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Conversion of Mechanical Force into Biochemical Signaling

2004; Elsevier BV; Volume: 279; Issue: 52 Linguagem: Inglês

10.1074/jbc.m406880200

1083-351X

Bing Han, Xiaohui Bai, Monika Lodyga, Jing Xu, Burton B. Yang, Shaf Keshavjee, Martin Post, Mingyao Liu,

Cellular transport and secretion

Physical forces play important roles in regulating cell proliferation, differentiation, and death by activating intracellular signal transduction pathways. How cells sense mechanical stimulation, however, is largely unknown. Most studies focus on cellular membrane proteins such as ion channels, integrins, and receptors for growth factors as mechanosensory units. Here we show that mechanical stretch-induced c-Src protein tyrosine kinase activation is mediated through the actin filament-associated protein (AFAP). Distributed along the actin filaments, AFAP can directly active c-Src through binding to its Src homology 3 and/or 2 domains. Mutations at these specific binding sites on AFAP blocked mechanical stretch-induced c-Src activation. Therefore, mechanical force can be transmitted along the cytoskeleton, and interaction between cytoskeletal associated proteins and enzymes related to signal transduction may convert physical forces into biochemical reactions. Cytoskeleton deformation-induced protein-protein interaction via specific binding sites may represent a novel intracellular mechanism for cells to sense mechanical stimulation. Physical forces play important roles in regulating cell proliferation, differentiation, and death by activating intracellular signal transduction pathways. How cells sense mechanical stimulation, however, is largely unknown. Most studies focus on cellular membrane proteins such as ion channels, integrins, and receptors for growth factors as mechanosensory units. Here we show that mechanical stretch-induced c-Src protein tyrosine kinase activation is mediated through the actin filament-associated protein (AFAP). Distributed along the actin filaments, AFAP can directly active c-Src through binding to its Src homology 3 and/or 2 domains. Mutations at these specific binding sites on AFAP blocked mechanical stretch-induced c-Src activation. Therefore, mechanical force can be transmitted along the cytoskeleton, and interaction between cytoskeletal associated proteins and enzymes related to signal transduction may convert physical forces into biochemical reactions. Cytoskeleton deformation-induced protein-protein interaction via specific binding sites may represent a novel intracellular mechanism for cells to sense mechanical stimulation. Sensing external and internal stimuli is a vital sign of living organisms. Physical forces, derived from or applied to cells, are essential signals in determining cellular structure, proliferation, differentiation, and survival. Physical forces are involved in the regulation of many physiological processes such as the maturation and branching of fetal lung (1Liu M. Post M. J. Appl. Physiol. 2000; 89: 2078-2084Crossref PubMed Scopus (124) Google Scholar), the formation of ventricles and atriums of the heart, angiogenesis (2Ingber D.E. Circ. Res. 2002; 91: 877-887Crossref PubMed Scopus (524) Google Scholar), microvascular remodeling (3Skalak T.C. Price R.J. Microcirculation. 1996; 3: 143-165Crossref PubMed Scopus (165) Google Scholar), and the maturation of bone (4Turner C.H. Owan I. Takano V. Am. J. Physiol. 1995; 269: E438-E442PubMed Google Scholar) and cartilages (5Grodzinsky A.J. Levenston M.E. Jin M. Frank E.H. Annu. Rev. Biomed. Eng. 2000; 2: 691-713Crossref PubMed Scopus (508) Google Scholar). Exposure to abnormal forces contributes to the pathogenesis of diseases such as ventilator-associated lung injury (6Pinhu L. Whitehead T. Evans T. Griffiths M. Lancet. 