P130Cas-associated protein (p140Cap) as a new tyrosine-phosphorylated protein involved in cell spreading

Abstract

Integrin-mediated cell adhesion stimulates a cascade of signaling pathways that control cell proliferation, migration, and survival, mostly through tyrosine phosphorylation of signaling molecules. p130Cas, originally identified as a major substrate of v-Src, is a scaffold molecule that interacts with several proteins and mediates multiple cellular events after cell adhesion and mitogen treatment. Here, we describe a novel p130Cas-associated protein named p140Cap (Cas-associated protein) as a new tyrosine phosphorylated molecule involved in integrin- and epidermal growth factor (EGF)-dependent signaling. By affinity chromatography of human ECV304 cell extracts on a MBP-p130Cas column followed by mass spectrometry matrix-assisted laser desorption ionization/time of flight analysis, we identified p140Cap as a protein migrating at 140 kDa. We detected its expression in human, mouse, and rat cells and in different mouse tissues. Endogenous and transfected p140Cap proteins coimmunoprecipitate with p130Cas in ECV304 and in human embryonic kidney 293 cells and associate with p130Cas through their carboxy-terminal region. By immunofluorescence analysis, we demonstrated that in ECV304 cells plated on fibronectin, the endogenous p140Cap colocalizes with p130Cas in the perinuclear region as well as in lamellipodia. In addition p140Cap codistributes with cortical actin and actin stress fibers but not with focal adhesions. We also show that p140Cap is tyrosine phosphorylated within 15 min of cell adhesion to integrin ligands. p140Cap tyrosine phosphorylation is also induced in response to EGF through an EGF receptor dependent-mechanism. Interestingly expression of p140Cap in NIH3T3 and in ECV304 cells delays the onset of cell spreading in the early phases of cell adhesion to fibronectin. Therefore, p140Cap is a novel protein associated with p130Cas and actin cytoskeletal structures. Its tyrosine phosphorylation by integrin-mediated adhesion and EGF stimulation and its involvement in cell spreading on matrix proteins suggest that p140Cap plays a role in controlling actin cytoskeleton organization in response to adhesive and growth factor signaling.

Figure 1.

Identification of KIAA1684 protein as a p130Cas-associated protein by affinity chromatography and mass spectrometry MALDITOF analysis. (A) Schematic diagram of the structural domains of full-length (FL) or truncated (ΔN) p130Cas molecules. (B) ECV304 cell lysates were loaded on MBP or MBP-p130CasΔN-Sepharose columns, and eluted fractions 4-6 were collected and run on a 5-10% SDS-PAGE gradient. Proteins migrating with molecular masses around 98-130 kDa were specifically detected by Coomassie Blue staining in the MBP-p130CasΔN eluted fractions (right line) but not in the MBP-eluted ones (left line). Bands 1-4 were excised and analyzed by mass spectrometry MALDI-TOF. Molecular mass markers are shown on the left. (C) List of peptide sequences derived from MALDI-TOF spectra of band 1 that led to identification of the KIAA1684 protein. The results are representative of three independent experiments.

Figure 2.

Expression of p140Cap protein in cells and tissues. (A) ECV304, T47D, FRT, HeLa epithelial cells, and N1E115 neuroblastoma cells were detergent extracted and analyzed by immunoblotting with anti-p140Cap-purified polyclonal antibodies. The blot was reprobed with p130Cas mAb (bottom). (B) Equal amounts of homogenates (100 μg of protein for lane) from the indicated mouse tissues were analyzed by immunoblotting by using the anti-p140Cap antibodies (top). Proteins migrating as a doublet at 140 and 116 kDa, indicated by the arrows, were specifically detected in brain, lung, kidney, mammary gland, and testis. The blot was reprobed with p130Cas mAb (bottom). p140Cap migration as a single band or a doublet was shown to be dependent on the length of the gels. The results are representative of five independent experiments.

Figure 4.

