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Positive regulation of cell-cell and cell-substrate adhesion by protein kinase A

Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA

View larger version (72K): [in a new window]   Fig. 1. MAb 12G10-induced cell-cell adhesion is dependent on PKA. The effects of (A) 100 nM staurosporine, 2 µM calphostin C and 150 µM genistein, (B) H89 and (C and F) MPKI peptide on HT-1080 cell-cell adhesion in the presence of 10 µg/ml 12G10. (D) HT-1080 cell-cell adhesion with no treatment, (E) 10 µg/ml 12G10 and (F) cells treated with 20 µM MPKI peptide prior to the addition of 10 µg/ml 12G10. CCAI was scored and defined as described in Materials and Methods. In (A), the data are expressed relative to the level of HT-1080 cell-cell adhesion with mAb 12G10 (100%). The dotted lines (A), (B) and (C) represent the level of basal HT-1080 cell-cell adhesion±s.d. Values shown are mean±s.d. (n=8). Bar, D-F, 100 µm.

View larger version (18K): [in a new window]   Fig. 2. Inhibition of cell attachment to collagen type IV with inhibitors of PKA and PKC. HT-1080 fibrosarcoma cells were assayed for cell adhesion to 0.2 µg/ml collagen type IV in the presence of no inhibitor (white bars), 20 µM MPKI peptide (grey bars) or 2 µM calphostin C (black bars). Cells were incubated with 10 µg/ml anti-ß1 integrin antibodies and allowed to attach to substrate for 20 minutes. The amount of nonspecific attachment, determined using wells coated with BSA alone (<5%), was subtracted from each point. Values shown are mean±s.d. (n=4).

View larger version (14K): [in a new window]   Fig. 3. Activation of ß1-integrins with mAb 12G10 causes an increase in intracellular cAMP levels and PKA activity. (A) HT-1080 cells were incubated with 10 µg/ml anti-ß1 integrin antibodies or 5 µM forskolin and assayed for cAMP as described in Materials and Methods. Asterisk indicates P<0.01 vs. no treatment. Values shown are mean±s.d. (n=4). (B) HT-1080 cells were incubated with 10 µg/ml anti-ß1 integrin antibodies or 10 µM Sp-cAMPS and assayed for PKA activity using the biotinylated kemptide peptide substrate as described in Materials and Methods. Asterisk indicates P<0.05 vs. no treatment. Values shown are mean±s.d. (n=2).

View larger version (15K): [in a new window]   Fig. 4. MAb 12G10-induced cell-cell adhesion is dependent on cAMP. The effect of Rp-cAMPS on HT-1080 cell-cell adhesion stimulated with 10 µg/ml 12G10. The dotted lines represent the level of basal HT-1080 cell-cell adhesion±s.d. (n=8). CCAI was scored and defined as described in Materials and Methods.

View larger version (13K): [in a new window]   Fig. 5. Integrin clustering and PKA activation are both required to induce cell-cell adhesion. (A) The effect of 10 µM Sp-cAMPS on HT-1080 cell-cell adhesion in the presence of no antibody or 0.3 µg/ml 12G10. (B) The effect of integrin clustering and 10 µM Sp-cAMPS on HT-1080 cell-cell adhesion. CCAI was scored and defined as described in Materials and Methods. Asterisk indicates P<0.01 vs. no treatment. Values shown are mean±s.d. (n=8).

View larger version (78K): [in a new window]   Fig. 6. Integrin clustering and F-actin polymerization are both dependent on PKA. HT-1080 cells were treated with 20 µM MPKI peptide for 30 minutes and plated on poly-D-lysine coated cover slips for another 20 minutes in the presence of no antibody or 10 µg/ml 12G10 directly labeled with AlexaTM 568. F-actin was visualized with fluorescently labeled phalloidin (green) and ß1 integrins were visualized with AlexaTM 568-labeled 12G10 (red). Fluorescent images were combined to determine colocalization (yellow). Bar, 10 µm.

View larger version (65K): [in a new window]   Fig. 7. Colocalization of PKA RII subunits with integrins expressing the 12G10 epitope following integrin activation. HT-1080 cells were treated with 10 µM Sp-cAMPS for 20 minutes, and plated on poly-D-lysine coated cover slips for another 20 minutes in the presence of no antibody or 10 µg/ml of 12G10 directly labeled with AlexaTM 568. PKA RII subunits were visualized by indirect immunofluorescence using fluorescently labeled donkey anti-goat IgG antibody (green) and ß1 integrins were visualized with AlexaTM 568-labeled 12G10 (red). Fluorescent images were combined to determine colocalization (yellow). All bars, 10 µm.

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