Mechanism of phosphotyrosine protection by LAT clustering. (A) TIRF microscopy revealed clustered and unclustered LAT and Grb2. Alexa Fluor 488–labeled LAT at 1,000 molecules/µm2 was incubated with 1 µM Grb2 + 500 nM Sos1 or 6 µM Grb2. 20% of Grb2 was labeled with Alexa Fluor 568. Similar Grb2 was recruited to the membrane in the two indicated conditions. Shown are mean ± SD; n = 3 independent experiments. Scale bar, 5 µm. (B) LAT clustering by Grb2 prevents LAT Y132 from being dephosphorylated by CD45 in vitro. pLAT, at 1,000 molecules/µm2, was incubated with 1 µM Grb2, 0.5 µM Sos1, or 1 µM Grb2. CD45 was added to dephosphorylate pLAT for 5 min. The reaction was terminated by adding SDS-PAGE loading buffer with 2 mM vanadate. The level of LAT pY132, after being normalized to total LAT, was quantified. Shown are mean ± SD; n = 3 independent experiments. MW, molecular weight. (C) CD45 is excluded by Grb2- or PLCγ1-mediated LAT clustering. pLAT–Alexa Fluor 488 (1,000 molecules/μm2) was incubated with 1 µM Sos1 and 1 µM Grb2 or 1 µM PLCγ1-SH2-2-3 fragment. The cytoplasmic domain of CD45-TMR (4 nM, with an N-terminal His10 tag) was added, and its localization was visualized by TIRF microscopy. Scale bar, 5 µm. (D) Quantification of fluorescence intensity of pLAT and CD45 along the line scan indicated by a white line in the top merged image.
