The Tyrosine Kinase Pyk2 Regulates Arf1 Activity by Phosphorylation and Inhibition of the Arf-GTPase-activating Protein ASAP1
2003; Elsevier BV; Volume: 278; Issue: 32 Linguagem: Inglês
10.1074/jbc.m302278200
ISSN1083-351X
AutoresAnamarija Kruljac‐Letunic, Jörg Moelleken, Anders Kallin, Felix Wieland, Andree Blaukat,
Tópico(s)Wnt/β-catenin signaling in development and cancer
ResumoProline-rich tyrosine kinase 2 (Pyk2), a non-receptor tyrosine kinase structurally related to focal adhesion kinase, has been implicated in the regulation of mitogen-activated protein kinase cascades and ion channels, the induction of apoptosis, and in the modulation of the cytoskeleton. In order to understand how Pyk2 signaling mediates these diverse cellular functions, we performed a yeast two-hybrid screening using the C-terminal part of Pyk2 that contains potential protein-protein interaction sites as bait. A prominent binder of Pyk2 identified by this method was the Arf-GTPase-activating protein ASAP1. Pyk2-ASAP1 interaction was confirmed in pull-down as well as in co-immunoprecipitation experiments, and contact sites were mapped to the proline-rich regions of Pyk2 and the SH3 domain of ASAP1. Pyk2 directly phosphorylates ASAP1 on tyrosine residues in vitro and increases ASAP1 tyrosine phosphorylation when co-expressed in HEK293T cells. Phosphorylation of tyrosine 308 and 782 affects the phosphoinositide binding profile of ASAP1, and fluorimetric Arf-GTPase assays with purified proteins revealed an inhibition of ASAP1 GTPase-activating protein activity by Pyk2-mediated tyrosine phosphorylation. We therefore provide evidence for a functional interaction between Pyk2 and ASAP1 and a regulation of ASAP1 and hence Arf1 activity by Pyk2-mediated tyrosine phosphorylation. Proline-rich tyrosine kinase 2 (Pyk2), a non-receptor tyrosine kinase structurally related to focal adhesion kinase, has been implicated in the regulation of mitogen-activated protein kinase cascades and ion channels, the induction of apoptosis, and in the modulation of the cytoskeleton. In order to understand how Pyk2 signaling mediates these diverse cellular functions, we performed a yeast two-hybrid screening using the C-terminal part of Pyk2 that contains potential protein-protein interaction sites as bait. A prominent binder of Pyk2 identified by this method was the Arf-GTPase-activating protein ASAP1. Pyk2-ASAP1 interaction was confirmed in pull-down as well as in co-immunoprecipitation experiments, and contact sites were mapped to the proline-rich regions of Pyk2 and the SH3 domain of ASAP1. Pyk2 directly phosphorylates ASAP1 on tyrosine residues in vitro and increases ASAP1 tyrosine phosphorylation when co-expressed in HEK293T cells. Phosphorylation of tyrosine 308 and 782 affects the phosphoinositide binding profile of ASAP1, and fluorimetric Arf-GTPase assays with purified proteins revealed an inhibition of ASAP1 GTPase-activating protein activity by Pyk2-mediated tyrosine phosphorylation. We therefore provide evidence for a functional interaction between Pyk2 and ASAP1 and a regulation of ASAP1 and hence Arf1 activity by Pyk2-mediated tyrosine phosphorylation. Pyk2 and FAK 1The abbreviations used are: FAK, focal adhesion kinase; GAPs, GTPase-activating proteins; FAT, focal adhesion targeting; PH, pleckstrin homology; SH, Src homology; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; GTPγS, guanosine 5′-3-O-(thio)triphosphate; aa, amino acids; DMEM, Dulbecco's modified Eagle's medium; PMA, phorbol 12-myristate 13-acetate; GST, glutathione S-transferase; PS, phosphatidylserine; PI, phosphatidylinositol; Arf, ADP-ribosylation factor; MEF, mouse embryonic fibroblasts. comprise a distinct family of non-receptor protein tyrosine kinases that are involved in transmission of extracellular signals to intracellular kinase cascades and regulation of diverse cellular responses such as adhesion, proliferation, differentiation, and apoptosis (1Avraham H. Park S.Y. Schinkmann K. Avraham S. Cell Signal. 