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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.

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Quantitation and  functional reconstitution analyses  of ZAP-70 GFP in stably transfected P116 subclones. (a) Cell  lysates of Jurkat, P116, or P116/ ZAP-70 GFP subclones were  immunoprecipitated with anti– ZAP-70. Immunoprecipitated  proteins were analyzed by SDS-PAGE and Western blot using  anti–ZAP-70 (2 × 106 cells/ lane). Levels of ZAP-70 GFP expression in each subclone, presented as a percent of endogenous ZAP-70 in Jurkat cells,  were as follows: 97% for C8,  143% for C11, 131% for H9, and  94% for F4. (b) Cells were lysed  directly or first stimulated for 2  min with OKT3 F(ab′)2 at 37°C,  and antiphosphotyrosine Western blot analysis of whole cellular  lysates (2 × 105 cells/lane) performed.
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Figure 4: Quantitation and functional reconstitution analyses of ZAP-70 GFP in stably transfected P116 subclones. (a) Cell lysates of Jurkat, P116, or P116/ ZAP-70 GFP subclones were immunoprecipitated with anti– ZAP-70. Immunoprecipitated proteins were analyzed by SDS-PAGE and Western blot using anti–ZAP-70 (2 × 106 cells/ lane). Levels of ZAP-70 GFP expression in each subclone, presented as a percent of endogenous ZAP-70 in Jurkat cells, were as follows: 97% for C8, 143% for C11, 131% for H9, and 94% for F4. (b) Cells were lysed directly or first stimulated for 2 min with OKT3 F(ab′)2 at 37°C, and antiphosphotyrosine Western blot analysis of whole cellular lysates (2 × 105 cells/lane) performed.

Mentions: To study the intracellular location and redistribution of ZAP-70 in T cells, we reconstituted a mutant Jurkat T cell line, P116, which lacks ZAP-70 (Williams, B.L., and R.T. Abraham, manuscript submitted for publication). Individual subclones were selected from stable bulk cultures by limiting dilution analysis and two, 2G1 and 1C2, were used for further study. These lines were further subcloned to derive C8, C11, H9 (from 2G1), and F4 (from 1C2). Anti–ZAP-70 IP and Western blot analysis (Fig. 4 a) with subsequent densitometry measurements indicated that the relative expression of ZAP-70 GFP compared to endogenous ZAP-70 in Jurkat cells was 97% for C8, 143% for C11, 131% for H9, and 94% for F4.


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)

Quantitation and  functional reconstitution analyses  of ZAP-70 GFP in stably transfected P116 subclones. (a) Cell  lysates of Jurkat, P116, or P116/ ZAP-70 GFP subclones were  immunoprecipitated with anti– ZAP-70. Immunoprecipitated  proteins were analyzed by SDS-PAGE and Western blot using  anti–ZAP-70 (2 × 106 cells/ lane). Levels of ZAP-70 GFP expression in each subclone, presented as a percent of endogenous ZAP-70 in Jurkat cells,  were as follows: 97% for C8,  143% for C11, 131% for H9, and  94% for F4. (b) Cells were lysed  directly or first stimulated for 2  min with OKT3 F(ab′)2 at 37°C,  and antiphosphotyrosine Western blot analysis of whole cellular  lysates (2 × 105 cells/lane) performed.
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Related In: Results  -  Collection

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Figure 4: Quantitation and functional reconstitution analyses of ZAP-70 GFP in stably transfected P116 subclones. (a) Cell lysates of Jurkat, P116, or P116/ ZAP-70 GFP subclones were immunoprecipitated with anti– ZAP-70. Immunoprecipitated proteins were analyzed by SDS-PAGE and Western blot using anti–ZAP-70 (2 × 106 cells/ lane). Levels of ZAP-70 GFP expression in each subclone, presented as a percent of endogenous ZAP-70 in Jurkat cells, were as follows: 97% for C8, 143% for C11, 131% for H9, and 94% for F4. (b) Cells were lysed directly or first stimulated for 2 min with OKT3 F(ab′)2 at 37°C, and antiphosphotyrosine Western blot analysis of whole cellular lysates (2 × 105 cells/lane) performed.
Mentions: To study the intracellular location and redistribution of ZAP-70 in T cells, we reconstituted a mutant Jurkat T cell line, P116, which lacks ZAP-70 (Williams, B.L., and R.T. Abraham, manuscript submitted for publication). Individual subclones were selected from stable bulk cultures by limiting dilution analysis and two, 2G1 and 1C2, were used for further study. These lines were further subcloned to derive C8, C11, H9 (from 2G1), and F4 (from 1C2). Anti–ZAP-70 IP and Western blot analysis (Fig. 4 a) with subsequent densitometry measurements indicated that the relative expression of ZAP-70 GFP compared to endogenous ZAP-70 in Jurkat cells was 97% for C8, 143% for C11, 131% for H9, and 94% for F4.

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