2003; 361: 332-340Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar), cardiac hypertrophy (7Epstein N.D. Davis J.S. Cell. 2003; 112: 147-150Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), atherosclerosis (8Shyy J.Y. Chien S. Circ. Res. 2002; 91: 769-775Crossref PubMed Scopus (458) Google Scholar), etc. Over the last two decades, mechanotransduction has become an important area of research. How cells sense mechanical signals and convert them into biochemical reactions for signal transduction is one of the fundamental questions.Specialized mechanosensory structures have been developed through evolution, such as touch receptors in Caenorhabditis elegans (9Chalfie M. Biol. Bull. 1997; 192: 125Crossref PubMed Scopus (14) Google Scholar, 10Tavernarakis N. Driscoll M. Annu. Rev. Physiol. 1997; 59: 659-689Crossref PubMed Scopus (183) Google Scholar) and bristle receptors in Drosophila (11Kernan M. Cowan D. Zuker C. Neuron. 1994; 12: 1195-1206Abstract Full Text PDF PubMed Scopus (258) Google Scholar, 12Walker R.G. Willingham A.T. Zuker C.S. Science. 2000; 287: 2229-2234Crossref PubMed Scopus (523) Google Scholar). In vertebrates, hair cells in the inner ear (13Gillespie P.G. Walker R.G. Nature. 2001; 413: 194-202Crossref PubMed Scopus (527) Google Scholar) and skin mechano-receptors (14Price M.P. Lewin G.R. McIlwrath S.L. Cheng C. Xie J. Heppenstall P.A. Stucky C.L. Mannsfeldt A.G. Brennan T.J. Drummond H.A. Qiao J. Benson C.J. Tarr D.E. Hrstka R.F. Yang B. Williamson R.A. Welsh M.J. Nature. 2000; 407: 1007-1011Crossref PubMed Scopus (421) Google Scholar) have been extensively studied. Recently, the cell membrane proteins polycystin-1 and polycystin-2 have been found to mediate mechanosensation in the primary cilium of kidney cells (15Nauli S.M. Alenghat F.J. Luo Y. Williams E. Vassilev P. Li X. Elia A.E. Lu W. Brown E.M. Quinn S.J. Ingber D.E. Zhou J. Nat. Genet. 2003; 33: 129-137Crossref PubMed Scopus (1603) Google Scholar). In these structures, transduction channels connected to intracellular and extracellular anchors control the entry of ions, thus converting a mechanical stimulus into an electrical signal, an alteration of membrane potential (13Gillespie P.G. Walker R.G. Nature. 2001; 413: 194-202Crossref PubMed Scopus (527) Google Scholar).It has been believed that all cells in the body can respond to mechanical stimulation. How regular cells sense mechanical signals is unclear. Stretch-activated and -inactivated ion channels have been found in many cell types (16Yang X-C. Sachs F. Science. 1989; 243: 1068-1071Crossref PubMed Scopus (694) Google Scholar). In response to mechanical forces, opening and closing of these channels can activate intracellular signal transduction (17Hamill O.P. Martinac B. Physiol. Rev. 2001; 81: 685-740Crossref PubMed Scopus (914) Google Scholar). However, Sawada and Sheetz used a Triton buffer to remove cytoplasm and apical cellular membrane and then applied a transient mechanical stretch to the Triton-insoluble cytoskeleton. They demonstrated a stretch-dependent binding of paxillin, focal adhesion kinase (pp125FAK), and p130CAS to the cytoskeleton (18Sawada Y. Sheetz M.P. J. Cell Biol. 2002; 156: 609-615Crossref PubMed Scopus (250) Google Scholar). Therefore, in addition to ion channels there are other mechanisms that enable cells to sense physical forces. Cells attach to the extracellular matrix (ECM) 1The abbreviations used are: ECM, extracellular matrix; AFAP, actin filament-associated protein; AFAP5Y, AFAP with mutants Y93F/Y94F/Y125F/Y451F/Y453F; AFAP71A, AFAP with mutant P71A; AFAP77A, AFAP with mutant