2000; 12: 123-133Crossref PubMed Scopus (409) Google Scholar). Pyk2 and FAK share about 45% amino acid identity and a similar domain structure: a unique N terminus containing a FERM (band 4.1/ezrin/radixin/moesin) domain, a central protein tyrosine kinase domain, two proline-rich sequences, and an FAT (focal adhesion targeting) domain in the C terminus. FAK is found in almost all tissues, whereas Pyk2 is mainly expressed in neuronal and hematopoietic cells. Whereas FAK is localized to focal adhesion sites in adherent cells, Pyk2 is rather diffused throughout the cytoplasm and concentrated in perinuclear regions (2Matsuya M. Sasaki H. Aoto H. Mitaka T. Nagura K. Ohba T. Ishino M. Takahashi S. Suzuki R. Sasaki T. J. Biol. Chem. 1998; 273: 1003-1014Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Both kinases can be regulated by integrins, growth factor receptors, and G-protein-coupled receptors. However, Pyk2 is unique in the way that its activation is triggered by intracellular calcium elevation and/or protein kinase C (3Dikic I. Batzer A.G. Blaikie P. Obermeier A. Ullrich A. Schlessinger J. Margolis B. J. Biol. Chem. 1995; 270: 15125-15129Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Together with Src that binds to autophosphorylated tyrosine 402 of Pyk2, it can function upstream of small G-proteins, such as Ras, Rac, and Rho, linking different transmembrane receptors with mitogen-activated protein kinase cascades (1Avraham H. Park S.Y. Schinkmann K. Avraham S. Cell Signal. 2000; 12: 123-133Crossref PubMed Scopus (409) Google Scholar, 3Dikic I. Batzer A.G. Blaikie P. Obermeier A. Ullrich A. Schlessinger J. Margolis B. J. Biol. Chem. 1995; 270: 15125-15129Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 4Blaukat A. Ivankovic-Dikic I. Gronroos E. Dolfi F. Tokiwa G. Vuori K. Dikic I. J. Biol. Chem. 1999; 274: 14893-14901Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). ADP-ribosylation factor (Arf) proteins constitute another family of small GTPases that were originally identified as co-factors required for the cholera toxin-catalyzed ADP-ribosylation of Gαs subunits (5Kahn R.A. Gilman A.G. J. Biol. Chem. 1986; 261: 7906-7911Abstract Full Text PDF PubMed Google Scholar). Arf1, the best characterized mammalian Arf, recruits coat proteins to membranes of the Golgi apparatus and has been implicated in intra-Golgi and Golgito-endoplasmic reticulum transport, endosome function, and synaptic vesicle formation (6Donaldson J.G. Klausner R.D. Curr. Opin. Cell Biol. 1994; 6: 527-532Crossref PubMed Scopus (232) Google Scholar, 7Rothman J.E. Nature. 1994; 372: 55-63Crossref PubMed Scopus (2011) Google Scholar, 8Sohn K. Orci L. Ravazzola M. Amherdt M. Bremser M. Lottspeich F. Fiedler K. Helms J.B. Wieland F.T. J. Cell Biol. 1996; 135: 1239-1248Crossref PubMed Scopus (181) Google Scholar). However, Arf GTPases are also involved in recruitment of paxillin to focal adhesions and remodeling of the cytoskeleton as cells change shape or move (9Norman J.C. Jones D. Barry S.T. Holt M.R. Cockcroft S. Critchley D.R. J. Cell Biol. 1998; 143: 1981-1995Crossref PubMed Scopus (121) Google Scholar). Arfs are distinct from other small GTPases in their strictly GTP-dependent recruitment to membranes and in the virtual absence of any intrinsic GTPase activity. Therefore, guanine-nucleotide exchange factors and GTPase-activating proteins (GAPs) are necessary for the completion of the full GTP-GDP cycle of Arfs (10Cukierman E. Huber I. Rotman M. Cassel D. Science. 