFP positive cells. *P < 0.01 and **P > 0.40 in comparison to the value from cells transfected with Pyk2 and a control vector (Minitab Release 10.5 Xtra).