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• W2053467415 abstract "The Src-activating and signaling molecule (Srcasm) is a recently described activator and substrate of Src-family tyrosine kinases (SFKs). When phosphorylated at specific tyrosines, Srcasm associates with Grb2 and p85, the regulatory subunit of phosphoinositide 3-kinase; however, little is known about the role of Srcasm in cellular signaling. Data presented here demonstrate that epidermal growth factor (EGF) receptor ligands promote the tyrosine phosphorylation of endogenous and adenovirally transduced Srcasm in keratinocytes, and that increased levels of Srcasm activate endogenous SFKs, with a preference for Fyn and Src. In addition, Srcasm potentiates EGF-dependent signals transmitted by SFKs in keratinocytes. Tyrosine phosphorylation of Srcasm is dependent on growth factors and the activity of EGFR and SFKs. Increased Srcasm expression enhances p44/42 mitogen-activated protein kinase activity and Elk-1-dependent transcriptional events. Elevated Srcasm levels inhibit keratinocyte proliferation while promoting specific aspects of keratinocyte differentiation. Lastly, Srcasm levels are decreased in human cutaneous neoplasia. Collectively, these data demonstrate that Srcasm plays a role in linking EGF receptor- and SFK-dependent signaling to differentiation in keratinocytes. The Src-activating and signaling molecule (Srcasm) is a recently described activator and substrate of Src-family tyrosine kinases (SFKs). When phosphorylated at specific tyrosines, Srcasm associates with Grb2 and p85, the regulatory subunit of phosphoinositide 3-kinase; however, little is known about the role of Srcasm in cellular signaling. Data presented here demonstrate that epidermal growth factor (EGF) receptor ligands promote the tyrosine phosphorylation of endogenous and adenovirally transduced Srcasm in keratinocytes, and that increased levels of Srcasm activate endogenous SFKs, with a preference for Fyn and Src. In addition, Srcasm potentiates EGF-dependent signals transmitted by SFKs in keratinocytes. Tyrosine phosphorylation of Srcasm is dependent on growth factors and the activity of EGFR and SFKs. Increased Srcasm expression enhances p44/42 mitogen-activated protein kinase activity and Elk-1-dependent transcriptional events. Elevated Srcasm levels inhibit keratinocyte proliferation while promoting specific aspects of keratinocyte differentiation. Lastly, Srcasm levels are decreased in human cutaneous neoplasia. Collectively, these data demonstrate that Srcasm plays a role in linking EGF receptor- and SFK-dependent signaling to differentiation in keratinocytes. Understanding the molecular mechanisms that regulate cellular proliferation and differentiation are fundamental biological questions important within metazoans, and tyrosine kinases play critical roles in regulating these processes (1Blume-Jensen P. Hunter T. Nature. 2001; 411: 355-365Crossref PubMed Scopus (3035) Google Scholar, 2Thomas S.M. Brugge J.S. Annu. Rev. Cell Dev. Biology. 1997; 13: 513-609Crossref PubMed Scopus (2126) Google Scholar, 3Walker J.L. Zhang L. Menko A.S. Dev. Dyn. 2002; 224: 361-372Crossref PubMed Scopus (39) Google Scholar). The Src family tyrosine kinases (SFKs) 1The abbreviations used are: SFK, Src family kinase; Ad, adenovirus; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; KGF, keratinocyte growth factor (also known as FGF-7); MAP, mitogen-activated protein; PHK, primary human keratinocyte; SCC, squamous cell carcinoma; SCIS, squamous cell carcinoma in situ; Srcasm, Src activating and signaling molecule; TGF-α, transforming growth factor α; BrdUrd, bromodeoxyuridine; aa, amino acid(s); PBS, phosphate-buffered saline; HA, hemagglutinin; h, human; m.o.i., multiplicity of infection; DAPI, 