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First published online October 11, 2005
doi: 10.1242/10.1242/jcs.02615

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Regulation of paxillin family members during epithelial-mesenchymal transformation: a putative role for paxillin

Department of Cell and Developmental Biology, State University of New York Upstate Medical University, 750 East Adams Street, Syracuse, NY 13210, USA

View larger version (30K): [in a new window]   Fig. 1. Paxillin Kozak sequence comparison and paxillin family domain structure. (A) Alignment of the paxillin and Kozak nucleotide sequence (Kozak, 1987) from various species. Nucleotides are labeled as follows: G, guanine; C, cytosine; A; adenine; U, uracil; R, purine; aug#, methionine start codon with amino acid position. Shaded residues are most highly conserved and required for ribosome recognition. (B) Avian paxillin , paxillin and Hic-5 domain structure. Paxillin is 559 amino acids and paxillin is 427 amino acids in length (amino acid numbers in parentheses represent position within paxillin ). Hic-5 Tyr60 residue is a potential Csk binding site (Thomas et al., 1999).

View larger version (58K): [in a new window]   Fig. 2. Verification of paxillin as an internal translation product and its localization to focal adhesions. (A) CHO.K1 cells were transfected with either pcDNA3.1 vector only, wild-type avian paxillin (wt pax), full-length avian paxillin with internal Kozak mutation (FL pax KM) or avian paxillin (pax ) (amino acid 133 to end). Immunoprecipitation with avian-specific paxillin polyclonal antiserum, Pax1, was performed on detergent-soluble lysates (DSL). Lysates and immunoprecipitates were resolved by 7.5% SDS-PAGE and transferred to nitrocellulose for western blot analysis. Paxillin monoclonal antibody PXC10 was utilized for western blotting. (B) Wild-type paxillin (a,d), full-length paxillin KM (b,e) and paxillin (c,f) were transiently transfected into CHO.K1 cells followed by respreading of the cells on fibronectin-coated coverslips. Cells were processed for indirect immunofluorescence microscopy utilizing the avian-specific Pax1 antibody (a,b,c) and double-labeled with an antibody to vinculin (d,e,f). Bar, 5 µm.

View larger version (52K): [in a new window]   Fig. 3. Reciprocal expression of paxillin and Hic-5 in cell lines of epithelial and fibroblast origin. Western blots were performed on total cell lysates and probed with paxillin-specific PXC10 and Hic-5 antibodies. -actinin expression was used as a measure of equivalent protein loading. Paxillin expression was restricted to cells exhibiting an epithelial phenotype (lanes 5-7) whereas Hic-5 expression was restricted to cells exhibiting a mesenchymal/fibroblastic phenotype (lanes 1-4). Paxillin was expressed in all cell types examined.

View larger version (65K): [in a new window]   Fig. 4. TGF-ß1-induced EMT in the NMuMG cell line regulates localization and expression of paxillin and Hic-5. (A) Hoffman modulation contrast and indirect immunofluorescence microscopy of NMuMG cell line either unstimulated (a,c,e) or stimulated with 2 ng/ml TGF-ß1 for 48 hours (b,d,e). Hoffman images (a,b), paxillin (clone 165 paxillin-specific monoclonal antibody) (c,d) and the actin cytoskeleton visualized using Rhodamine-conjugated phalloidin (e,f). Induction of an EMT leads to the relocalization of paxillin from a mainly cytoplasmic distribution to a predominant focal adhesion localization at the ends of actin stress fibers. (B) Induction of Hic-5 expression and focal adhesion localization following an EMT. Indirect immunofluorescence microscopy of NMuMG cells either unstimulated (a,c) or stimulated with 2 ng/ml TGF-ß1 for 48 hours (b,d). Hic-5 (a,b), actin cytoskeleton (c,d). Scale bar, 10 µm.

View larger version (45K): [in a new window]   Fig. 5. TGF-ß1-induced EMT in NMuMG cell line reciprocally modulates paxillin and Hic-5 protein expression. A, Detergent soluble lysates from cells either unstimulated (–) or stimulated (+) with 2 ng/ml TGF-ß1 for 48 hours were processed for western blot analysis. Paxillin monoclonal antibody PXC10 was used to recognize both and forms of paxillin. Paxillin expression is suppressed while Hic-5 expression increases following a TGF-ß1-induced EMT in the NMuMG cell line. E-cadherin and N-cadherin expression were monitored to verify the induction of an EMT. (B) Western blot analysis of detergent-soluble lysates obtained from NMuMG cells either unstimulated (–) or stimulated (+) with 2 ng/ml TGF-ß1 for 48 hours. Phospho-specific antibodies to paxillin Y31 and Y118 residues as well as to FAK Tyr397 were utilized. Paxillin Y31 and Y118 phosphorylation occurs concurrent with the suppression of paxillin following an EMT.