1995; 270: 1999-2002Crossref PubMed Scopus (271) Google Scholar, 11Moss J. Vaughan M. J. Biol. Chem. 1995; 270: 12327-12330Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). A number of Arf-GAPs have been identified of which some are co-regulated by acidic lipids and phosphoinositides. One of those Arf-GAPs is ASAP1 (Arf-GAP containing SH3, ankyrin repeats and PH domain) that binds to Src and displays phospholipid-dependent activity toward Arf1 and Arf5. Upon binding, Src can phosphorylate ASAP1, but the functional consequence of this phosphorylation has remained unclear (12Brown M.T. Andrade J. Radhakrishna H. Donaldson J.G. Cooper J.A. Randazzo P.A. Mol. Cell. Biol. 1998; 18: 7038-7051Crossref PubMed Scopus (196) Google Scholar). More recently, FAK has also been shown to interact with ASAP1, and an involvement of ASAP1 in cell spreading and focal adhesion localization of FAK as well as paxillin has been suggested (13Liu Y. Loijens J.C. Martin K.H. Karginov A.V. Parsons J.T. Mol. Biol. Cell. 2002; 13: 2147-2156Crossref PubMed Scopus (128) Google Scholar). However, FAK does not phosphorylate ASAP1 nor modulate its activity. Pyk2 can also interact with an Arf-GAP called PAPα (Pyk2 C-terminal associated protein), but again the functional consequence of this interaction has remained elusive (14Andreev J. Simon J.P. Sabatini D.D. Kam J. Plowman G. Randazzo P.A. Schlessinger J. Mol. Cell. Biol. 1999; 19: 2338-2350Crossref PubMed Scopus (146) Google Scholar). We have identified ASAP1 as a new binding partner of Pyk2 and mapped the interaction to the SH3 domain of ASAP1 and the proline-rich regions of Pyk2. Furthermore, we could demonstrate a direct phosphorylation of ASAP1 by Pyk2 and a subsequent regulation of the ASAP1 GTPase activity. We therefore suggest a role of Pyk2 in regulating Arf function and potentially vesicular trafficking. Our results provide a first molecular mechanism how tyrosine kinases can modulate signaling of small Arf GTPases. Materials—[γ-32P]ATP (110 TBq/mmol), [32P]orthophosphate (360 MBq/ml), and GST-Sepharose were from Amersham Biosciences; phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) was from Calbiochem, and other lipids were from Avanti Polar Lipids; bradykinin was from Bachem; aprotinin (Trasylol©) was from Bayer; PMA was from Calbiochem; lipid arrays were from Echelon Research Laboratories; Pefabloc™ was from Fluka; LipofectAMINE™ and protein markers were from Invitrogen; cellulose TLC plates were from Merck; sequencing grade trypsin was from Promega; leupeptin, anti-hemagglutinin (12CA5) monoclonal antibody, and chemiluminescence substrate were from Roche Applied Science; nitrocellulose membranes were from Schleicher & Schuell; epidermal growth factor, GTP, GTPγS, anti-FLAG M2 affinity agarose, anti-FLAG (M2) monoclonal antibody, and phosphoamino acid standards were from Sigma; anti-ASAP1 polyclonal antibody (sc-11539), anti-phosphotyrosine (PY99, sc-7020) monoclonal antibody, and anti-c-Src polyclonal antibody (sc-018) were from Santa Cruz Biotechnology; protein A-agarose was from Zymed Laboratories Inc.. All tissue culture reagents were from Invitrogen, and all other chemicals were of analytical grade and purchased from Applichem, ICN, Merck, and Sigma. cDNA Constructs and Mutagenesis—For yeast two-hybrid studies, a C-terminal portion of Pyk2 (aa 781–1009) or FAK (aa 693–1053) cDNA was amplified by PCR and subcloned as an EcoRI/BamHI fragment in-frame with the LexA sequence in pBTM116. The pGEX-PRNK (proline-rich non-kinase) vector encoding the fusion protein GST-PRNK was constructed by insertion of PRNK (aa 781–1009 of Pyk2) into the EcoRI/NotI sites of pGEX-4T. FLAG-tagged ASAP1 was a gift from P. Randazzo (NCI, National Institutes of Health, Bethesda). To obtain the FLAG-ASAP1-δSH3 