P77A; cAFAP, chicken AFAP; EGF, epidermal growth factor; GFP, green fluorescent protein; GST, glutathione S-transferase; hAFAP, human AFAP; SH, Src homology; siRNA, small interfering RNA. 1The abbreviations used are: ECM, extracellular matrix; AFAP, actin filament-associated protein; AFAP5Y, AFAP with mutants Y93F/Y94F/Y125F/Y451F/Y453F; AFAP71A, AFAP with mutant P71A; AFAP77A, AFAP with mutant P77A; cAFAP, chicken AFAP; EGF, epidermal growth factor; GFP, green fluorescent protein; GST, glutathione S-transferase; hAFAP, human AFAP; SH, Src homology; siRNA, small interfering RNA. via integrins that link to the cytoskeleton. This complex provides a structural connection allowing the transmission of physical forces from the ECM to the cell interior. There is increasing evidence to support the belief that integrins are mechanosensors (2Ingber D.E. Circ. Res. 2002; 91: 877-887Crossref PubMed Scopus (524) Google Scholar, 8Shyy J.Y. Chien S. Circ. Res. 2002; 91: 769-775Crossref PubMed Scopus (458) Google Scholar), but how physical forces are converted into biochemical signals through the ECM-integrin-cytoskeleton complex is not addressed by this model system. Recently, Tschumperlin et al. reported that compressive stress shrinks the lateral intercellular space surrounding epithelial cells and triggers cellular signaling via autocrine binding of epidermal growth factor (EGF) family ligands to the EGF receptor (19Tschumperlin D.J. Dai G. Maly I.V. Kikuchi T. Laiho L.H. McVittie A.K. Haley K.J. Lilly C.M. So P.T. Lauffenburger D.A. Kamm R.D. Drazen J.M. Nature. 2004; 429: 83-86Crossref PubMed Scopus (270) Google Scholar). This observation again supports the importance of cellular membrane proteins in mechanosensory processes.We have previously found that mechanical stretch rapidly activates c-Src in fetal rat lung cells and that blocking stretch-induced activation of protein tyrosine kinases reduces stretch-mediated fetal lung cell proliferation (20Liu M. Qin Y. Liu J. Tanswell A.K. Post M. J. Biol. Chem. 1996; 271: 7066-7071Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). We also noted that mechanical stretch increases the binding of c-Src to the actin filament-associated protein (AFAP) (20Liu M. Qin Y. Liu J. Tanswell A.K. Post M. J. Biol. Chem. 1996; 271: 7066-7071Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Based on the molecular structure of c-Src and AFAP, we hypothesized that the cytoskeletal structure not only can transmit physical forces intracellularly but can also convert physical forces into biochemical reactions for signaling via specific binding sites. The present study tested this concept with supportive evidence that mechanical signals can be sensed intracellularly through protein-protein interaction.EXPERIMENTAL PROCEDURESCell Culture, Transfection, and Mechanical Stretch—The pCMV1 expression constructs for c-Src, chicken AFAP (cAFAP), and its mutants (AFAP71A, AFAP77A, and AFAP5Y) were gifts from Dr. Flynn (West Virginia University) (21Guappone A.C. Weimer T. Flynn D.C. Mol. Carcinog. 1998; 22: 110-119Crossref PubMed Scopus (29) Google Scholar). The full-length human AFAP (hAFAP) coding sequence was subcloned into the pCMV1 vector by replacing the resident KpnI-XbaI fragment of the pCMV-AFAP construct (21Guappone A.C. Weimer T. Flynn D.C. Mol. Carcinog. 