1998;140:861C871. maximum after 45 min of activation; in contrast, PYK-2 activation peaked at 30 min, declining after 60 min. Upon polarization of lymphoblasts, FAK and PYK-2 redistributed from a diffuse localization in the cytoplasm to a region close to the microtubule-organizing center in these cells. FAK and PYK-2 activation was clogged when lymphoblasts were pretreated with actin and tubulin cytoskeleton-interfering providers, indicating its cytoskeletal dependence. Our results demonstrate that connection of the 2-integrin LFA-1 with its ligand intercellular adhesion molecule 1 induces redesigning of T lymphocyte morphology and activation and redistribution of the cytoplasmic tyrosine kinases FAK BCDA and PYK-2. Intro Integrins are heterodimeric cell surface proteins that function in cell adhesion, cytoskeleton anchorage, and the transduction of cellular stimuli into cytoplasmic signals (Clark and Brugge, 1995 ; Schwartz COMOS graphical user interface and software. Quantitative Time-Lapse Video Microscopy BCDA of T Lymphoblast Motility Plastic dishes (35 mm) were precoated with recombinant ICAM-1Fc protein and clogged with BSA as indicated above. T lymphoblasts were plated immediately before video recording in RPMI 1640 medium supplemented with 1% FCS in the presence or absence of the relevant stimuli for LFA-1 activation. Time-lapse video films of cells were generated like a sequence of individual digital images (frames) that were acquired every 10 s for 2.30 h in an (Thornwood, NY) Axiovert 135 video microscope using the IP-Lab Spectrum software (Signal Analytics, Vienna, VA). The cellular random migration songs, distances, and average speeds of individual cells for each experimental condition were acquired using the Cell Tracking software extension for IP-Lab Spectrum developed by Tim Hutton (Confocal Microscopy and Digital Image Unit, Imperial Malignancy Research Account). Immunoprecipitation T lymphoblasts (100 106 or 2.5 106 cells to immunoprecipitate FAK or PYK-2, respectively, unless otherwise stated) were washed twice with RPMI 1640, plated on dishes coated with either BSA or ICAM-1, and, after 15 min on ice, stimulated with 10 g/ml mAb KIM-127 for 60 min. The activation was terminated by solubilizing the cells in 1 ml of ice-cold lysis buffer (10 mM Tris-HCl, pH 7.65, 5 mM EDTA, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM NaF, 2 mM sodium orthovanadate, 1% Triton X-100, 50 g/ml aprotinin, 50 g/ml leupeptin, 5 g/ml pepstatin, and BCDA 1 mM PMSF). Lysates were clarified by centrifugation at 14,000 rpm for 10 min, and the pellets were discarded. TRIB3 After centrifugation, supernatants were transferred to new tubes, and proteins were immunoprecipitated at 4C over BCDA night with either protein A-agaroseClinked rabbit polyclonal anti-FAK antibodies (C-20 or A-17) or protein G-agaroseClinked mAbs directed against FAK (2A7 or a-FAK mAbs) or against Tyr(P) proteins (PY20 and PY72 mAbs) or protein G-agaroseClinked goat polyclonal anti-PYK-2 antibody (C-19). Immunoprecipitates were washed three times with lysis buffer and either utilized for in vitro kinase reactions (observe below) or extracted in 2 SDS-PAGE sample buffer (200 mM Tris-HCl, pH 6.8, 0.1 mM sodium orthovanadate, 1 mM EDTA, 6% SDS, 2 mM EDTA, 4% 2-mercaptoethanol, and 10% glycerol), by boiling 5 min, fractionated by one-dimensional SDS PAGE, and further analyzed as explained in RESULTS and figure legends. In Vitro Kinase Reactions Reactions were performed as explained (Rodrguez-Fernndez and Rozengurt, 1996 , 1998 ). Briefly, immunoprecipitates were washed and pelleted (2500 rpm 10 min in the chilly) three times in lysis buffer and twice with kinase buffer (20 mM HEPES and 3 mM MnCl2, pH 7.35). Pellets were dissolved in 40 l of kinase buffer, and reactions were started by adding 10 Ci of [-32P]ATP. The reactions were carried out at 30C.