deletion mutant, ASAP1 lacking the SH3 domain (aa 1–1054) was subcloned in EcoRI/XhoI sites of pcDNA3-FLAG. Point mutants of mouse ASAP1 were generated using the QuickChange® site-directed mutagenesis kit (Stratagene) following the manufacturer's instructions. Tyr-308 was replaced by phenylalanine using the following oligonucleotide primers: 5′-GAT CCG AAA GAA GTA GGT GGT TTA TTT GTT GCT AGC AGG GCT AAC AGT TCT-3′ and 5′-AGA ACT GTT AGC CCT GCT AGC AAC AAA TAA ACC ACC TAC TTC TTT CGG ATC-3′. Thereafter ASAP1-Y308F as template and the following oligonucleotide primers were used for generation of Y308F/Y782F mutant: 5′-GAC AAG CAG CGG CTC TCC TTC GGC GCC TTC ACC AAC-3′ and 5′-GTT GGT GAA GGC GCC GAA GGA GAG CCG CTG CTT GTC-3′. Pyk2 variants P717A, P859A, and P717A/P859A have been described previously (15Ivankovic-Dikic I. Gronroos E. Blaukat A. Barth B.U. Dikic I. Nat. Cell Biol. 2000; 2: 574-581Crossref PubMed Scopus (184) Google Scholar, 16Aoto H. Sasaki H. Ishino M. Sasaki T. Cell Struct. Funct. 2002; 27: 47-61Crossref PubMed Scopus (46) Google Scholar) and were generated using a similar mutagenesis strategy. All mutations were confirmed by DNA sequencing. Yeast Two-hybrid Screening—An L40 yeast reporter strain carrying pBTM116-PRNK, in which PRNK, a C-terminal fragment of Pyk2, was expressed as a fusion protein with the LexA DNA-binding domain, was transformed with a day 9.5 mouse embryonic cDNA library fused to the VP16 activation domain (17Vojtek A.B. Cooper J.A. Hollenberg S.M. Bartel P.L. Fields S. The Yeast Two-hybrid System. Oxford University Press, Oxford, UK1997: 29-42Google Scholar). Transformation was performed by the lithium acetate method (18Gietz R.D. Schiestl R.H. Yeast. 1991; 7: 253-263Crossref PubMed Scopus (368) Google Scholar). Approximately 3 × 106 transformants were screened for growth on Trp–Leu–His– plates supplemented with 20 mm 3-aminotriazole. His+ colonies were subsequently subjected to nitrocellulose filters on which a β-galactosidase assay was performed to verify the bait-prey interaction. Plasmid DNAs isolated from positive yeast clones were introduced into Escherichia coli HB101, and amplified and recovered library plasmids were sequenced. To reconfirm the LacZ+ phenotype, recovered library plasmids were re-transformed in L40 yeast carrying pBTM116-PRNK, and β-galactosidase activity of re-transformants was tested in yeast lysates with a liquid β-galactosidase assay (17Vojtek A.B. Cooper J.A. Hollenberg S.M. Bartel P.L. Fields S. The Yeast Two-hybrid System. Oxford University Press, Oxford, UK1997: 29-42Google Scholar). Cell Culture and Transfections—HEK293T cells were cultured in DMEM supplemented with 10% fetal bovine serum, 10 μg/ml penicillin, and 0.25 μg/ml streptomycin. For transfection experiments, cells were grown until 60–80% confluence and transfected with the indicated amounts of plasmid DNA, using a modified calcium phosphate method (MBS; Stratagene). 48 h after transfection, cells were lysed in 1 ml/10-cm dish of ice-cold lysis buffer (1% Triton X-100, 20 mm HEPES, pH 7.5, 150 mm NaCl, 2 mm EDTA, 10% glycerol) including protease (0.5 mm Pefabloc™, 10 μg/ml pepstatin A, 10 μg/ml leupeptin, 1% Trasylol® (aprotinin)) and phosphatase inhibitors (25 mm NaF, 1 mm sodium orthovanadate). Lysates were incubated by gentle rocking for 45 min at 4 °C and subsequently centrifuged at 4 °C for 20 min at 14,000 rpm. Supernatants were used for in vitro binding assay, affinity purification, and co-immunoprecipitation studies. PC12 cells were cultured in DMEM supplemented with 3% fetal bovine serum, 7% horse serum, 10 μg/ml penicillin, and 0.25 μg/ml streptomycin. MEF cells were cultured in DMEM supplemented with 10% fetal bovine serum, non-essential amino acids, 10 μg/ml penicillin, and 