1998; 22: 110-119Crossref PubMed Scopus (29) Google Scholar). The pEGFP-C3 eukaryotic expression vector (Clontech) was used to express cAFAP and its mutants fused to green fluorescent protein (GFP) by shuttling cAFAP, AFAP71A, AFAP77A, and AFAP5Y sequences from pCMV vectors to pEGFP-C3 vector, respectively, using EcoRI subcloning sites (22Qian Y. Baisden J.M. Zot H.G. Van Winkle W.B. Flynn D.C. Exp. Cell Res. 2000; 255: 102-113Crossref PubMed Scopus (38) Google Scholar). Small interfering RNA (siRNA) was designed according to the nucleotides 311-331 of the human AFAP sequence (GenBank™ access number AF188700). The AFAP siRNA duplex with a sense sequence of 5′-GCUCCGAAUACAUCACAU(dTdT)-3′ and a non-specific control double-stranded RNA sequence of 5′-GAAUCCGCUGAUAAGUGAC(dTdT)-3′ were synthesized by Dharmacon (Lafayette, CO).Cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum in a humidified atmosphere at 37 °C and 5% CO2. For transfection, cells were seeded in 6-well plates (2 × 105 cells/well) overnight. The cells were transfected using Lipofectamine reagent for DNA and Oligofectamine for RNA transfections (Invitrogen) following the manufacturer's protocols. Transfected cells were maintained in Dulbecco's modified Eagle's medium containing 5% fetal bovine serum and harvested 60 h later. Cells, with or without transfection, cultured on collagen I- or ProNectin (a synthetic peptide fragment of fibronectin)-coated BioFlex plates were subjected to mechanical stretch (25% elongation, 60 cycles/min for 10 min) with a Flexercell Strain Unit, FX-2000™ (Flexcell International, Hillsborough, NC). Cells cultured on the same type of plates served as non-stretch controls.Western Blotting Analysis and Immunoprecipitation—Cells were lysed with modified radioimmune precipitation assay buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 2 mm EGTA, 2 mm EDTA, and 1% Triton X-100) containing 10 μg ml-1 each aprotinin, leupeptin, pepstatin, 1 mm phenylmethylsulfonyl fluoride, 1 mm Na3VO4, and 10 mm NaF. For isolating the Triton-insoluble cytoskeletal fraction, whole cell lysates were centrifuged at 4 °C for 10 min. The insoluble pellets were dissolved in Laemmli sample buffer. Protein content was determined by a standard protein assay (Bio-Rad). Lysates containing equal amount of total protein were boiled in Laemmli sample buffer, subjected to SDS-PAGE, and then transferred to nitrocellulose membranes. Immunoblotting was conducted with antibodies against protein phosphotyrosine (4G10), Src (GD11) (Upstate Biotechnology, Lake Placid, NY), Src phosphotyrosines 416 and 527 (Biosource International, Camarillo, CA), and actin (AC-40) (Sigma) at 1:1,000 dilution. Polyclonal antibody against AFAP (F1) was a gift from Dr. Flynn and was used at 0.5 μg ml-1. Blots were detected with horseradish peroxidase-conjugated secondary antibodies and developed with an enhanced chemiluminescence detection kit (Amersham Biosciences). Membranes were stripped off and re-probed with other antibodies.For immunoprecipitation, whole cell lysates containing 1 mg of total protein were adjusted to equal volume (1 ml) and incubated with designated antibody (2 μg) at 4 °C overnight. Immune complexes were recovered by incubating with 50 μl of protein A-Sepharose beads (20%; w/v) for polyclonal antibodies or 100 μl of protein G-Sepharose beads (10%; w/v) for monoclonal antibodies for 1 h at 4 °C under gentle agitation. The immunoprecipitates were washed twice with radioimmune precipitation assay buffer. Proteins were eluted by boiling the precipitates in sample buffer and then subjected to SDS-PAGE and immunoblotted as described above.c-Src Kinase Assay—After cell transfection, 10 μl of cell lysate from each sample was used for Src kinase assay as described previously (20Liu M. Qin Y. Liu J. Tanswell A.K. Post M. J. Biol. Chem. 1996; 271: 7066-7071Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). To determine whether AFAP can directly activate c-Src, an in vitro reconstitution assay was performed (23Ma Y.C. Huang J. Ali S. Lowry W. Huang X.Y. Cell. 2000; 102: 