Limits...
Regulation of ZAP-70 intracellular localization: visualization with the green fluorescent protein.

Sloan-Lancaster J, Zhang W, Presley J, Williams BL, Abraham RT, Lippincott-Schwartz J, Samelson LE - J. Exp. Med. (1997)

Bottom Line: Subsequent studies in T cells confirmed this phenotype.Intriguingly, a large amount of ZAP-70, both chimeric and endogenous, resides in the nucleus of quiescent and activated cells.Nuclear ZAP-70 becomes tyrosine phosphorylated upon stimulation via the T cell receptor, indicating that it may have an important biologic function.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, Bethesda, Maryland 20892, USA.

ABSTRACT
To investigate the cellular dynamics of ZAP-70, we have studied the distribution and regulation of its intracellular location using a ZAP-70 green fluorescent protein chimera. Initial experiments in epithelial cells indicated that ZAP-70 is diffusely located throughout the quiescent cell, and accumulates at the plasma membrane upon cellular activation, a phenotype enhanced by the coexpression of Lck and the initiation of ZAP-70 kinase activity. Subsequent studies in T cells confirmed this phenotype. Intriguingly, a large amount of ZAP-70, both chimeric and endogenous, resides in the nucleus of quiescent and activated cells. Nuclear ZAP-70 becomes tyrosine phosphorylated upon stimulation via the T cell receptor, indicating that it may have an important biologic function.

Show MeSH

Related in: MedlinePlus

Biochemical isolation of ZAP-70 from nuclei of Jurkat cells,  and demonstration of its increased tyrosine phosphorylation after anti-TCR stimulation. (a) Cytosol/membrane (1.2 × 107 cells) and nuclear  fractions (2.6 × 107 cells) of Jurkat or P116 cells, immunoprecipitated  with anti–ZAP-70, were analyzed by antiphosphotyrosine Western blotting (top). The stripped membrane was reprobed with anti–ZAP-70 (bottom). Specific activity of ZAP-70 after cellular stimulation was 2.4 for cytosolic/membrane ZAP-70 and 1.4 for nuclear ZAP-70. A nonspecific  band appears in the 4G10 blot of P116 cells, which migrates more slowly  than the phosphorylated ZAP-70. (b) Whole lysate samples from the purified material (6 × 105 cells/lane) was immunoblotted with anti–ZAP-70,  and then reprobed with anti–IRP-1. Densitometric analysis confirmed  that there was a 5.3 fold enrichment of ZAP-70 in the nuclear fraction as  compared to IRP-1. This was confirmed in 3 independent experiments,  with enrichment of ZAP-70 as compared to IRP-n1 ranging from 5 fold  to 6.2 fold.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199132&req=5

Figure 9: Biochemical isolation of ZAP-70 from nuclei of Jurkat cells, and demonstration of its increased tyrosine phosphorylation after anti-TCR stimulation. (a) Cytosol/membrane (1.2 × 107 cells) and nuclear fractions (2.6 × 107 cells) of Jurkat or P116 cells, immunoprecipitated with anti–ZAP-70, were analyzed by antiphosphotyrosine Western blotting (top). The stripped membrane was reprobed with anti–ZAP-70 (bottom). Specific activity of ZAP-70 after cellular stimulation was 2.4 for cytosolic/membrane ZAP-70 and 1.4 for nuclear ZAP-70. A nonspecific band appears in the 4G10 blot of P116 cells, which migrates more slowly than the phosphorylated ZAP-70. (b) Whole lysate samples from the purified material (6 × 105 cells/lane) was immunoblotted with anti–ZAP-70, and then reprobed with anti–IRP-1. Densitometric analysis confirmed that there was a 5.3 fold enrichment of ZAP-70 in the nuclear fraction as compared to IRP-1. This was confirmed in 3 independent experiments, with enrichment of ZAP-70 as compared to IRP-n1 ranging from 5 fold to 6.2 fold.

Mentions: For preparation of cytosolic/membrane and nuclear fractions, Jurkat cells (4 × 107) were suspended in homogenization buffer (10 mM Tris, pH 7.6, 0.5 mM MgCl2, 1 mM PMSF, 10 μg/ml Aprotinin, 10 μg/ml Leupeptin, 5 mM EDTA, 1 mM Na3VO4), with (see Fig. 9 a) or without (see Fig. 9 b) 0.1% Tween 20, and incubated on ice for 20 min. Swollen cells were then homogenized by aspirating 50 times using a 25-gauge needle and 1-ml syringe, and the percentage of broken cells was >95%. NaCl (150 mM final) and Hepes (pH 7.5, 50 mM final) were added, and cells were centrifuged at 2,000 rpm for 5 min to pellet the nuclei. Supernatants were considered the cytosolic/membrane fraction. Nuclear pellets were washed three times with homogenization buffer with (Fig. 9 a) or without (Fig. 9 b) 0.1% Triton X-100, resuspended in the same buffer (400 μl), and sonicated (four times, 30 s each). Samples were boiled for 15 min, centrifuged at 15,000 rpm × 10 min, and supernatants collected as purified nuclear material. A portion of each cellular fraction was used to determine enrichment of nuclear material and percent contamination from cytosol. The remainder of each was immunoprecipitated with anti– ZAP-70 antiserum after adding Brij 97 to 1% final concentration.


