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2, 5, 431–437 (2001)
doi:10.1093/embo-reports/kve086
Figures and Tables
Growth defects induced by perturbation of  1-integrin function in the mammary gland epithelium result from a lack of MAPK activation via the Shc and Akt pathways
Marisa M. Faraldo, Marie-Ange Deugnier, Jean Paul Thiery & Marina A. Glukhova
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Figure 1
Growth defects and low levels of MAPK phosphorylation in mammary gland epithelium from 2-day-lactating MMTV-1-cyto transgenic mice. (A) BrdU incorporation and TUNEL assay. The microphotographs show representative fields from sections of 2-day-lactating glands from transgenic and wild-type mice subjected to BrdU incorporation analysis to detect proliferating cells and TUNEL assay to detect apoptotic cells. BrdU-positive nuclei are dark grey to black, the tissue was slightly counterstained with haematoxylin; apoptotic nuclei are brown, the corresponding tissue sections were counterstained with methyl green. Bar, 40  m. (B) Comparison of MAPK phosphorylation levels in 2-day-lactating wild-type and MMTV-1-cyto glands. At first, phosphorylated ERK1 and ERK2 (upper panel) and phosphorylated JNKs, p46 and p54, (lower panel) were detected in the gland extracts by western blotting using corresponding antibodies. The blots were then stripped and reprobed for total ERK and total JNK. An additional non-specific band of 50–52 kDa was detected with the antibody against total JNK. Each lane corresponds to a mammary gland extract from an individual animal (three wild-type, followed by three transgenic). An aliquot of protein extract (100 g per lane) was loaded.
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Figure 2
Analysis of the phosphorylation status of the signalling molecules implicated in MAPK activation. Each lane corresponds to a mammary gland extract from an individual animal; representative data for two wild-type and two transgenic glands are shown in each case. (A) Tyrosine phosphorylation levels of FAK, paxillin and p130cas. Immunoprecipitation of FAK, paxillin and p130cas was performed by incubating 2 mg of mammary gland protein extract with the corresponding antibodies, and the immunoprecipitates were analysed by western blotting, first with an anti-P-Tyr-antibody and, after stripping, with anti-FAK, anti-paxillin and anti-p130cas antibodies. Bars in the diagrams represent P-Tyr levels relative to FAK, paxillin and p130cas, respectively. The phosphorylation level of FAK Tyr-925 was estimated by western blotting using 100 g of gland protein extract. In the diagram, levels relative to the total amount of FAK are presented. The bars show the mean values for three or four animals  SEM. (B) Analysis of Shc phosphorylation levels and Grb2 recruitment. Shc was immunoprecipitated from lactating mammary gland extracts (0.5 mg of protein) with anti-Shc mAb, and the amounts of P-Tyr-Shc, total Shc, and co-precipitating Grb2 in the immunoprecipitate were determined by western blotting. (C) Analysis of the phosphorylation status of PI3K signalling pathway components. First, phosphorylation levels of Akt Ser 473 and Thr 308, of FKHR Ser256, and of Bad Ser136 were analysed by western blotting using the corresponding specific antibodies; blots were then stripped and reprobed for total Akt, FKHR and Bad. A 100 g aliquot of 2-day-lactating mammary gland extract was loaded per lane. (D) Analysis of EGFR phosphorylation. The phosphorylation levels of EGFR were estimated by western blotting using 100 g of gland protein extract per lane. The blots were first probed with the antibody specific for phosphorylated EGFR, then stripped and probed for total EGFR. The bars indicate the values obtained for phosphorylated EGFR relative to the total amount of EGFR. Mean values for three or four animals SEM are shown.
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Figure 3
Comparison of the phosphorylation status of the signalling molecules in lactating and involuting wild-type glands. (A and B) ERK and JNK phosphorylation levels were determined by western blotting of 2-day-lactating and 4-day-involuting wild-type glands with the phospho-protein specific antibodies, as described in the legend to Figure 1. (C and D) Shc and FAK phospho-Tyr levels were determined in the immunoprecipates obtained with the corresponding antibodies. Protein extracts (0.5 mg) of 2-day-lactating and 2-day-involuting glands were used to immunoprecipitate Shc, whereas 0.5 mg of 2-day-lactating and 1 mg of 2-day-involuting gland extracts were used to immunoprecipitate FAK. (E) Phosphorylation of Akt at Ser473 was analysed by western blotting of 2-day-lactating and 4-day involuting gland extracts (100 g). Throughout the figure, each lane corresponds to a mammary gland extract from an individual animal; three or four animals were analysed in each case.
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Figure 4
Signalling pathways regulated by integrins in concert with Tyr-kinase growth factor receptors (from Giancotti and Ruoslahti, 1999, with modification). The molecules whose activation was affected by perturbation of 1-integrin-mediated adhesion in the lactating mammary gland epithelium are shown in red. PI3K and RAS are shown in italic as their activation was not directly estimated in this study, although the data presented herein suggest their impaired activity.
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