635-646Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar). Briefly, 20 ng of purified and C-terminal Src kinase-inactivated c-Src (a gift from Dr. X. Huang, Cornell University) in Src kinase buffer (30 mm HEPES, pH 7.4, 5 mm MgCl2, 5 mm MnCl2, and 10 μm ATP) was incubated with 2 μg of Src substrate peptide (24Cheng H.C. Nishio H. Hatase O. Ralph S. Wang J.H. J. Biol. Chem. 1992; 267: 9248-9256Abstract Full Text PDF PubMed Google Scholar) and 10 μCi of [γ-32P]ATP with purified recombinant cAFAP or AFAP71A (22Qian Y. Baisden J.M. Zot H.G. Van Winkle W.B. Flynn D.C. Exp. Cell Res. 2000; 255: 102-113Crossref PubMed Scopus (38) Google Scholar) in a total 20-μl reaction volume at 30 °C for 15 min. The reaction was terminated by adding Laemmli sample buffer. After denaturing at 90 °C for 5 min, the samples were subjected to 20% SDS-PAGE to separate the substrate peptide. The gel was dried, autoradiographed, and quantified with a GS-690 densitometer (Bio-Rad).Immunofluorescent Staining and Microscopy—Immunofluorescent staining was conducted at room temperature. After different treatments, cells were fixed in 3.7% formaldehyde for 10 min, permeabilized with 0.25% Triton X-100 for 5 min, and then stained with indicated primary antibody for 60 min and the proper secondary antibody at 1:1,000 dilution (v/v) for 30 min in the dark. Slides were mounted with an anti-fading reagent, SlowFade, (Molecular Probes, Eugene, OR), followed by fluorescent microscopic examination (Nikon Canada, Toronto, Canada). The specificity of staining was determined by replacing the primary antibodies with nonspecific rabbit or mouse IgG (Sigma).For detecting the distribution of endogenous AFAP, A549 or C3H10T1/2 cells were cultured on glass cover slips and stained with anti-AFAP antibody (F1; 5 μg ml-1) and fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). For confirming the intracellular distribution of GFP-AFAP and its mutants, cells were cultured on glass coverslips and transfected with either pEGFP vector alone or pEGFP vectors containing fused cAFAP or its mutants (AFAP71A, AFAP77A, and AFAP5Y) for 48 h. Cells were co-stained with rhodamine-phalloidin (Molecular Probe) at 1:1,000 dilution (v/v) for 30 min to image actin filaments.For evaluating the role of AFAP in stretch-induced c-Src activation, cells were cultured on collagen-coated BioFlex plates and transfected with either pEGFP vector alone or pEGFP vectors containing fused AFAP71A, AFAP77A, and AFAP5Y mutants for 48 h and then exposed to mechanical stretch or static control, as described above. The transfected cells were identified by GFP. The stretch-induced activation of Src was shown by staining with anti-Src phosphotyrosine 416 antibody followed by goat anti-mouse IgG conjugated with Alex 594 (Molecular Probe).Statistical Analysis—Data are expressed as mean ± S.D. from at least three experiments and analyzed by one-way analysis of variance followed by a Student-Neuman-Keuls test with significance defined as p < 0.05.RESULTSMechanical Stretch-induced c-Src Activation and c-Src/AFAP Interaction—C3H10T1/2, C3H10T1/2-5H, NIH3T3, COS7, and A549 cells were cultured on collagen I-coated flexible plates and subjected to mechanical stretch (25% elongation, 60 cycles/min for 10 min) with a Flexcell Strain Unit. Autophosphorylation of Src Tyr-416 at the activation loop is a critical step leading to full activation of c-Src (25Xu W. Doshi A. Lei M. Eck M.J. Harrison S.C. Mol. Cell. 1999; 3: 629-638Abstract Full Text Full Text PDF PubMed Scopus (723) Google Scholar), which was increased in these cells by stretch regardless of the expression levels of total Src protein (Fig. 1a). Receptor protein tyrosine phosphatase α has been reported as a