p140Cap tyrosine phosphorylation depends mainly on two tyrosine residues. A.

<p>Schematic representation of p140Cap structure and localization of FYELE, EPLYA, EGLYA, and ADPYG sequences into the tyrosine rich region. These four tyrosine residues have been mutated to phenylalanine. <b>B.</b> cDNAs encoding GFP, GFP-p140Cap full length (p140 WT) and its single (p140 EPL<b>Y/F</b>A, p140 EGL<b>Y/F</b>A), double (p140 EPL<b>Y/F</b>A, EGL<b>Y/F</b>A; p140 F<b>Y/F</b>ELE, ADP<b>Y</b>/<b>F</b>G) and triple (p140 EPL<b>Y/F</b>A, EGL<b>Y/F</b>A, ADP<b>Y</b>/<b>F</b>G) mutants were used to transfect HEK-293 cells. 48 hours after transfection cells were treated for 5 minutes with 100 micromolar pervanadate solution. Cell extracts were immunoprecipitated with a specific antibody to p140Cap and immunocomplexes were analysed by western blotting using monoclonal antibodies to phosphotyrosine (PY99), p140Cap and Tubulin respectively. The results are representative of six independent experiments.</p

Effects of E2 stimulation on EGF− and FGF-dependent branching morphogenesis.

<p>(A) Brightfield images of representative wt and p130Tg organoids, cultured for 5 days in the absence or presence of E2, EGF, or the combination of both. (B) Quantification of primary and secondary branching of wt vs. p130Tg organoids upon treatment with either E2, EGF or EGF+E2 for 5 days. Experiments were repeated at least five times. Two-way ANOVA analysis show a significant effect of both treatment (EGF vs. EGF+E) and genotype (wild type vs. transgenic) for Acinar Structures, secondary branching and enlarged spheroids (P<0.0001 for both treatment and genotype). For primary branching only treatment has a significant effect (P<0.0001). Interaction between treatment and genotype is significant for all variables (Acinar: P<0.0065; primary branching: P<0.0001; secondary branching: P<0.0001; enlarged spheroids: P<0.0001). (C) Quantification of spherical organoids size in untreated and EGF+E2 treated wt and p130Tg organoids was performed by using ImageJ software. Statistical significance is indicated (** p<0.001).</p

Identification of Abl as the tyrosine kinase responsible of p140Cap tyrosine phosphorylation on EPLYA and EGLYA sequences. A–B.

<p>For each recombinant kinase, bar charts of the mean values of the triplicate activity raw counts of kinase activity and the means of the corresponding background values of the synthetic peptides with (black bars) or without (white bars) enzyme are indicated. The synthetic peptides contain respectively EPLYA (A) and EGLYA (B) sequences. A. cDNAs encoding GFP and GFP-p140Cap full length (p140 WT) were used to transfect HEK-293 cells. After 24 hours, cells were starved and treated with 10 micromolar Src inhibitor SU6656 or Abl inhibitor Imatinib for 16 hours. Cell extracts were immunoprecipitated with a specific antibody to p140Cap and analysed by western blotting using monoclonal antibodies for phosphotyrosine and p140Cap. <b>B.</b> Left panel. cDNAs encoding GFP, GFP-p140Cap full length (p140 WT) and its double mutant (p140 EPLY/FA, EGLY/FA) were used to transfect HEK-293 cells together with cDNA encoding for active BCR-Abl. Extracts were immunoprecipitated with a specific antibody to p140Cap and analysed by western blotting using monoclonal antibodies to phosphotyrosines (PY99) and p140Cap. Right panel. cDNAs encoding GFP, GFP-p140Cap full length (p140 WT) and its double mutant (p140 EPLY/FA, EGLY/FA) were used to transfect HEK-293 cells. Cells were treated with 100 micromolar pervanadate solution for five minutes and extracts were processed as in the left panel.</p

Effects of p130Cas expression on branching morphogenesis in primary mouse mammary gland organoids grown in 3D Matrigel cultures.

<p>(A) Let panel: qRT-PCR expression analysis of p130Cas using total RNA isolated from the 4th inguinal mammary gland of at least three FVB wt mice during different stages of puberty. The amount of p130Cas mRNA was normalized to 18S rRNA. Right panel: Analysis of p130Cas protein expression from wt mice (three mice/stage) at the same stages of puberty as in the left panel. (B) Analysis of p130Cas protein expression from 5 day untreated wt and p130Tg organoids (upper panel) and quantification analysis expressed as the mean of p130Cas/tubulin ± standard deviation (SD) (*p<0.05) (lower panel). (C) Brightfield images of representative wt and p130Tg organoids, after 1, 4 and 5 days of culture in presence of EGF (35 ng/ml) or FGF-2 (35 ng/ml) in the culture medium. Images were captured by using the Axio Observer Z1 microscope. (D) Quantitative analyses of branching morphogenesis after treatment of wt and p130Tg organoids with EGF and FGF-2 at 5 days. The plot represents the mean number of overall branching response in at least three wells from 4 independent experiments. Similar results were obtained with a second p130Cas transgenic line (data not shown). Quantification of the mean number of branched organoids buds was significantly higher in EGF and FGF-2 treated p130Tg organoids compared to wt organoids (*p<0.002).</p

Prediction of tyrosine phosphorylation and relative score.

<p>Prediction of tyrosine phosphorylation and relative score.</p

Amino acid sequence of p140Cap and spectra of <i>in vivo</i> phosphorylated peptides. A.