4′,6-diamidino-2-phenylindole.1The abbreviations used are: SFK, Src family kinase; Ad, adenovirus; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; KGF, keratinocyte growth factor (also known as FGF-7); MAP, mitogen-activated protein; PHK, primary human keratinocyte; SCC, squamous cell carcinoma; SCIS, squamous cell carcinoma in situ; Srcasm, Src activating and signaling molecule; TGF-α, transforming growth factor α; BrdUrd, bromodeoxyuridine; aa, amino acid(s); PBS, phosphate-buffered saline; HA, hemagglutinin; h, human; m.o.i., multiplicity of infection; DAPI, 4′,6-diamidino-2-phenylindole. have been implicated in promoting differentiation in a number of cell types such as skin and lens keratinocytes, oligodendrocytes, and endometrial cells (3Walker J.L. Zhang L. Menko A.S. Dev. Dyn. 2002; 224: 361-372Crossref PubMed Scopus (39) Google Scholar, 4Maruyama T. Yoshimura Y. Yodoi J. Sabe H. Endocrinology. 1999; 140: 2632-2636Crossref PubMed Scopus (28) Google Scholar, 5Osterhout D.J. Wolven A. Wolf R.M. Resh M.D. Chao M.V. J. Cell Biol. 1999; 145: 1209-1218Crossref PubMed Scopus (198) Google Scholar, 6Calautti E. Missero C. Stein P.L. Ezzell R.M. Dotto G.P. Genes & Dev. 1995; 9: 2279-2291Crossref PubMed Scopus (103) Google Scholar, 7Gniadecki R. Biochem. Pharmacol. 1998; 55: 499-503Crossref PubMed Scopus (20) Google Scholar, 8Shimizu Y. Yamamichi N. Saitoh K. Watanabe A. Ito T. Yamamichi-Nishina M. Mizutani M. Yahagi N. Suzuki T. Sasakawa C. Yasugi S. Ichinose M. Iba H. Oncogene. 2003; 22: 884-893Crossref PubMed Scopus (16) Google Scholar). In addition, increased activity of the SFKs has been associated with a variety of epithelial tumors, including colonic adenocarcinoma, mammary carcinoma, and murine cutaneous squamous cell carcinoma (9Park J. Meisler A.I. Cartwright C.A. Oncogene. 1993; 8: 2627-2635PubMed Google Scholar, 10Muthuswamy S.K. Muller W.J. Oncogene. 1995; 11: 1801-1810PubMed Google Scholar, 11Cartwright C.A. Meisler A.I. Eckhart W. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 558-562Crossref PubMed Scopus (279) Google Scholar, 12Frame M.C. Biochim. Biophys. Acta. 2002; 1602: 114-130PubMed Google Scholar, 13Matsumoto T. Jiang J. Kiguchi K. Carbajal S. Rho O. Gimenez-Conti I. Beltran L. DiGiovanni J. Mol. Carcinog. 2002; 33: 146-155Crossref PubMed Scopus (19) Google Scholar). Given the importance of SFKs in cellular physiology, cells have evolved a variety of mechanisms to regulate their activity. Structural analyses have shown that SFKs can form an inactive “closed” configuration, with the SH2 domain bound to the phosphorylated C-terminal tyrosine (Tyr-527) and the SH3 domain associated with a polyproline motif in the linker region; this configuration prevents phosphorylation at tyrosine 416 in the activation loop rendering the kinase inactive (14Xu W. Doshi A. Lei M. Eck M.J. Harrison S.C. Mol. Cell. 1999; 3: 629-638Abstract Full Text Full Text PDF PubMed Scopus (714) Google Scholar, 15Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1229) Google Scholar). Given these structural data, it has been hypothesized that molecules containing ligands for the SH2 and SH3 domains of SFKs may disrupt the SH2-dependent and SH3-dependent intramolecular interactions and promote opening of the “closed” configuration, phosphorylation of tyrosine 416, and activation of the kinase (14Xu W. Doshi A. Lei M. Eck M.J. Harrison S.C. Mol. Cell. 1999; 3: 629-638Abstract Full Text Full Text PDF PubMed Scopus (714) Google Scholar, 15Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1229) Google Scholar). Theoretically, if a molecule contained a stronger ligand for the SFK SH2 domain than the C-terminal pTyr-527 motif, then such a molecule could activate Src kinases regardless of Csk activity (the kinase that induces Tyr-527 phosphorylation), thereby independently promoting important regulatory signals (2Thomas S.M. Brugge J.S. Annu. Rev. Cell Dev. Biology. 