transducer of mechanical force on integrin-cytoskeleton linkage (26von Wichert G. Jiang G. Kostic A. De Vos K. Sap J. Sheetz M.P. J. Cell Biol. 2003; 161: 143-153Crossref PubMed Scopus (181) Google Scholar), which can activate c-Src by reducing its phosphorylation on the Tyr-527 residue. In the present study, the phosphorylation of c-Src Tyr-527 was not decreased after cell stretch (Fig. 1a), suggesting that the c-Src activation is not secondary to the activation of protein tyrosine phosphatases. Similar results were obtained when these cells were cultured on fibronectin fragment-coated plates (data not shown). Therefore, c-Src activation appears to be a common phenomenon in different cell types responding to mechanical stimuli on different ECMs.Using COS7 cells as an example, we demonstrated that stretch for 10 min induced an increase of c-Src in a Tritoninsoluble cytoskeletal fraction (Fig. 1b). AFAP was first cloned from chicken. It contains an actin-binding motif and is localized to actin filaments, the cortical actin matrix, and along the leading edge of the cell (27Flynn D.C. Leu T.H. Reynolds A.B. Parsons J.T. Mol. Cell. Biol. 1993; 13: 7892-7900Crossref PubMed Scopus (108) Google Scholar). This distribution pattern suggests that AFAP is suitable for transmitting physical forces along the actin filaments and that it may mediate c-Src translocation to the cytoskeletal fraction through protein binding. Using C3H10T1/2-5H cells, we demonstrated that the binding of c-Src and AFAP, as determined by co-immunoprecipitation followed by immunoblotting, increased after mechanical stretch (Fig. 1c). Similar results were obtained with COS7 cells (data not shown).Crystallographic structure studies have revealed that the kinase activity of c-Src is maintained at a low basal level by two major intramolecular interactions; one is the binding of its SH3 domain to the linker between the SH2 domain and the kinase domain, and the other is the binding of its SH2 domain to the phosphorylated tyrosine residue 527 in its C-terminal tail (28Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1242) Google Scholar, 29Williams J.C. Weijland A. Gonfloni S. Thompson A. Courtneidge S.A. Superti-Furga G. Wierenga R.K. J. Mol. Biol. 1997; 274: 757-775Crossref PubMed Scopus (220) Google Scholar, 30Sicheri F. Kuriyan J. Curr. Opin. Struct. Biol. 1997; 7: 777-785Crossref PubMed Scopus (328) Google Scholar) (Fig. 1d). Interruption of these interactions with high affinity ligands for either the Src SH2 or SH3 domain may activate the enzyme. AFAP contains Src SH3 and SH2 binding motifs (21Guappone A.C. Weimer T. Flynn D.C. Mol. Carcinog. 1998; 22: 110-119Crossref PubMed Scopus (29) Google Scholar). Therefore, it may competitively bind to the c-Src SH2 and SH3 domains and induce an alteration of c-Src configuration, leading to c-Src activation. The interaction between AFAP and c-Src may be an important mechanism for mechanical stretch-induced c-Src activation (Fig. 1d).Cloning and Characterization of Human AFAP Protein—To determine the molecular structure of AFAP in mammalian cells, we cloned the hAFAP gene from human lung epithelial (A549) cells. The GenBank™ accession number of hAFAP is AF188700. The identity of peptide sequences between human and chicken AFAP is 87%. All of the functional motifs and domains described for chicken AFAP protein (31Qian Y. Baisden J.M. Westin E.H. Guappone A.C. Koay T.C. Flynn D.C. Oncogene. 