In vivo interaction between p130Cas and p140Cap. (A) ECV304 cell lysates were immunoprecipitated both with preimmune serum (PI), p130Cas mAb, and p140Cap polyclonal antibodies. Western blotting analysis was performed using anti-p140Cap (top) or p130Cas antibodies (bottom). (B) HEK293 cells either untransfected (-) or transfected with p140Cap cDNA (+) were immunoprecipitated with PI, p130Cas, or p140Cap antibodies. Immunoprecipitates were run on a 6% SDS-PAGE and Western blotted with anti-p140Cap (top) and anti-p130Cas antibodies (middle). The blots were reprobed with anti-Crk antibody (bottom). The results are representative of seven independent experiments.

Figure 3.

Genomic organization of p140Cap mouse gene. (A) Amino acid sequence of p140Cap. Indicated are the predicted glycine-rich region (underlined in dark gray), the proline-rich domains (boxed), the putative SH3 binding regions (dashed black underlined), the coiled-coil domains (underlined in white), and the charged sequence (underlined in light gray). (B) p140Cap gene localized on chromosome 11 consists of 24 exons (top). The first two exons, 1a and 1b, are subjected to alternative splicing on exon 2 resulting in mRNA isoforms coding for two proteins that differ in their N-terminal portion (underlined). The sequence of exon 2 is only partially reproduced. (C) RT-PCR products of the two isoforms of the p140Cap gene was performed on total RNA extracted from mouse brain. cDNA was subjected to PCR by using specific oligonucleotides on 1a, 1b, and 2 exon sequences. The molecular weights of 1a + 2 (348-base pair) and 1b + 2 (350-base pair) amplicons are shown. Note the absence of an amplicon of 651 base pairs that would have been represented 1a + 1b + 2. DNA molecular weight ladder is shown on the left. The results are representative of two independent experiments.

Figure 5.

p140Cap binds to p130Cas through the carboxy-terminal domain. A, left, a schematic representation of full-length p140Cap and of recombinant GST-p140Cap fragments. Right, pull down of p130Cas from HEK293 cell lysates on immobilized GST, GST-CrkSH2 (+), and GST-P140Cap fusion proteins. Bound proteins were eluted and analyzed by 8% SDS-PAGE and immunoblotted by using p130Cas mAb. The results are representative of two independent experiments. (B) Multiple alignment of human (Hu), mouse (Mo), and fugu (Fu) amino acids of the Cap5 region. ^*, single, fully conserved residues,:, conservation of strong groups;., conservation of weak groups. Box: highly conserved proline-rich region. (C) HEK293 cells were transfected either with full-length p140Cap or with p140-ΔCT mutant deleted of the region associating with p130Cas. Immunoprecipitation was performed using anti-p130Cas antibodies and immunoprecipitates were run on a 6% SDS-PAGE and blotted with anti-myc antibodies (D) Left, schematic diagram of different domains of full-length p130Cas and of GST-p130CasΔ, GST-YXXP, GST-Ab1-Ab2, and GST-CT (from top to bottom) used for pull-down assays. Left, pull down of p140Cap from p140Cap-transfected HEK293 cell lysates bound to GST, GST-YXXP, GST-Ab1-Ab2 and GST-CT fusion proteins immobilized on glutathione-Sepharose beads. Bound proteins were eluted and analyzed by 8% SDS-PAGE and immunoblotted using p140Cap polyclonal antibodies.

Figure 5.

p140Cap binds to p130Cas through the carboxy-terminal domain. A, left, a schematic representation of full-length p140Cap and of recombinant GST-p140Cap fragments. Right, pull down of p130Cas from HEK293 cell lysates on immobilized GST, GST-CrkSH2 (+), and GST-P140Cap fusion proteins. Bound proteins were eluted and analyzed by 8% SDS-PAGE and immunoblotted by using p130Cas mAb. The results are representative of two independent experiments. (B) Multiple alignment of human (Hu), mouse (Mo), and fugu (Fu) amino acids of the Cap5 region. ^*, single, fully conserved residues,:, conservation of strong groups;., conservation of weak groups. Box: highly conserved proline-rich region. (C) HEK293 cells were transfected either with full-length p140Cap or with p140-ΔCT mutant deleted of the region associating with p130Cas. Immunoprecipitation was performed using anti-p130Cas antibodies and immunoprecipitates were run on a 6% SDS-PAGE and blotted with anti-myc antibodies (D) Left, schematic diagram of different domains of full-length p130Cas and of GST-p130CasΔ, GST-YXXP, GST-Ab1-Ab2, and GST-CT (from top to bottom) used for pull-down assays. Left, pull down of p140Cap from p140Cap-transfected HEK293 cell lysates bound to GST, GST-YXXP, GST-Ab1-Ab2 and GST-CT fusion proteins immobilized on glutathione-Sepharose beads. Bound proteins were eluted and analyzed by 8% SDS-PAGE and immunoblotted using p140Cap polyclonal antibodies.