1997; 13: 513-609Crossref PubMed Scopus (2126) Google Scholar, 16Okada M. Nada S. Yamanashi Y. Yamamoto T. Nakagawa H. J. Biol. Chem. 1991; 266: 24249-24252Abstract Full Text PDF PubMed Google Scholar). Some SFK-activating molecules have been identified, including FAK and Sin; however, these molecules do not contain the highest affinity ligands for the SFK SH2 domain, a pYEEI motif (17Schaller M.D. Hildebrand J.D. Parsons J.T. Mol. Biol. Cell. 1999; 10: 3489-3505Crossref PubMed Scopus (178) Google Scholar, 18Alexandropoulos K. Baltimore D. Genes Dev. 1996; 10: 1341-1355Crossref PubMed Scopus (221) Google Scholar, 19Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Ratnofsky S. Lechleiter R.J. SH2 domains recognize specific phosphopeptide sequences.Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2362) Google Scholar). The Src-activating and signaling molecule (Srcasm) is a recently described activator and substrate of SFKs that contains the optimal ligands for the SH2 and SH3 domains of SFKs, and thus can activate SFKs in vitro (20Seykora J.T. Mei L. Dotto G.P. Stein P.L. J. Biol. Chem. 2002; 277: 2812-2822Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). In addition, phosphorylated Srcasm can associate in vitro with Grb2 and p85 (the regulatory subunit of phosphoinositide 3-kinase), molecules that regulate important signaling pathways involving RAS-MAP kinases and Akt, respectively (19Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Ratnofsky S. Lechleiter R.J. SH2 domains recognize specific phosphopeptide sequences.Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2362) Google Scholar, 20Seykora J.T. Mei L. Dotto G.P. Stein P.L. J. Biol. Chem. 2002; 277: 2812-2822Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 21Songyang Z. Shoelson S.E. McGlade J. Oliver P. Pawson T. Bustelo X.R. Barbacid M. Sabe H. Hanafusa H. Yi T. Mol. Cell. Biol. 1994; 14: 2777-2785Crossref PubMed Scopus (826) Google Scholar). Given the molecular attributes of Srcasm, we speculated that it may represent an important regulator of SFKs in cells. In this report, we provide evidence to support such a role of Srcasm in keratinocytes. Srcasm is an SFK substrate downstream of the EGF receptor, and it activates endogenous cellular SFKs with a preference for Fyn and Src. Tyrosine phosphorylation of Srcasm is dependent on the presence of EGFR ligands, EGFR activity, and SFK activity. In addition, Srcasm appears to modulate p44/42 MAP kinase activity in a manner dependent on EGF stimulation. Srcasm also promotes downstream signaling events such as stimulation of Elk-1-dependent transcription, and it decreases the S-phase fraction of keratinocytes under a variety of conditions. Increased levels of Srcasm promoted expression of differentiation markers in primary keratinocytes, and Srcasm protein levels were found to be decreased in lesions of keratinocytic neoplasia. The data presented provide a molecular context for Srcasm signaling in keratinocytes and suggest that Srcasm may play an important role regulating SFKs and differentiation in epithelial cells. Adenovirus Construction—Srcasm adenovirus (Ad-Srcasm) was made by the adenovirus vector core at the University of Pennsylvania by cloning hemagglutinin-tagged Srcasm (HA-Srcasm) into a shuttle vector containing a cytomegalovirus promoter. The expression cassette was excised and ligated into an adenoviral backbone vector (pAdX), and the intactness of the coding region was performed by restriction analysis. DNA was amplified and linearized with PacI, then transfected into 293 cells. Viral induced cytopathic change was confirmed microscopically, and the virus was amplified then purified by