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First published online 13 December 2005
doi: 10.1242/jcs.02712

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Cell adhesion regulates Ser/Thr phosphorylation and proteasomal degradation of HEF1

Center for Cell Biology and Cancer Research, Albany Medical College, Albany, NY 12208, USA

View larger version (65K): [in a new window]   Fig. 1. Proteasome inhibitors prevent HEF1 degradation and result in p115 HEF1 accumulation in A1-F and MG-63 cells. (A) MG-63 and A1-F cells were plated onto FN-coated tissue culture dishes in DMEM supplemented with 10% FBS and grown to confluence. Cell layers were treated with 10 µM lactacystin (LAC), 50 µM LLnL, 50 µM ALLM or carrier solvent DMSO (Cont) for 14 hours. (B) MG-63 cells on FN-coated dishes were serum-starved for 6 hours and then treated with TGF-ß1 (2.5 ng/ml) for 12 hours to induce HEF1 expression (0 hours). TGF-ß1-containing medium was removed and cell layers were washed twice with serum-free medium. Cells were treated with 20 µg/ml cycloheximide (CHX) in the presence or absence of 10 µM lactacystin for indicated times. Cell lysates were prepared and processed for immunoblotting with anti-HEF1 pAb. Nitrocellulose membranes were stripped and reprobed with anti-p130Cas mAb as a loading control.

View larger version (48K): [in a new window]   Fig. 2. The interconversion of p105 and p115 HEF1 is regulated by cell adhesion and detachment. (A) A1-F monolayers plated on FN-coated dishes were serum-starved overnight and treated with TGF-ß1 (2.5 ng/ml) for 4 hours to induce HEF1 expression (0 hours). Cells were then detached and suspended in serum-free medium for indicated times. (B) After suspension for 2 hours (indicated as 0 hours of adhesion), A1-F cells were plated onto FN-coated dishes for indicated times. (C) After having been treated with TGF-ß1 (2.5 ng/ml) for 4 hours, A1-F monolayers were detached and suspended in serum-free medium for 1 hour. Suspended cells were then plated onto tissue culture dishes that had been coated with polylysine (PL), fibronectin (FN), collagen (Coll), laminin (LM) or vitronectin (VN) and incubated for 2 hours. Cell lysates were prepared and processed for immunoblotting with anti-HEF1 pAb. Nitrocellulose membranes were stripped and reprobed with anti-p130Cas mAb as loading control.

View larger version (76K): [in a new window]   Fig. 3. The effects of cytoskeleton-disruptive agents on the conversion of p115HEF1 to p105HEF1. A1F cells were serum-starved overnight, detached and resuspended in DMEM containing 0.1% heat-inactivated BSA and 2.5 ng/ml TGF-ß1. (A) Suspended cells were then plated onto FN-coated dishes and incubated for 4 hours. They were then treated for 2 hours with the indicated concentrations of nocodazole, colchicine, cytochalasin D (Cyto D) or latrunculin A. Cells treated with vehicle only were set as control (0). Cell lysates were generated and processed for immunoblotting with anti-HEF1 pAb. Nitrocellulose membranes were stripped and reprobed with anti-p130Cas mAb as loading control. (B) Suspended cells were seeded onto FN-coated coverslips for 4 hours and treated for 2 hours with the indicated concentration of nocodazole, cytochalasin D or the same volume of vehicle as control. Cells were then triple-stained for F-actin, microtubules and vimentin. Actin was visualized using Texas-Red-conjugated phalloidin (red); microtubules were visualized by staining the cell layers with anti-ß-tubulin mAb followed by the Alexa Fluor-350-conjugated secondary antibody (blue); and intermediate filaments were stained with anti-vimentin pAb followed by Alexa Fluor-488-conjugated secondary antibody (green).

View larger version (51K): [in a new window]   Fig. 4. Cytochalasin-D-induced conversion of p115HEF1 to p105HEF1 is not due to selective degradation of p115 HEF1. A1-F monolayers on FN-coated dishes were serum-starved overnight and pre-treated with TGF-ß1 (2.5 ng/ml) for 4 hours in the presence of 10 µM lactacystin (LAC) or 50 µM ALLM as indicated. 2 µM cytochalasin D (CD) or equal volumes of carrier solvent DMSO (control) were then directly added into pre-treatment medium, and cells were further incubated for 6 hours. Cell lysates were generated and processed for immunoblotting with anti-HEF1 pAb. Nitrocellulose membranes were stripped and reprobed with anti-p130Cas mAb as loading control.

View larger version (41K): [in a new window]   Fig. 5. Cell suspension or cytochalasin D treatment inhibit HEF1 degradation. MG-63 cells on FN-coated dishes were serum-starved for 6 hours and then treated with TGF-ß1 (2.5 ng/ml) for 12 hours to induce HEF1 expression. TGF-ß1-containing medium was removed and cell layers were washed twice with serum-free medium. (A) Cell layers were then treated with 20 µg/ml cycloheximide in the absence (adhesion) or presence (adhesion + CD) of 2 µM cytochalasin D for indicated times, or detached and plated onto agar-coated dishes in the presence 20 µg/ml cycloheximide (suspension). Times indicate hours after addition of cytochalasin D or placement in suspension. Aliquots of cell lysates containing equal amounts of protein were processed for immunoblotting with anti-HEF1 antibody. Membranes were stripped and reprobed for p130 Cas, which served as a loading control. (B) The levels of total HEF1 as shown in A were analyzed by densitometry and the time at 0 hours was set as 100%.

View larger version (46K): [in a new window]   Fig. 6. The effects of Ser/Thr phosphatase inhibitors on the cytochalasin-D-induced loss of p115HEF1. (A-C) A1-F cells were plated onto FN-coated dishes, serum-starved overnight and then incubated for 4 hours with DMEM containing 0.1% heat-inactivated BSA and 2.5 ng/ml TGF-ß1 (Contr). After incubation of cells with indicated concentration of calyculin A for 0.5 hour (A), or okadaic acid for 2 hours (B), or tautomycin for 14 hours (C), the cells were treated with 2 µM cytochalasin D for 2 hours in the presence of the inhibitors. Aliquots of cell extracts containing equal amount of protein were analyzed by western blotting with anti-HEF1 pAb. Nitrocellulose membranes were stripped and reprobed with anti-actin pAb to ensure equal loading.

View larger version (28K): [in a new window]   Fig. 7. PP2A, rather than PP1, is responsible for the dephosphorylation of p115HEF1 in human dermal fibroblasts. (A) The activities of PP1 and PP2A in the cell extracts obtained as described in Fig. 6 were determined by measuring the generation of free 32Pi from 32P-phosphorylase-a and differentiated by their sensitivities to inhibitor-2 as described in Materials and Methods. The activity of control was set as 100%. The data represent the mean ± s.d. of duplicate assays from three separate experiments. (B) To ensure equal amounts of protein phosphatases in the sample shown in A, cell extracts containing the same amount of protein were processed for western blotting with anti-PP2A monoclonal antibody. Nitrocellulose membrane was stripped and reprobed with anti-PP1 monoclonal antibody. (C) The cell extracts were obtained as described in Fig. 6. The PP1 and PP2A activities in cell extracts were determined as described above, and expressed as percentage of control. The data represent the mean ± s.d. of triplicate assays from two separate experiments. (D) Cell extracts containing the same amount of protein were analyzed by western blotting with anti-PP1 monoclonal antibody. Nitrocellulose membrane was stripped and reprobed with anti-PP2A monoclonal antibody.

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