Involvement of LAT in CD2 signaling of human peripheral blood T cells.
(A) LAT is tyrosine phosphorylated and associates with a number of tyrosine phosphorylated proteins on CD2 stimulation of natural human T cells. Human PBLs were isolated, as described in “Materials and methods,” and either left unstimulated or stimulated for the indicated periods of time with either OKT3 1 μg, T112plus T113 (1 μL each), or a combination of anti-CD3 and anti-CD2 mAbs. PNLs were either subjected to immunoprecipitation with the rabbit polyclonal anti-LAT antibody or incubated with protein A–agarose alone as indicated, separated by SDS-PAGE, and immunoblotted with 4G10 (upper panel). Precipitation of protein A–agarose alone was included to control for carryover of the stimulating antibody. Note the coprecipitation with LAT of other tyrosine phosphorylated proteins; tyrosylphosphorylated bands in the range of 70 to 203 kd are revealed in a longer exposure of the membrane (middle panel). The membrane was stripped and reblotted with anti-LAT antibody (lower panel). (B and C) CD2 induces association of tyrosine phosphorylated LAT with Grb-2 in human PBLs. A segment of the membrane in panel A is shown here to demonstrate LAT tyrosine phosphorylation (upper panel) and Grb-2 association. Grb-2 coprecipitation was demonstrated by stripping and reblotting the same membrane with anti-Grb-2 antibody (lower panel). In panel C, quantification of LAT tyrosine phosphorylation and Grb-2 coprecipitation was determined by densitometry using the Imagequant software. (D and E) CD2 induces Erk1/2 activation in human PBLs. Thirty μL of postnuclear lysate (0.6 × 106 cell equivalents) from each sample of the experiment described in panel A was set aside prior to immunoprecipitation, separated by SDS-PAGE, and immunoblotted with antiphospho-ERK1/2 antibody (D, upper panel). The membrane was stripped and reblotted with an anti-ERK1/2–specific antibody to control for protein loading (D, lower panel). Quantification of Erk2 (p42 MAPK) phosphorylation was determined by densitometry using the Imagequant software, and expressed as phospho-Erk2/Erk2 ratio (E).