CsCl gradient centrifugation. The control adenovirus (Ad-Con) contains the green fluorescent protein coding region driven by a bacterial LacZ promoter. Hence, it expresses only in bacterial cells, and serves as a control for adenoviral infection. Primary Human Keratinocyte Culture, Infections/Transfections, and Pharmacological Manipulations—Cultures of neonatal human keratinocytes were obtained from foreskins. After isolation, the cells were cultured in MCDB-153 medium, supplemented with 0.1 mm ethanolamine (Sigma), 0.1 mmO-phosphoethanolamine (Sigma), 10 μg/liter hEGF (Invitrogen), 5 × 10-7m hydrocortisone (Sigma), 5 mg/liter insulin (Sigma), bovine pituitary extract (BPE) (150 μg/ml), 100 units/ml penicillin, and 100 mg/liter streptomycin, 70 μm calcium, and maintained at 37 °C with an atmosphere of 5% CO2. Growth factor depletion was conducted in MCDB base lacking insulin, EGF, and BPE. All keratinocytes analyzed were less than passage 4. Keratinocytes at ∼60–70% confluence were infected with Ad-Srcasm or Ad-Con at the indicated m.o.i. under the culture conditions described above and usually analyzed or manipulated 16 h after infection. Luciferase assays utilized the Pathdetect Elk-1 trans-reporting assay (Stratagene) according to the manufacturer's recommendations. Cells were transfected in MCDB (without penicillin/streptomycin) using a ratio of 1 μg of DNA to 5 μl of Lipofectamine (Invitrogen) according to the manufacturer's instruction. Cells were allowed to recover for 18 h before virus infection and subsequent experimentation. Prior to EGF, TGF-α, KGF stimulation, cells were deprived of growth factors for 24 h, then incubated with hEGF (10 or 100 ng/ml, Invitrogen), hTGF-α (0.1 ng/ml, Sigma #T7924), hKGF (10 ng/ml, Research Diagnostics Inc.) for the indicated times. Some cells were incubated with Src family kinase-selective inhibitor PP2 (10 μm), or its negative control PP3 (10 μm), or specific EGFR kinase inhibitor AG112 (20 μm)or its negative control AG9 (20 μm), or the MEK 1/2 inhibitors U0126 (10 μm) or its negative control U0124 (10 μm) 60 min before cell lysis (all from Calbiochem). Antibodies—α-Phosphotyrosine (Upstate Biotechnology, 4G10) was used at 1/1000 for Western blotting. α-Phosphotyrosine immunoprecipitation of endogenous Srcasm was performed overnight at 4 °C using 1.5 mg of protein lysate and 2.5 μg of antibody; α-phosphotyrosine immunoprecipitation for transduced Srcasm was performed using 0.5 mg of lysate for 15 min with 1.5 μg of antibody using the Catch and Release Reversible Immunoprecipitation system (Upstate Biotechnology). High affinity α-HA antibody (clone 3F10, Roche Molecular Biochemicals) was used at 1/1000 for Western blotting and at 2 μg/ml for immunoprecipitations. α-Activated Src family kinase antibody (phosphor-Tyr-416) (Cell Signaling) was used at 1/1000 for Western blotting. α-p44/42 MAP kinase antibody and α-phospho-p44/42 MAP kinase antibody (Cell Signaling) were used at 1/1000 to detect total or phosphorylated p44/42 MAP kinase. α-Fyn (SC-16), α-Src (SC-19), and α-Yes (SC-14, all from Santa Cruz Biotechnology) were all used at 1:1000 for Western blotting, and 3 μg was used for immunoprecipitation. α-Src2 antibody (SC-8056) that detects Src, Fyn, and Yes was used at 1:500. α-Srcasm antibody is a polyclonal antibody that was generated by incubating rabbits with three purified glutathione S-transferase fusion proteins spanning murine Srcasm (aa 1–200, aa 150–400, and aa 389–474). For Western blotting, the Srcasm antisera was diluted 1:1000. For endogenous Srcasm immunoprecipitation, IgG was purified from the crude serum via Protein A affinity chromatography; 6 μg of this IgG fraction was used for each immunoprecipitation. The specificity of the α-Srcasm antibody was demonstrated by Western blotting