View larger version (94K): [in a new window]   Fig. 6. Paxillin expression is maintained following differentiation of NMuMG cells in a collagen I gel. NMuMG cells were cultured under normal 2D conditions or cultured in a 3D collagen I gel for a period of 7 days. (A) Hoffman modulation contrast images (20x) were taken of NMuMG cells cultured under 2D conditions (a) and under 3D collagen gel conditions (b). (B) Detergent-soluble lysates (DSL) were prepared from 2D and 3D gel-cultured NMuMG cells and were processed for western blot analysis. Appropriate antibodies were utilized to detect the indicated proteins and their phosphorylated isoforms. (C) Paxillin was immunoprecipitated from 2D and 3D gel lysates and immunoblotted for paxillin and phospho-Y118 paxillin. Although paxillin expression levels remain unchanged, basal paxillin phospho-Y118 was absent from cells cultured under 3D collagen gel conditions. Bar, 10 µm.

View larger version (26K): [in a new window]   Fig. 7. Paxillin overexpression attenuates paxillin phosphorylation at Y118 during respreading on fibronectin in the CHO.K1 cell line. (A) CHO.K1 cells were transiently transfected with either full-length paxillin KM (FL pax KM; lane 1) or paxillin (pax ; lane 2). Total cell lysates were probed with paxillin 165 monoclonal antibody to paxillin to demonstrate equal expression levels. (B) CHO.K1 cells transfected with full-length paxillin KM (a,b) or paxillin (c,d) were respread on fibronectin-coated coverslips (10 µg/ml) for 120 minutes and processed for immunofluorescence microscopy. Phosphorylation of paxillin Y118 was visualized with a phospho-specific antibody (b,d). Transfected cells were visualized by cotransfection with pEGFPc1 vector (a,c). Overexpression of paxillin attenuates paxillin Y118 phosphorylation in focal adhesions during cell spreading on fibronectin. (C) Quantification of the suppression of paxillin phosphorylation at Y118. Results are expressed as the percentage of transfected cells showing a reduction in paxillin pY118 focal adhesion staining visualized by a phospho-specific antibody after respreading on fibronectin for 60, 120 and 240 minutes. Results are from three independent experiments with error bars representing the calculated s.d. Bar, 5 µm.

View larger version (72K): [in a new window]   Fig. 8. Paxillin overexpression inhibits NMuMG cell motility. NMuMG cells were retrovirally transduced with GFP, GFP-FL pax and GFP pax . (A) Indirect immunofluorescence microscopy (a-f) of NMuMG cells expressing either GFP, GFP-FL pax or GFP-pax . Arrows (b,d,f) indicate phospho-paxillin Y118 staining in focal adhesions along the periphery of epithelial islands is suppressed in GFP-pax expressing cells. (B) Detergent-soluble lysates of retrovirally transduced NMuMG cells untreated (–) or treated with TGF-ß1 (+) for 48 hours expressing either GFP, GFP-FL pax or GFP-pax were prepared and run on standard SDS-PAGE. Western blot analysis was performed utilizing antibodies to the specified proteins. (C) NMuMG cells retrovirally transduced and treated with TGF-ß1 for 48 hours followed by GFP immunoprecipitation. Immunoblot analysis was performed using antibodies to GFP, phosphotyrosine (clone 4G10) or phospho-paxillin Y118. Arrowhead indicates GFP-FL pax and arrow indicates GFP-pax . (D) Western blot and modified Boyden chamber migration assays were performed with retrovirally transduced NMuMG cells expressing GFP, GFP-FL pax, GFP-pax or GFP-Hic-5. Immunoblots were performed with antibodies to GFP and FAK. Cells were allowed to migrate to 10 µg/ml fibronectin for 16 hours followed by fixation, staining and quantification of cells on the underside of the filter. Experiments were performed in triplicate. Migration is represented as a percentage of the GFP control with significance levels *P<0.05 and **P<0.001. Bar, 5 µm.

View larger version (42K): [in a new window]   Fig. 9. In vivo association of paxillin and Crk in NMuMG cells. Lysates were prepared from either untreated (–) or TGF-ß1-treated (+) NMuMG cells for coimmunoprecipitation experiments. (A) Detergent soluble lysates (DSL) were run alongside control (IgG) or Crk immunoprecipitates on standard SDS-PAGE. Immunoblots were performed using specific antibodies to the indicated proteins and as indicated to their phosphorylated isoforms. (B) DSL and p120RasGAP immunoprecipitates were loaded and run on standard SDS-PAGE. Immunoblots were performed using specific antibodies to the indicated proteins. (C) Detergent-soluble lysates (DSL) were run alongside control (IgG) and paxillin (clone 165 antibody) immunoprecipitates, prepared from NMuMG cells treated with TGF-ß1 for 48 hours, on standard SDS-PAGE. Immunoblots were performed using specific antibodies to the indicated proteins.

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