Figure 6.

Subcellular localization of p140Cap and colocalization with p130Cas. (A) ECV304 cells were detached from culture dishes and plated for 12 h on fibronectin-coated coverslips in presence of serum. Some coverslips were treated with 100 nM PMA for 1 h (g-i). Cells were then fixed, permeabilized, and double labeled using anti-p140Cap purified polyclonal antibody (b, e, and h), phalloidin (Phd) (a), or anti-p130Cas mAb (d and g). p140Cap colocalization with stress fibers is indicated in the inset of the overlay (c), with a higher magnification. d, e, g, and h show confocal analysis of cells double stained with p130Cas and p140Cap in growing conditions (d and e) or in presence of PMA (g and h). The overlay staining is shown in c, f, and i. In the overlay (i), the inset represents an enlargement of the area indicated by the arrow. The figures are representative of four independent experiments.

Figure 7.

p140Cap is tyrosine phosphorylated in response to adhesion, serum, or EGF treatment. (A) p140Cap-transfected HEK293 cells, serum-deprived for 24 h, were detached and plated for different times on dishes coated with mAb L230 against the αv integrin subunit or kept in suspension for 30 min. Cells were detergent extracted at the indicated times, and extracts were immunoprecipitated using antibodies to p140Cap. The immunoprecipitates were blotted with anti-phosphotyrosine antibody (top) and reblotted with antibodies to p140Cap (bottom). (B) Cells treated as described in A, kept in suspension or plated on FN for the indicated times, were extracted and immunoprecipitated using antibodies to phosphotyrosine (p-Tyr), and the immunoprecipitates were blotted with anti-p140Cap antibodies (top) and reblotted with antibodies to p130Cas (bottom). (C) Cells were treated with 20% FCS or with 50 ng/ml human recombinant EGF for 30 min. (D) Cells were treated with 50 ng/ml human recombinant EGF for 30 min in the presence of 250 nM AG1478. The p140Cap immunoprecipitates were blotted with anti-phosphotyrosine antibody (top) and reblotted with antibodies to p140Cap (bottom). The results are representative of three independent experiments.

Figure 8.

Overexpression of p140Cap affects cell spreading in NIH3T3 cells. (A) NIH3T3 cells were transfected with pEGFP-N2 (a-c), pEGFP-p140Cap (d-f), or pEGFP-p140-ΔCT cDNAs (g-i). Forty-eight hours after transfection, cells were detached from culture dishes and plated for 1.5 h on fibronectin-coated glass coverslips. Cells were than fixed, permeabilized, and stained using rhodamine-labeled phalloidin (Phd). The overlay staining is shown in c, f, and i. The images are representative of three independent experiments. (B) NIH3T3 cells were transfected with pCDNA3.1/Myc-Hys-p140Cap (l-n) or with mutant pCDNA3.1/Myc-Hys-p140-ΔCT expression vectors (o-q). Forty-eight hours after transfection, cells were detached from culture dishes and plated for 1.5 h on fibronectin-coated glass coverslips and then double stained using fluorescein-labeled phalloidin (l and o) or and rhodamine-labeled Myc antibodies (m and p). The overlay staining is shown in n and q.

Figure 9.

Overexpression of p140Cap affects cell spreading in ECV304 cells. (A and B) ECV 304 cells were treated, transfected, and stained as described in Figure 8, A and B.