COS protein lysates overexpressing Srcasm; preincubating the antisera with the 1 μm Srcasm fusion proteins for 30 min at room temperature ablates detection of Srcasm (Fig. 8A). Immunoblotting and Immunoprecipitation—Cell lysates were prepared using radioimmune precipitation assay lysis buffer (150 mm NaCl, 50 mm Tris-HCl, pH 8.0,1% Triton X-100, 0.1% SDS, 0.5% deoxycholate, 5 mm EDTA, 10 mm NaF, 10 mm sodium pyrophosphate, 1 μg/ml aprotinin, 100 μm leupeptin, 1 mm phenylmethylsulfonyl fluoride, 1 mm NaVO4). Tissue samples of normal skin and squamous cell carcinoma tissue were obtained after Moh's surgery and frozen at -80 °C until use (Internal Review Board protocols 707777 and 706409). The tissue samples were diced and homogenized in radioimmune precipitation assay buffer. The lysates were incubated on ice for 10 min and cleared by centrifugation at 14,000 × g for 10 min at 4 °C. The supernatants were collected and assayed for protein content using the MicroBCA protein assay kit (Pierce Chemical Co.). For immunoprecipitation with α-HA antibody, typically 1 mg of pre-cleared lysates was sequentially incubated with α-HA, rabbit α-Rat IgG, and protein A-agarose. Immunoprecipitation with α-pY antibody was carried out with the Catch and Release reversible immunoprecipitation system (Upstate). For α-Srcasm immunoprecipitation of Srcasm, typically 1.5 mg of keratinocyte lysate were utilized, and the immunoprecipitations were carried out for ∼16 h. Equal amounts of lysates or washed immunoprecipitates were separated by SDS-PAGE and transferred to PolyScreen polyvinylidene difluoride transfer membrane (PerkinElmer Life Sciences). For filaggrin detection, Western blots were conducted in a standard manner with the indicated antibodies and developed using an enhanced chemiluminescence kit as described by the manufacturer (Lumilight Plus, Roche Molecular Biochemicals). Immunofluorescence Analysis—Cells were cultured in 2-well Lab-Tek chamber slides (Nalge Nunc International) and infected with Ad-Srcasm or Ad-Con (m.o.i. 200). Cells were fixed with ice-cold 4% paraformaldehyde in PBS for 10 min on ice then permeabilized with 0.2% Triton/PBS for 10 min. Fixed cells were washed with PBS and then incubated with primary antibodies for 60 min at room temperature, followed by washing, and incubation with the appropriate secondary antibodies in the same manner. Primary antibodies used at a 1:100 dilution included: phospho-Y416 (Cell Signaling Technology), phospho p44/42 (Cell Signaling Technology), HA-high affinity (Roche Applied Science), Src (SC-19, Santa Cruz Biotechnology), Fyn (SC-16, Santa Cruz Biotechnology), and Yes (SC-14, Santa Cruz Biotechnology). α-Filaggrin (PRB-417P, Covance) was used at 1:200. α-BrdUrd (#1299964, Roche Applied Science) was used at 1:50. Secondary antibodies were species-specific for the primary antibodies and conjugated with fluorescein isothiocyanate or Texas Red; these antibodies were used at 1:100 (Jackson ImmunoResearch). Nuclei were counterstained using DAPI. Imaging was performed on an IX-81 inverted fluorescence scope under oil coupled with a Cooke Sensicam digital camera. Where indicated, deconvolved, confocal images (nearest neighbors algorithm) and image overlay were performed using Slide-book, version 4, at × 600 magnification. In Vitro Kinase Assays—PHKs were infected with Ad-Con or Ad-Srcasm at m.o.i. 200 and cultured in MCDB complete media. Sixteen hours post-infection, cells were lysed in radioimmune precipitation assay buffer, and 1 mg of cell lysate was subjected to immunoprecipitation with 3 μg of α-Fyn, α-Src, or α-Yes for 16 h at 4 °C. The immunoprecipitated Fyn, Src, and Yes were subjected to in vitro kinase assays according to manufacturer's specifications (Src Assay kit #17-131, Upstate Biotechnology). Phosphocellulose