0.25 μg/ml streptomycin. Within 3 day after seeding, cells were serum-starved for 24 h in DMEM containing 0.2% bovine serum albumin and then treated as indicated. In Vitro Binding Assays—GST fusion proteins were expressed in E. coli DH5α and purified using glutathione-Sepharose beads according to the manufacturer's instructions (Amersham Biosciences). Equal amounts of GST fusion proteins or GST alone (about 5 μg) were incubated with 200-μl cell lysates from transfected HEK293T cells (100–200 μg) at 4 °C for 3 h on a rotating platform. Beads were washed three times with lysis buffer, and associated proteins were released with SDS sample buffer (62.5 mm Tris, pH 6.8, 2% SDS, 25 mm dithiothreitol, 30% glycerol, 0.02% bromphenol blue) and 5 min of incubation at 98 °C. Samples were subjected to SDS-PAGE and analyzed by Western blotting with a Pyk2-specific polyclonal antibody (antibody 694 (3Dikic I. Batzer A.G. Blaikie P. Obermeier A. Ullrich A. Schlessinger J. Margolis B. J. Biol. Chem. 1995; 270: 15125-15129Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar)) or an anti-FLAG monoclonal antibody to detect FLAG-tagged ASAP1 constructs. Immunoprecipitation and Western Blotting—Equal amounts of lysates (100–200 μg) were subjected to immunoprecipitation at 4 °C for 3 h on a rotating platform by using anti-Pyk2 (antibody 598 (3Dikic I. Batzer A.G. Blaikie P. Obermeier A. Ullrich A. Schlessinger J. Margolis B. J. Biol. Chem. 1995; 270: 15125-15129Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar)) or anti-FLAG antibodies. Immune complexes were isolated using protein A-Sepharose or primary antibodies pre-coupled to agarose beads. Beads were washed three times with lysis buffer, and proteins were released with SDS sample buffer and 5 min of incubation at 98 °C. Samples were subjected to SDS-PAGE and analyzed by Western blotting with indicated antibodies. Two-dimensional Phosphopeptide Mapping—FLAG-tagged ASAP1 was immunoprecipitated, and in vitro kinase reactions were performed in 10 mm Tris, pH 7.5, 10 mm MgCl2, 100 μm ATP including 5 μCi of [γ-32P]ATP at 30 °C for 30 min. Reactions were stopped by addition of SDS sample buffer and incubation at 98 °C for 5 min. In vivo 32P labeling was performed using 1 mCi/ml in phosphate-free DMEM for 6 h as detailed previously (19Blaukat A. Pizard A. Breit A. Wernstedt C. Alhenc-Gelas F. Muller-Esterl W. Dikic I. J. Biol. Chem. 2001; 276: 40431-40440Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Radiolabeled FLAG-ASAP1 was isolated by immunoprecipitation and processed identically to in vitro labeled species. Proteins were separated by 8% SDS-PAGE, transferred to nitrocellulose, and analyzed using a PhosphorImager (Molecular Imager FX Pro, Bio-Rad). Two-dimensional phosphopeptide mapping was performed as described previously (19Blaukat A. Pizard A. Breit A. Wernstedt C. Alhenc-Gelas F. Muller-Esterl W. Dikic I. J. Biol. Chem. 2001; 276: 40431-40440Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Briefly, 32P-labeled ASAP1 bands were identified and in situ subjected to trypsin digestion. Phosphopeptides were separated on cellulose TCL plates in two dimensions by electrophoresis and chromatography, visualized by PhosphorImager analysis, and extracted from the matrix. Thereafter, phosphoamino acid composition was determined, and positions of phosphorylated residues were determined by Edman sequencing. By using this information, phosphorylation sites were predicted and confirmed by site-directed mutagenesis and two-dimensional phosphopeptide mapping of mutants. Purification of Arf and FLAG-ASAP1—Arf1 was expressed in E. coli BL21 (DE3) together with N-myristoyltransferase and purified in the myristoylated form in three steps as follows: (i) precipitation with 35% ammonium