<p>Amino acid sequence of p140Cap (NCBI Reference Sequence: NP_079524.2; REFSEQ: accession NM_025248.2) showing, the peptides found to be phosphorylated by MS analysis (phosphoserine: blank open box; phosphotyrosine: grey box) and the EPIYA-like motifs (underlined). All the tyrosine residues present in p140Cap are indicated in Bold. Note that the phosphoserines in position 45, 987 and 1003 are conserved among human and murine sequences. <b>B.</b> The phosphopeptide 392-GEGLpYADPYGLLHEGR-407 with m/z 913.9091 (z: +2) was sequenced by LC-MS². The signals of ions within 350–1030 and 1100–1685 m/z were enhanced of 2 and 5 times, respectively. The signal enhancement facilitated the labeling of the ions. Only the most relevant fragment ion signals are labeled in the MS² spectrum. *, Ions containing phosphorylated tyrosine-396 (pY396). <b>C.</b> MS³ spectrum of the phosphopeptide 985-RGpSDELTVPR-994. In MS² mode the parent ion at m/z 605.2843 Th (z: 2+) lost phosphoric acid generating an ion at m/z 556.2868 Th, which was subjected to MS³ fragmentation. Delta indicates the loss of phosphoric acid. <b>D.</b> MS³ spectrum of the peptide 43-RFpSNVGLVHTSER-55. <b>E.</b> MS² spectrum of 997-TEKPSKpSPPPPPPR-1010. *, Ions containing phosphorylated serine-1003 (pS1003). PPP-CO indicates an internal fragment ion containing three proline residues which has lost carbon monoxide (28 Da).</p

p140Cap tyrosine phosphorylation on EPLYA and EGLYA sequences regulate Csk binding.

<p><b>A.</b> cDNAs encoding GFP, GFP-p140Cap full length (p140 WT) and its double mutant (p140 EPL<b>Y/F</b>A, EGLY<b>/F</b>A) were used to transfect HEK-293 cells. Cells were treated with 100 micromolar pervanadate solution as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054931#pone-0054931-g002" target="_blank">Figure 2B</a> and extracts were immunoprecipitated with a specific antibody to p140Cap and analysed by western blotting using monoclonal antibodies PY99, p140Cap, and Csk. The results are representative of six independent experiments. <b>B.</b> HEK-293 cells transfected as in A for 48 hours, were starved overnight and treated for 0, 5, 15 minutes with 20%FBS. Cell extracts were immunoprecipitated with a specific antibody to p140Cap. Immunocomplexes were analysed by western blotting using monoclonal antibodies specific for phosphotyrosines, p140Cap, Csk and Vinculin.</p

E2 treatment of p130Tg organoids alters myoepithelial-luminal architecture and lumen clearance.

<p>(A) Confocal images representing immunofluorescence staining for K14 (red; myoepithelium) and K18 (green; luminal epithelium) of wt and p130Tg organoids stimulated with EGF and E2 at day 5 of culture. Scale bar = 0,50 micron. (B) Confocal z-stacks of DAPI staining of wt and p130Tg organoids at day 5 of culture to visualize lumen clearance. Images in A and B were acquired using a Leica TCS-SP5 II confocal microscope. Images in A and B are representative of four independent experiments.</p

p130Cas Over-Expression Impairs Mammary Branching Morphogenesis in Response to Estrogen and EGF

<div><p>p130Cas adaptor protein regulates basic processes such as cell cycle control, survival and migration. p130Cas over-expression has been related to mammary gland transformation, however the <em>in vivo</em> consequences of p130Cas over-expression during mammary gland morphogenesis are not known. In ex vivo mammary explants from MMTV-p130Cas transgenic mice, we show that p130Cas impairs the functional interplay between Epidermal Growth Factor Receptor (EGFR) and Estrogen Receptor (ER) during mammary gland development. Indeed, we demonstrate that p130Cas over-expression upon the concomitant stimulation with EGF and estrogen (E2) severely impairs mammary morphogenesis giving rise to enlarged multicellular spherical structures with altered architecture and absence of the central lumen. These filled acinar structures are characterized by increased cell survival and proliferation and by a strong activation of Erk1/2 MAPKs and Akt. Interestingly, antagonizing the ER activity is sufficient to re-establish branching morphogenesis and normal Erk1/2 MAPK activity. Overall, these results indicate that high levels of p130Cas expression profoundly affect mammary morphogenesis by altering epithelial architecture, survival and unbalancing Erk1/2 MAPKs activation in response to growth factors and hormones. These results suggest that alteration of morphogenetic pathways due to p130Cas over-expression might prime mammary epithelium to tumorigenesis.</p> </div

p130Cas over-expression enhances cell proliferation and survival in EGF and EGF+E2-treated primary organoids.

<p>(A) Quantification of nuclear Ki67 staining from total nuclei count of each organoid at day 2. Quantification analysis of Ki67 staining as mean of Ki67 positive cells/total number of cells ±SD and significance are reported (*p<0.05, **p<0.001). More than 20 organoids from each condition were counted in three independent experiments. (B) Western blot analysis of cleaved caspase-3 from 2 and 5 day of EGF, E2 and EGF+E2-treated wt and p130Tg cultured organoids. Quantification analysis at day 2 and day 5 of cleaved-caspase 3 are reported on the right (*p<0.05). (C) TUNEL analysis from 2 day treated wt and p130Cas cultured organoids. TUNEL positive cells in the organoids are stained in red. The plot represents the quantification of the results of three independent experiments in which apoptotic cells are counted and normalized to the total number of nuclei/organoid for at least 10 organoids from each condition ± standard deviation (*p<0.05).</p