filters with bound 32P-labeled substrate were assessed by liquid scintillation using Econofluor-2 (Packard Instrument Co., Cat. no. 6NE9699) in a Beckman LS-6500 instrument. Background cpm activity (from a no kinase control reaction) was subtracted from all values. cpm values of assays of SFKs from control cells was set equal to 1; values of assays from Ad-Srcasm cells were divided by the corresponding value from control infected cells to obtain -fold stimulation. Data presented are from two independent experiments. Luciferase Assays—Human primary keratinocytes were plated in complete MCDB media, allowed to reach 50% confluency, and then transfected with plasmids from the Pathdetect trans-reporting system (Stratagene), specific for assaying Elk-1-dependent transcription from serum-response elements. Primary keratinocytes plated at 5 × 104 per 35-mm well were transfected using Lipofectamine (5 μl/μg of DNA) with 0.5 μg of pFA2-Elk-1 and 1.0 μg of pFR-Luc. Approximately 16 h after transfection, cells were not infected or infected with Ad-Srcasm or Ad-Con at an m.o.i. of 200. After 18 h of infection, some cells were stimulated for 5 h with 100 μg/liter hEGF. Luciferase activity in equivalent amounts of cell lysates was determined using the firefly luciferase assay system (Promega) in a Monolight 3096 luminometer (BD Biosciences) equipped with Simplicity 2.0 software. Relative luciferase units reflect the numerical values obtained from the luminometer; values represent the averages of three experiments with the standard deviation indicated by bars. Equivalent amounts of cell lysate were subjected to Western blotting to detect Srcasm. Flow Cytometry Proliferation Assay—Sub-confluent PHKs infected with Ad-Srcasm or Ad-Con (m.o.i. 200) 18 h prior were labeled with 10 μm BrdUrd for 3h under standard MCDB culture conditions (see above). Then, cells were harvested and stained with fluorescein isothiocyanate conjugated anti-BrdUrd antibody and 7-amino-actinomycin D following the BrdUrd flow kit instruction (BD Pharmingen); the analysis parameters used are those recommended by the manufacturer. The stained cells were analyzed by FACScan machine (BD Pharmingen); the cell populations were sorted into G0/G1 (gate R2), G2/M (gate R3), and S phase (gate R4) according to the manufacturer's specifications. In Fig. 6C, subconfluent PHKs were infected as above and deprived of growth factors for 16 h, and then some cells were stimulated with EGF (10 ng/ml) for the indicated times. These cells were collected, washed in PBS, and fixed in 70% ethanol, overnight at 4 °C. Cells were pelleted then washed in PBS with 2% fetal calf serum, and subjected to passage through a 0.2-μm filter. Cells were treated with RNase A and stained with propidium iodide. Cells were analyzed on a FACScan machine using ModFit LT 3.1 (Verity Software House) to determine S-phase fraction. Data are representative of two experiments. BrdUrd Labeling and Filaggrin Blotting—Cells were cultured in 2-well Lab-Tek chamber slides (Nalge Nunc International) and infected with Ad-Srcasm or Ad-Con (m.o.i. 200) in MCDB complete media. 24 h after infection, cells were labeled with BrdUrd (10 μm) for 3 h and then fixed and permeabilized with 50 mm glycine, 70% ethanol, pH 2, for 20 min at 4 °C. Cells were stained for BrdUrd, filaggrin, or Srcasm as indicated under “Immunofluorescence Analysis.” Cells stained for filaggrin and Srcasm were fixed and permeabilized also as indicated under “Immunofluorescence Analysis.” For filaggrin Western blotting, PHKs were plated on collagen-coated dishes and infected with Ad-Srcasm or Ad-Con (m.o.i. 200). Cells were cultured in MCDB complete media. At the times indicated (post-infection), cells were lysed in radioimmune precipitation assay, and equivalent protein