sulfate saturation; (ii) DEAE-Sepharose chromatography; (iii) MonoS chromatography as described previously (20Franco M. Chardin P. Chabre M. Paris S. J. Biol. Chem. 1995; 270: 1337-1341Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). To isolate FLAG-ASAP1 from transiently transfected HEK293T cells, affinity purification with anti-FLAG M2 affinity matrix following the manufacturer's instructions (Sigma) was performed. Briefly, lysates obtained from 10 10-cm dishes were incubated with 400 μl of anti-FLAG matrix for 3 h on a rotating platform at 4 °C. The matrix was washed three times with 10 ml of lysis buffer and once with 10 ml of TBS (50 mm Tris, pH 7.5, 150 mm NaCl). Specifically bound proteins were eluted at 4 °C by incubation with TBS containing 150 μg/ml 3× FLAG peptide. Eluates were divided into aliquots, shock-frozen, and stored at –80 °C. Purity of proteins and phosphorylation status were analyzed by Coomassie or silver staining and Western blotting with corresponding antibodies. Protein-Lipid Overlay Assay—Protein-lipid overlay assays with affinity-purified FLAG-tagged ASAP1 were performed according to Dowler et al. (21Dowler S. Kular G. Alessi D.R. Sci. STKE. 2002; 129: PL6Google Scholar). Phospholipids and blank sample immobilized on membranes were blocked with 3% fatty acid-free bovine serum albumin in TBS containing 0.05% Tween 20 (TBST) for1hat room temperature. Thereafter membranes were incubated for 1 h in the same solution containing 0.5 μg/ml of FLAG-tagged ASAP1 and washed with TBST for 30 min. Following incubation with anti-FLAG monoclonal antibody and anti-mouse-horseradish peroxidase conjugate, proteins bound to phospholipids were detected by the enhanced chemiluminescence method. Fluorimetric GTPase Assay—To follow Arf1-GTPase activity, we used a previously described fluorimetric assay based on the change of intrinsic tryptophan fluorescence of Arf1 during its transition from GTP- to GDP-bound state (22Antonny B. Beraud-Dufour S. Chardin P. Chabre M. Biochemistry. 1997; 36: 4675-4684Crossref PubMed Scopus (281) Google Scholar). The assay was performed with a Jasco FP6500 fluorimeter in a cylindrical cuvette at 37 °C under stirring conditions. In a typical experiment, the cuvette initially contained a suspension of liposomes with or without 1 mol % PI(4,5)P2 in reaction buffer (25 mm HEPES, pH 7.4, 150 mm KCl, 2 mm MgCl2). Liposomes were prepared as described (22Antonny B. Beraud-Dufour S. Chardin P. Chabre M. Biochemistry. 1997; 36: 4675-4684Crossref PubMed Scopus (281) Google Scholar) and contained 43 mol % phosphatidylcholine (PC), 19 mol % phosphatidylethanolamine, 5 mol % phosphatidylserine (PS), 10 mol % phosphatidylinositol (PI), 7 mol % sphingomyelin, 16 mol % cholesterol. Then Arf1-GDP (4 μg) and GTP (10 μm) or the non-hydrolyzable analogue GTPγS were injected, and GDP/GTP exchange was accelerated by addition of 4 mm EDTA (23Paris S. Beraud-Dufour S. Robineau S. Bigay J. Antonny B. Chabre M. Chardin P. J. Biol. Chem. 1997; 272: 22221-22226Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). GTP-loaded Arf1 was stabilized by addition of 4 mm MgCl2, and Arf1-GTPase activity was followed after injection of purified FLAG-tagged ASAP1 (∼2 μg) or control proteins. Tryptophan fluorescence was recorded at 340 nm (bandwidth 20 nm) upon excitation at 297.5 nm (bandwidth 5 nm). Identification of ASAP1 as an Interaction Partner of Pyk2—In order to improve understanding of how Pyk2 mediates its diverse cellular functions, we performed a yeast two-hybrid screening using the C-terminal part of Pyk2 (PRNK) that contains several potential protein-protein interaction sites as bait (Fig. 1A). We screened 3 × 106 clones of an embryonic mouse cDNA library using the LexA system (17Vojtek