1998; 16: 2185-2195Crossref PubMed Scopus (44) Google Scholar) are present in hAFAP in the same order (data not shown). The proline-rich Src SH3 binding motif, five putative tyrosines for Src SH2 binding, and an actin binding region are presented in Fig. 2a. The hAFAP gene is localized on human chromosome 4p16, as confirmed by PCR-based sequence-tagged sites serving as landmarks of genomic maps and by fluorescent in situ hybridization (data not shown). The steady state mRNA levels of hAFAP are differentially expressed in human tissues (Fig. 2b). The endogenous hAFAP protein is co-localized with actin filaments in A549 cells (Fig. 2c), as determined by double staining with rhodamine-conjugated phalloidin to decorate actin stress fibers and the antibody for AFAP followed by fluorescein isothiocyanate-conjugated anti-rabbit IgG to highlight hAFAP.Fig. 2Molecular cloning and characterization of human AFAP. a, schematic diagram for Src SH3, SH2, and actin binding motifs (Bmf.) in AFAP. b, differential expression of hAFAP in various human tissues. Manufacturer-made membranes cross-linked with equal amounts of human RNA (<10% variation) were probed for expression of hAFAP by Northern blotting. c, co-localization of hAFAP with actin filaments. A549 cells were stained with an anti-AFAP antibody (a) and rhodamine-phalloidin for the actin filament (b). The merged image shows the alignment of hAFAP along with actin filaments (c). d, tyrosine phosphorylation of hAFAP and its binding with c-Src. A549 cell lysates were immunoprecipitated (IP) with the indicated antibodies, and the blots (IB) were probed with the indicated antibodies. pTyr, phosphotyrosine; NRS, nonspecific rabbit serum. e, binding of hAFAP to the Src SH2 and SH3 domains. A549 cell lysates were incubated with the GST protein alone or the GST protein fused with the Src SH2, SH3, or SH3/SH2 domains, respectively. The precipitates were analyzed by immunoblotting with anti-AFAP antibody (top). For protein loading control, after transferring proteins to the blotting membranes the gel was stained with Coomassie Blue (bottom).View Large Image Figure ViewerDownload Hi-res image Download (PPT)The endogenous hAFAP protein in A549 cells is tyrosine-phosphorylated and binds to c-Src as determined by immunoprecipitation and immunoblotting (Fig. 2d). To determine the binding affinity of Src SH3 and SH2 domains to hAFAP, bacterial GST fusion proteins containing either or both Src SH3 and SH2 domains and captured on glutathione beads were incubated with A549 cell lysates, and the bound proteins were resolved by SDS-PAGE and blotted with anti-AFAP antibody. hAFAP binds most efficiently to GST-Src SH2/SH3 beads, more so to GST-Src SH2 than to Src SH3 beads, and fails to bind to GST-only beads (Fig. 2e).Overexpression of AFAP Activates c-Src—To determine whether AFAP can activate c-Src, either hAFAP or cAFAP was co-expressed with c-Src in COS7 cells. Tyrosine phosphorylation of multiple proteins (Fig. 3a, top section) and autophosphorylation of c-Src at the Tyr-416 residue (Fig. 3a, second section from top, and b) were increased in the presence of both AFAP and c-Src. These effects are dependent on c-Src kinase activity, because the co-expression of c-Src-KD (a kinase inactive mutant of c-Src) with hAFAP or cAFAP did not have similar effects (Fig. 3a). The phosphorylation of c-Src Tyr527 was not reduced when hAFAP or cAFAP was co-expressed with c-Src (Fig. 3a, third section from top). The total Src protein levels in kinase-inactive c-Src-KD-transfected cells were higher (Fig. 3a, fourth section from top). It has been shown that Src activation leads to its degradation via an ubiquitin-dependent mechanism (32Hakak Y. Martin G.S. Curr. Biol. 1999; 9: 1039-1042Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Thus, in kinase inactive c-Src-KD-transfected cells the lack of kinase activity may stabilize c-Src. We further measured c-Src kinase activity with a specific Src substrate