amounts were subjected to Western blot analysis. Immunohistochemical Detection of Srcasm in Formalin-fixed Biopsy Specimens—Formalin-fixed, paraffin-embedded tissue samples of actinic keratosis, squamous cell carcinoma, in situ, invasive squamous cell carcinoma and unremarkable skin were collected from the dermato-pathology archives of the University of Pennsylvania Department of Dermatology with Internal Review Board approval under protocol 704450. Biopsy specimens containing unremarkable epidermis adjacent to lesional skin were selected as normal skin provides an internal control. For immunohistochemical staining, tissue samples were blocked for 1 h at room temperature with 10% horse serum, then incubated for 1 h with affinity-purified rabbit polyclonal anti-Srcasm antibody (4 μg/ml). The antibody was affinity-purified by incubating anti-sera with glutathione S-transferase immobilized on polyvinylidene difluoride membranes followed by the Srcasm fusion proteins immobilized on polyvinylidene difluoride membranes. The anti-Srcasm antibody was eluted and handled as previously discussed (22Rosen A. Keenan K.F. Thelen M. Nairn A.C. Aderem A. J. Exp. Med. 1990; 172: 1211-1215Crossref PubMed Scopus (165) Google Scholar). Control preimmune IgG was purified using protein A-agarose and used at the same concentration as the affinity purified antibody. Immunostaining was performed using the avidin-biotin complex technique (Roche Applied Science). Increases or decreases in expression were made by comparing lesions with adjacent normal skin from the same tissue section and with normal skin from separate biopsies. Statistical Analysis—Values are expressed as the average ± S.D. of the indicated number of samples for each experiment. Student's t test was used to compare data between two groups. p < 0.05 was considered statistically significant. EGFR Ligands and SFKs Promote Tyrosine Phosphorylation of Srcasm in Primary Human Keratinocytes—Because EGF stimulation of cells can activate Src kinases, the effect of EGF on the tyrosine phosphorylation of endogenous Srcasm was evaluated in primary human keratinocytes (Fig. 1) (23Osherov N. Levitzki A. Eur. J. Biochem. 1994; 225: 1047-1053Crossref PubMed Scopus (262) Google Scholar, 24Stover D.R. Becker M. Liebetanz J. Lydon N.B. J. Biol. Chem. 1995; 270: 15591-15597Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). Immunoprecipitation with α-Srcasm followed by Western blotting with α-phosphotyrosine demonstrated increased levels of Srcasm tyrosine phosphorylation in EGF-treated keratinocytes (Fig. 1A). Similarly, immunoprecipitation with α-phosphotyrosine from EGF-treated cells followed by Western blotting using α-Srcasm demonstrated more phospho-Srcasm present in EGF-treated cells (Fig. 1B). Parallel experiments with TGF-α and KGF were performed; TGF-α treatment of keratinocytes increased tyrosine phosphorylation of Srcasm, whereas KGF treatment did not (Fig. 1B). These results show that EGF and TGF-α, both EGFR ligands, induce tyrosine phosphorylation of endogenous Srcasm. The behavior of adenovirally transduced HA-Srcasm was examined in PHKs. PHKs transduced with HA-Srcasm adenovirus and cultured in complete media exhibited readily detectable levels of tyrosine-phosphorylated Srcasm (Fig. 1C), whereas PHKs deprived of growth factors (EGF, insulin, and bovine pituitary extract) for 24 h contained very low levels of tyrosine-phosphorylated Srcasm. PHKs stimulated with EGF demonstrated a rapid and significant increase in tyrosine-phosphorylated Srcasm (Fig. 1C); these results for transduced Srcasm parallel those for endogenous Srcasm. Upstream signaling events necessary for tyrosine phosphorylation of Srcasm were explored using pharmacological inhibitors of EGFR and SFKs. Treatment of keratinocytes with