A.B. Cooper J.A. Hollenberg S.M. Bartel P.L. Fields S. The Yeast Two-hybrid System. Oxford University Press, Oxford, UK1997: 29-42Google Scholar) and identified 30 clones that were positive in growth and filter as well as liquid β-galactosidase assays. Three independent clones encoded cDNAs covering the SH3 domain of ASAP1, a phospholipid-dependent GTPase-activating protein for small Arf GTPases. ASAP1 consist of a PH domain followed by an Arf GAP domain, three ankyrin repeats, three proline-rich sequences, eight E/DLPPKP repeats, and an SH3 domain (Fig. 1A). It is a 130-kDa protein that is expressed in many tissues but is most abundant in testis, brain, lung, and spleen (12Brown M.T. Andrade J. Radhakrishna H. Donaldson J.G. Cooper J.A. Randazzo P.A. Mol. Cell. Biol. 1998; 18: 7038-7051Crossref PubMed Scopus (196) Google Scholar). Activation of ASAP1 involves PI(4,5)P2 binding to the PH domain and may be implicated in regulating the actin cytoskeleton (24Kam J.L. Miura K. Jackson T.R. Gruschus J. Roller P. Stauffer S. Clark J. Aneja R. Randazzo P.A. J. Biol. Chem. 2000; 275: 9653-9663Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 25Randazzo P.A. Andrade J. Miura K. Brown M.T. Long Y.Q. Stauffer S. Roller P. Cooper J.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4011-4016Crossref PubMed Scopus (165) Google Scholar). The Pyk2-ASAP1 interaction and its specificity were confirmed by re-tranformation of the empty library vector, ASAP1 SH3 domain, and a β-catenin in LexA, LexA-PRNK, as well as in LexA-LEF yeast, subsequent growth (Fig. 1B), and β-galactosidase assay (Fig. 1C). The β-catenin-LEF interaction (26Behrens J. von Kries J.P. Kuhl M. Bruhn L. Wedlich D. Grosschedl R. Birchmeier W. Nature. 1996; 382: 638-642Crossref PubMed Scopus (2605) Google Scholar) and a C-terminal FAK fragment (FRNK, FAK-related nonkinase) that has been shown previously (13Liu Y. Loijens J.C. Martin K.H. Karginov A.V. Parsons J.T. Mol. Biol. Cell. 2002; 13: 2147-2156Crossref PubMed Scopus (128) Google Scholar) to interact with ASAP1 SH3 domain served as positive controls. Only the PRNK-ASAP1 SH3 domain and positive controls resulted in growth of yeast and expression of β-galactosidase (Fig. 1, B and C). Altogether, our yeast two-hybrid data suggest a specific and tight interaction between the C-terminal Pyk2 fragment PRNK and the ASAP1 SH3 domain. In Vitro binding of PRNK and Pyk2 to the SH3 Domain of ASAP1—To confirm the Pyk2-ASAP1 interaction found in the yeast two-hybrid screen and to more precisely map domains that mediate binding, we expressed Pyk2, PRNK, a mutant form PRNK-P859A in which the proline-rich region is disturbed, and the SH3 domain of ASAP1 as GST fusion proteins in E. coli, purified them on glutathione-Sepharose, and used these baits to pull down mammalian proteins expressed in HEK293T cells. In support of our yeast two-hybrid data, the GST-PRNK fusion protein bound to full-length ASAP1 expressed in HEK293T cells, whereas interaction was abrogated when proline 859 in GST-PRNK was mutated to alanine or GST alone was used as bait (Fig. 2A). These results were confirmed in a reverse experiment using a GST fusion protein of the ASAP1 SH3 domain that bound to full-length Pyk2 and PRNK but not to PRNK-P859A expressed in HEK293T cells (Fig. 2B). Comparable levels of the different GST baits were used for pull downs as demonstrated by Ponceau S staining of nitrocellulose membranes before proceeding with Western blots (Fig. 2, A and B, lower panel). Furthermore, expression of corresponding binding proteins in HEK293T cells was confirmed by Western blotting using either anti-FLAG or anti-Pyk2/PRNK antibodies (Fig. 2, A, upper panel, and B, middle panel). In summary, these data from in vitro interaction assays suggest specific binding of ASAP1 to proline-rich sequences in Pyk2 surrounding proline 859. SH3 domain-mediated interactions are frequently of moderate affinity and specificity (27Rickles R.J. Botfield M.C. Weng Z. Taylor J.A. Green O.M. Brugge J.S. Zoller M.J. EMBO J. 1994; 13: 5598-5604Crossref PubMed Scopus (223) Google Scholar, 28Yu H. Chen J.K. Feng S. Dalgarno D.C. Brauer A.W. Schreiber S.L. Cell. 1994; 76: 933-945Abstract Full Text PDF PubMed Scopus (876) Google Scholar, 29Pawson T. Nature. 