Regulation of ZAP-70 intracellular localization: visualization with the green fluorescent protein.

Sloan-Lancaster J, Zhang W, Presley J, Williams BL, Abraham RT, Lippincott-Schwartz J, Samelson LE - J. Exp. Med. (1997)

Biochemical isolation of ZAP-70 from nuclei of Jurkat cells,  and demonstration of its increased tyrosine phosphorylation after anti-TCR stimulation. (a) Cytosol/membrane (1.2 × 107 cells) and nuclear  fractions (2.6 × 107 cells) of Jurkat or P116 cells, immunoprecipitated  with anti–ZAP-70, were analyzed by antiphosphotyrosine Western blotting (top). The stripped membrane was reprobed with anti–ZAP-70 (bottom). Specific activity of ZAP-70 after cellular stimulation was 2.4 for cytosolic/membrane ZAP-70 and 1.4 for nuclear ZAP-70. A nonspecific  band appears in the 4G10 blot of P116 cells, which migrates more slowly  than the phosphorylated ZAP-70. (b) Whole lysate samples from the purified material (6 × 105 cells/lane) was immunoblotted with anti–ZAP-70,  and then reprobed with anti–IRP-1. Densitometric analysis confirmed  that there was a 5.3 fold enrichment of ZAP-70 in the nuclear fraction as  compared to IRP-1. This was confirmed in 3 independent experiments,  with enrichment of ZAP-70 as compared to IRP-n1 ranging from 5 fold  to 6.2 fold.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199132&req=5

Figure 9: Biochemical isolation of ZAP-70 from nuclei of Jurkat cells, and demonstration of its increased tyrosine phosphorylation after anti-TCR stimulation. (a) Cytosol/membrane (1.2 × 107 cells) and nuclear fractions (2.6 × 107 cells) of Jurkat or P116 cells, immunoprecipitated with anti–ZAP-70, were analyzed by antiphosphotyrosine Western blotting (top). The stripped membrane was reprobed with anti–ZAP-70 (bottom). Specific activity of ZAP-70 after cellular stimulation was 2.4 for cytosolic/membrane ZAP-70 and 1.4 for nuclear ZAP-70. A nonspecific band appears in the 4G10 blot of P116 cells, which migrates more slowly than the phosphorylated ZAP-70. (b) Whole lysate samples from the purified material (6 × 105 cells/lane) was immunoblotted with anti–ZAP-70, and then reprobed with anti–IRP-1. Densitometric analysis confirmed that there was a 5.3 fold enrichment of ZAP-70 in the nuclear fraction as compared to IRP-1. This was confirmed in 3 independent experiments, with enrichment of ZAP-70 as compared to IRP-n1 ranging from 5 fold to 6.2 fold.
Mentions: For preparation of cytosolic/membrane and nuclear fractions, Jurkat cells (4 × 107) were suspended in homogenization buffer (10 mM Tris, pH 7.6, 0.5 mM MgCl2, 1 mM PMSF, 10 μg/ml Aprotinin, 10 μg/ml Leupeptin, 5 mM EDTA, 1 mM Na3VO4), with (see Fig. 9 a) or without (see Fig. 9 b) 0.1% Tween 20, and incubated on ice for 20 min. Swollen cells were then homogenized by aspirating 50 times using a 25-gauge needle and 1-ml syringe, and the percentage of broken cells was >95%. NaCl (150 mM final) and Hepes (pH 7.5, 50 mM final) were added, and cells were centrifuged at 2,000 rpm for 5 min to pellet the nuclei. Supernatants were considered the cytosolic/membrane fraction. Nuclear pellets were washed three times with homogenization buffer with (Fig. 9 a) or without (Fig. 9 b) 0.1% Triton X-100, resuspended in the same buffer (400 μl), and sonicated (four times, 30 s each). Samples were boiled for 15 min, centrifuged at 15,000 rpm × 10 min, and supernatants collected as purified nuclear material. A portion of each cellular fraction was used to determine enrichment of nuclear material and percent contamination from cytosol. The remainder of each was immunoprecipitated with anti– ZAP-70 antiserum after adding Brij 97 to 1% final concentration.

Bottom Line: Subsequent studies in T cells confirmed this phenotype.Intriguingly, a large amount of ZAP-70, both chimeric and endogenous, resides in the nucleus of quiescent and activated cells.Nuclear ZAP-70 becomes tyrosine phosphorylated upon stimulation via the T cell receptor, indicating that it may have an important biologic function.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, Bethesda, Maryland 20892, USA.

ABSTRACT
To investigate the cellular dynamics of ZAP-70, we have studied the distribution and regulation of its intracellular location using a ZAP-70 green fluorescent protein chimera. Initial experiments in epithelial cells indicated that ZAP-70 is diffusely located throughout the quiescent cell, and accumulates at the plasma membrane upon cellular activation, a phenotype enhanced by the coexpression of Lck and the initiation of ZAP-70 kinase activity. Subsequent studies in T cells confirmed this phenotype. Intriguingly, a large amount of ZAP-70, both chimeric and endogenous, resides in the nucleus of quiescent and activated cells. Nuclear ZAP-70 becomes tyrosine phosphorylated upon stimulation via the T cell receptor, indicating that it may have an important biologic function.

Show MeSH
Related in: MedlinePlus