Plasmids. The expression vectors pRK5-DAPK and pRK5-DAPK42A were described previously (Jang et al., 2002). The cDNA for DAPKΔCaM (Cohen et al., 1997) was constructed by the Quick-Change site-directed mutagenesis kit (Strategene) and then cloned to pRK5. The cDNAs for DAPK, DAPK42A and DAPKΔCaM were cloned to pBabepuro3 to generate retroviral vectors. The expression vector for CD2-FAK (Chan et al., 1994) was a gift from Gena Whitney.
Cell culture, transformation and retroviral infection. 293T and NIH3T3 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum (FCS). BT474 cells were cultured in DMEM/F12 medium containing 5%
FCS. MCF10A cells were grown in DMEM/F12 medium supplemented with 5%
horse serum, 5 g/ml of insulin, 0.01 g/ml of EGF, 0.1 g/ml of cholera toxin, and 0.5 g/ml of hydrocortisone. Transfection was performed by the calcium phosphate method. Generation of recombinant retrovirus and infection of NIH3T3 cells was carried out following procedures described previously (Tsai et al., 2000). For infection of MCF10A cells, the viral stock was supplemented with 2 g/ml of polybrene and incubated with cells for 18 h. Two days after the beginning of the infection, cells were selected in culture medium containing 1 g/ml of puromycin.
Two days after selection, the uninfected cells were virtually eliminated and the surviving cells were used for various analyses.
Antibodies and reagents. The polyclonal antibody to DAP-kinase was described previously (Jang et al., 2002). The hybridoma for TS2/16 was from ATCC and the antibody was purified by affinity chromatography using HiTrap protein G column (Amersham Pharmacia Biotech). The mouse 1 integrin activating antibody 9EG7, the antibody HUTS-21 for activated human 1, antibodies to human 5-integrin (Vc5), 6-integrin (GoH3), and p53 were from Pharmigen. The human 1-integrin antibody AIIB2 was a gift from Caroline Damsky. The antibody B44 for the
activated human 1 integrin and the antibody MB1.2 for mouse 1 integrin were from Chemicon. Antibodies to FAK, paxillin and phosphotyrosine were from
Transduction Laboratories, whereas antibody to FAK phosphorylated at tyrosine 397 was from Biosource.
Adhesion assays. Assays of cell adhesion on plates coated with 10 ng/l of fibronectin, 30 ng/l of laminin, 2 mg/ml of poly-L-lysine or 1% BSA in
phosphate-buffered saline (PBS) were performed essentially as described (Mould et
al., 1995). For experiments with blocking or activating antibodies, cells were pre-incubated with 5 g/ml of purified antibodies on ice for 30 min, and adhesion assays were performed in the presence of antibodies. For experiments with Mn2+, 2 mM MnCl2 was added into cell suspension before the adhesion assays. To estimate the reference value for 100 % attachment, cells were seeded on plates pre-coated with 20 ng/l of fibronectin or 50 ng/l of laminin and incubated for 3 to 4 h at 37°C.
After incubation, cells were fixed immediately and approximately 90 to 100 % of input cells were recovered.
Flow cytometry analysis. To assess cell surface expression of specific integrins, cells were washed with PBS, and resuspended in blocking solution containing 5 % dissociation buffer (Invitrogen) and 2% goat serum in PBS. Cells were then incubated with anti-integrin antibody for 1 h at 4°C, washed with blocking solution and labeled with FITC-conjugated secondary antibody for 30 min at 4°C. Cells were then washed and analyzed on a Becton Dickison FACScan device. For detecting the activated 1 integrin, cells transfected with various constructs were incubated in blocking solution with or without 2 mM MnCl2 at 37°C for 30 min., followed by incubating with B44 or HUTS-21 antibody for 45 min at 4°C, and then labeled with secondary antibody as described above.
Immunoprecipitations. Cells were lysed in buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 1% NP40, 1% sodium deoxycholate, 0.1% SDS, supplemented with 1 mM PMSF, 10 g/ml aprotinin, 10 g/ml leupeptin, 1 mM sodium vanadate, 4 mM sodium pyrophosphate, and 20 mM NaF. Lysates containing equal amount of proteins were subjected to immunoprecipitations as described previously (Tsai et al., 2000).
Apoptosis and caspase activity assays. Cells transfected with various DAP-kinase expression constructs or infected with recombinant retrovirus expressing DAP-kinase or its mutants were seeded on plates pre-coated with 25 ng/l of fibronectin, 0.1%
poly-L-lysine or 0.5 ml of 12 mg/ml poly-HEMA and cultured for various time periods in the presence or absence of integrin activating antibody. For all apoptosis-related assays, both detached and adherent cells were harvested and
combined. DNA fragmentation and Caspase 3 activities were measured as described previously (Jang et al., 2002). For determining population of cells with sub-G1 DNA content, cells were stained with propidium iodide and then analyzed by flow cytomerty. Annexin V staining was performed using the Annexin V-FITC Apoptosis Detection Kit (Oncogene).
Reporter assays. To analyze transcriptional activity of p53, the p53-TA-luc reporter (Clonetech) and the pRK5-gal plasmid were transfected into NIH3T3 cells in the presence or absence of various DAP-kinase expression constructs. Cells were detached at 36 h after transfection, replated onto dishes coated with fibronectin and cultured under serum-starved conditions for 12 h. For experiments using the 9EG7 antibody, cells were fed with serum-free medium at 36 h after transfection. Eight hours later, cells were detached and pre-incubated with 5 g/ml of 9EG7 in DMEM containing 1% BSA at 37°C for 30 min. Cells were then plated on fibronectin and cultured for another 4 h. In all cases, both attached and detached cells were
harvested and combined. Luciferase and -galactosidase activities were quantitated as described (Jang et al., 2002).