1995; 373: 573-580Crossref PubMed Scopus (2234) Google Scholar). We therefore sought to test the specificity of the ASAP1-Pyk2 interaction by using a panel of different SH3 domains fused to GST in pull-down assays with lysates from PRNK and Pyk2 expressing HEK293T cells. GST alone served as a negative control, and GST-Grb2 that can bind to Pyk2 via multiple interactions was used as a positive control (4Blaukat A. Ivankovic-Dikic I. Gronroos E. Dolfi F. Tokiwa G. Vuori K. Dikic I. J. Biol. Chem. 1999; 274: 14893-14901Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). Comparable levels of the different GST baits were applied for the pull down as demonstrated by Ponceau S staining of nitrocellulose membranes before proceeding with Western blots (Fig. 3, lower panels) and by probing membranes with an anti-GST antibody (not shown). In this setting, PRNK and Pyk2 did bind only to Grb2 and ASAP1 baits but not to the SH3 domains of Ras-GAP, Abl, Src, phospholipase-Cγ, p85, and Crk suggesting a high degree of specificity of the SH3 domain-mediated association of ASAP1 and Pyk2 (Fig. 3, upper panel). Co-immunoprecipitation of Pyk2 and ASAP1—To confirm the interaction of full-length proteins in living cells and to map further the contact sites we used, HEK293T cells were cotransfected with different FLAG-ASAP1 and Pyk2 constructs. Isolation of ASAP1 by immunoprecipitation and Western blotting with anti-Pyk2 antibodies revealed binding of full-length FLAG-ASAP1 to Pyk2 in HEK293T cells (Fig. 4A, left upper panel). No corresponding band was seen when Pyk2 cDNA was omitted from the transfections and when an ASAP1 construct lacking the SH3 domain (ASAP1-δSH3) was co-expressed with Pyk2. In a reverse experiment, FLAG-ASAP1 but not FLAG-ASAP1-δSH3 could be detected in Pyk2 immunoprecipitates from co-transfected cells (Fig. 4A, right upper panel). Immunoprecipitation of equal amounts of Pyk2 and ASAP1 (Fig. 4A, lower panels) and expression levels of Pyk2, ASAP1, and ASAP1-δSH3 in HEK293T cells (Fig. 4B) were verified by Western blotting using anti-Pyk2 or anti-FLAG antibodies. For a more precise mapping of the region in Pyk2 that interacts with the ASAP1 SH3 domain, we disrupted both proline-rich regions of Pyk2 individually or in combination by site-directed mutagenesis. Co-immunoprecipitation experiments either with anti-FLAG or with anti-Pyk2 antibodies revealed a moderate reduction of ASAP1 binding to Pyk2-P859A where the binding motif of the second proline-rich region was mutated (Fig. 4C, upper panels). A more dramatic effect was observed upon disruption of the first proline-rich domain of Pyk2 (Pyk2-P717A), and binding of ASAP1 was completely abolished in the corresponding double mutant. Quality of the immunoprecipitation was controlled by re-probing blots with antibodies against bait proteins (Fig. 4C, lower panels), and comparable quantities of FLAG-ASAP1 and Pyk2 proteins in cell lysates were demonstrated by Western blotting using corresponding antibodies (Fig. 4D). These experiments demonstrate binding of ASAP1 via its SH3 domain to the second and more effectively to the first proline-rich domain of Pyk2. To validate our co-immunoprecipitation strategy, we also reproduced
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