peptide, and this activity was significantly increased when hAFAP or cAFAP was co-expressed with c-Src (Fig. 3c).Fig. 3AFAP directly activates c-Src. a, co-expression of AFAP and c-Src increased total protein tyrosine phosphorylation and Src Tyr-416 phosphorylation. Constructs containing vector, hAFAP, cAFAP, or c-Src alone or hAFAP (or cAFAP) together with c-Src (or c-Src-KD, kinase inactive c-Src) were transfected into COS7 cells. The protein tyrosine phosphorylation (pTyr) status, the phosphorylation of the Tyr-416 (Src-pY416) or Tyr-527 (Src-pY527) of c-Src, and the expression of c-Src and AFAP protein levels were revealed by Western blotting. b, the Tyr-416 phosphorylation of c-Src was significantly increased when co-expressed with either hAFAP or cAFAP in COS7 cells. Data were quantified by densitometry from independent experiments. *, p < 0.05 versus other groups. c, the Src kinase activity was increased in hAFAP/c-Src or cAFAP/c-Src co-transfected cells. *, p < 0.05 versus c-Src alone. d, recombinant cAFAP directly activates c-Src in vitro in a dose-dependent fashion. An in vitro reconstitution Src kinase assay was conducted with C-terminal Src kinase-treated inactive c-Src and increasing doses of recombinant cAFAP. The phosphorylation of the substrate peptide was measured by densitometry. *, p < 0.05; **, p < 0.01 versus control (no cAFAP).View Large Image Figure ViewerDownload Hi-res image Download (PPT)To determine whether AFAP-induced c-Src activation is a direct effect, various amounts of purified, recombinant cAFAP were incubated with an inactivated c-Src protein, a Src substrate peptide, together with [γ-32P]ATP. Phosphorylated substrate peptide was separated by 20% SDS-PAGE and exposed to an x-ray film (23Ma Y.C. Huang J. Ali S. Lowry W. Huang X.Y. Cell. 2000; 102: 635-646Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar). Recombinant cAFAP increased phosphorylation of the Src substrate in a dose-dependent fashion (Fig. 3d).Effects of SH2 and SH3 Binding on AFAP-induced c-Src Activation—Binding to SH3 domains is mediated through proline-rich sequences (33Pawson T. Adv. Cancer Res. 1994; 64: 87-110Crossref PubMed Google Scholar). Src-SH3-specific binding uses a sequence of seven amino acids of the consensus RPLPXXP (34Alexandropoulos K. Cheng G. Baltimore D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3110-3114Crossref PubMed Scopus (250) Google Scholar). There are two stretches of proline-rich amino acids in the N terminus of AFAP. The first stretch (MPLPEIP) is a consensus Src-SH3 binding motif (Fig. 4a). Mutation of the last proline to alanine at position 71 (AFAP71A) significantly decreased total protein tyrosine phosphorylation and c-Src Tyr-416 phosphorylation (Fig. 4, b and c). In contrast, mutation from proline to alanine at position 77 (AFAP77A) in the second stretch of the proline-rich region had no such effects (Fig. 4, a-c). There are five putative Src SH2 binding tyrosines in AFAP (21Guappone A.C. Weimer T. Flynn D.C. Mol. Carcinog. 1998; 22: 110-119Crossref PubMed Scopus (29) Google Scholar). We co-expressed c-Src with a mutant (AFAP5Y), of which tyrosines were replaced with phenylalanines at positions 93, 94, 125, 451, and 453 (Fig. 4a). This mutant also decreased significantly protein tyrosine phosphorylation and the phosphorylation of c-Src at Tyr-416 (Fig. 4, b and c). AFAP71A and AFAP5Y had no significant effect on phosphorylation of the Tyr-527 of c-Src (Fig. 4b). Similarly to our finding, Lerner and Smithgall also showed that human immunodeficiency virus-1 Nef protein-induced Hck (another Src family member) activation is not associated with a decrease in the phosphorylation of the conserved C-terminal tyrosine (