inhibitors of SFKs (PP2) and EGFR (AG112) ablated the EGF-induced tyrosine phosphorylation of Srcasm (Fig. 1D), demonstrating that EGF-induced tyrosine phosphorylation of Srcasm requires the activity of both EGFR and SFKs. In addition, these data show that adenoviral HA-Srcasm appears to be a useful model for characterizing the effect of increased Srcasm levels on keratinocyte signaling. Srcasm Promotes Cellular Tyrosine Phosphorylation and Activates SFKs in Keratinocytes—EGF treatment and elevated Srcasm levels correlate with increased tyrosine phosphorylation of a number of cellular proteins (Fig. 2A). Notably, a doublet at ∼70 kDa show increased tyrosine phosphorylation with EGF treatment or increased Srcasm levels. Because Srcasm can activate SFKs in vitro, the effect of Srcasm on endogenous keratinocyte SFK activation was evaluated. In non-infected PHKs, the levels of activated SFKs were low (Fig. 2B); similar results were seen with control adenovirus. In contrast, infection of the keratinocytes with Ad-Srcasm led to significantly higher levels of activated SFKs (Fig. 2B). Increased levels of Srcasm also potentiated the ability of EGF to activate Src-kinases (Fig. 2B). These results show that increased levels of Srcasm are associated with activation of endogenous SFKs in PHKs and that increased Srcasm level can lead to higher levels of activated SFKs secondary to EGF treatment. The ability of Srcasm to activate SFKs is dose-dependent; the level of activated SFKs is proportional to the level of cellular Srcasm (Fig. 2C). Association of increased Srcasm levels with SFK activation was evaluated at the cellular level via immunofluorescence. Cells containing higher amounts of Srcasm have higher levels of activated SFKs (Fig. 2D). Srcasm Differentially Activates SFKs in Primary Keratinocytes—Because keratinocytes express Fyn, Src, and Yes, the effect of increased Srcasm levels on each SFK family member was evaluated. Immunoprecipitation of Fyn from keratinocytes containing higher levels of Srcasm followed by Western blotting to assess levels of activated kinase demonstrated increased Fyn activation when compared with control cells (Fig. 3A). Activation of Src was also seen in similar experiments. Interestingly, Yes showed decreased activation in Ad-Srcasm-infected cells relative to controls (Fig. 3A). The effect of increased Srcasm levels on SFK activity was also evaluated using in vitro kinase assays. Both Fyn and Src showed increased kinase activity in PHKs infected with Ad-Srcasm compared with control cells (Fig. 3B). The kinase activity of Yes was mildly decreased in Ad-Srcasm-infected PHKs compared with control cells (Fig. 3B). Using two different experimental methods, increased Srcasm levels are associated with increased Fyn and Src activity but with decreased Yes activity. Immunofluorescence studies demonstrated more prevalent co-localization of Fyn and Src with Srcasm, as indicated by increased yellow color in the merged confocal images (Fig. 3C). In contrast, relatively little co-localization of Yes and Srcasm was seen in similar experiments (Fig. 3C). Therefore, the degree of cellular co-localization between Srcasm and Fyn, Src, or Yes" @default.
• W2053467415 created "2016-06-24" @default.
• W2053467415 creator A5002421935 @default.
• W2053467415 creator A5003193460 @default.
• W2053467415 creator A5014576610 @default.
• W2053467415 creator A5041187400 @default.
• W2053467415 creator A5044318950 @default.
• W2053467415 creator A5064309595 @default.
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• W2053467415 date "2005-02-01" @default.
• W2053467415 modified "2023-09-27" @default.
• W2053467415 title "Srcasm Modulates EGF and Src-kinase Signaling in Keratinocytes" @default.
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