Figures & data
Figure 1. Dynamic S-acylation of Lck. (A) The S-acylation (represented by thick grey lines) of Lck at two cysteines near an N-terminal myristoylation (represented by thin black line). A protein acyl transferase (PAT) mediates the addition of palmitate from palmitoyl coenzyme A (PalCoA) whereas a thioesterase (TE) catalyzes the hydrolysis of palmitic acid (PalCOOH) from Lck. (B) Accumulation of proteins with phosphorylated tyrosines in lysates from human blast T cells exposed to 1 mM pervanadate (PV) for the times indicated, analyzed by phosphotyrosine (PY) Western blotting. (C) Lck immunoprecipitated from human T lymphoblast cells pulsed with 3H-palmitate, and chased for the indicated times. Pervanadate (PV) treatment causes increased activation of Lck, seen as increasing amounts of active, serine-phosphorylated Lck (p60 SP) and also increases the palmitate turnover on Lck compared to control cells not treated with pervanadate (ctrl). Method: T lymphoblasts were a kind gift of Dr Julian Ng and Dr Doreen Cantrell at CRUK-LRI. They were prepared from outdated blood bags and maintained for up to two weeks in RPMI 1640 medium containing 10% foetal calf serum and Interleukin 2 (IL2) (20 ng/ml) at 50–200×104 cells/ml Citation[94]. Cells were quiesced by removal of IL2 for 2–3 days, spun down and resuspended in labelling medium containing 5 mM pyruvate, then labelled with 3H palmitic acid (30–60 Ci/mmol, approx. 200 µCi/ml) and incubated at 37°C for 45 min, then spun down and resuspended in chase medium (labelling medium containing 80 µM palmitic acid, approx. 20-fold excess over tritiated palmitic acid, with or without 1 mM PV). This was followed by rapid washing with ice-cold PBS, lysis, immunoprecipitation, separation by SDS-PAGE and fluorography to detect 3H-palmitate labelled bands.
![Figure 1. Dynamic S-acylation of Lck. (A) The S-acylation (represented by thick grey lines) of Lck at two cysteines near an N-terminal myristoylation (represented by thin black line). A protein acyl transferase (PAT) mediates the addition of palmitate from palmitoyl coenzyme A (PalCoA) whereas a thioesterase (TE) catalyzes the hydrolysis of palmitic acid (PalCOOH) from Lck. (B) Accumulation of proteins with phosphorylated tyrosines in lysates from human blast T cells exposed to 1 mM pervanadate (PV) for the times indicated, analyzed by phosphotyrosine (PY) Western blotting. (C) Lck immunoprecipitated from human T lymphoblast cells pulsed with 3H-palmitate, and chased for the indicated times. Pervanadate (PV) treatment causes increased activation of Lck, seen as increasing amounts of active, serine-phosphorylated Lck (p60 SP) and also increases the palmitate turnover on Lck compared to control cells not treated with pervanadate (ctrl). Method: T lymphoblasts were a kind gift of Dr Julian Ng and Dr Doreen Cantrell at CRUK-LRI. They were prepared from outdated blood bags and maintained for up to two weeks in RPMI 1640 medium containing 10% foetal calf serum and Interleukin 2 (IL2) (20 ng/ml) at 50–200×104 cells/ml Citation[94]. Cells were quiesced by removal of IL2 for 2–3 days, spun down and resuspended in labelling medium containing 5 mM pyruvate, then labelled with 3H palmitic acid (30–60 Ci/mmol, approx. 200 µCi/ml) and incubated at 37°C for 45 min, then spun down and resuspended in chase medium (labelling medium containing 80 µM palmitic acid, approx. 20-fold excess over tritiated palmitic acid, with or without 1 mM PV). This was followed by rapid washing with ice-cold PBS, lysis, immunoprecipitation, separation by SDS-PAGE and fluorography to detect 3H-palmitate labelled bands.](/cms/asset/8f80a0d9-4946-4a73-8e1f-adb94c0e38f0/imbc_a_363102_f0001_b.gif)