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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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Related in: MedlinePlus

In vitro functional  activity and apparent molecular  weight of the ZAP-70 GFP fusion protein. (a) Schematic of the  ZAP-70 GFP fusion protein,  showing the position of the GFP  (F64L, S65T) molecule at the  COOH terminus of ZAP-70,  with a predicted molecular  weight of ∼97 kD. (b) Cos 7  cells expressing pEGFP/ZAP-70 and pSXSRαLck F505, were  incubated for 10 min with or  without PV as indicated. Immunoprecipitation of lysed cells was  performed with either anti– ZAP-70 antiserum or A2B4 mAb,  and the phosphorlyated proteins  from an in vitro kinase assay analyzed (top). An anti–ZAP-70  Western blot of the same membrane is shown (bottom).
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Figure 1: In vitro functional activity and apparent molecular weight of the ZAP-70 GFP fusion protein. (a) Schematic of the ZAP-70 GFP fusion protein, showing the position of the GFP (F64L, S65T) molecule at the COOH terminus of ZAP-70, with a predicted molecular weight of ∼97 kD. (b) Cos 7 cells expressing pEGFP/ZAP-70 and pSXSRαLck F505, were incubated for 10 min with or without PV as indicated. Immunoprecipitation of lysed cells was performed with either anti– ZAP-70 antiserum or A2B4 mAb, and the phosphorlyated proteins from an in vitro kinase assay analyzed (top). An anti–ZAP-70 Western blot of the same membrane is shown (bottom).

Mentions: The ZAP-70 GFP fusion protein was constructed in a mammalian expression vector fusing the GFP coding sequence to the COOH terminus of ZAP-70 kinase (Fig. 1 a). To assess the enzymatic activity of the chimeric protein, Cos 7 cells were transfected with ZAP-70 GFP and Lck F505, left untreated, or stimulated with PV, and an in vitro kinase assay was performed on the anti–ZAP-70 IPs (Fig. 1 b, top). Enzymatic activity was measured by detection of autophosphorylation and phosphorylation of cfb3, a substrate for ZAP-70. ZAP-70 GFP showed significant kinase activity, which was increased upon cellular stimulation, indicating that the fusion protein retained the enzymatic properties of the native ZAP-70 molecule (8). An anti–ZAP-70 immunoblot of the membrane demonstrated the amounts of protein in the lanes and confirmed that the antigenic properties of ZAP-70 had been retained (Fig. 1 b, bottom). Anti-GFP antiserum also identified this single species (data not shown), whereas an irrelevant mAb failed to IP the chimeric protein (Fig. 1 b).


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)

In vitro functional  activity and apparent molecular  weight of the ZAP-70 GFP fusion protein. (a) Schematic of the  ZAP-70 GFP fusion protein,  showing the position of the GFP  (F64L, S65T) molecule at the  COOH terminus of ZAP-70,  with a predicted molecular  weight of ∼97 kD. (b) Cos 7  cells expressing pEGFP/ZAP-70 and pSXSRαLck F505, were  incubated for 10 min with or  without PV as indicated. Immunoprecipitation of lysed cells was  performed with either anti– ZAP-70 antiserum or A2B4 mAb,  and the phosphorlyated proteins  from an in vitro kinase assay analyzed (top). An anti–ZAP-70  Western blot of the same membrane is shown (bottom).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: In vitro functional activity and apparent molecular weight of the ZAP-70 GFP fusion protein. (a) Schematic of the ZAP-70 GFP fusion protein, showing the position of the GFP (F64L, S65T) molecule at the COOH terminus of ZAP-70, with a predicted molecular weight of ∼97 kD. (b) Cos 7 cells expressing pEGFP/ZAP-70 and pSXSRαLck F505, were incubated for 10 min with or without PV as indicated. Immunoprecipitation of lysed cells was performed with either anti– ZAP-70 antiserum or A2B4 mAb, and the phosphorlyated proteins from an in vitro kinase assay analyzed (top). An anti–ZAP-70 Western blot of the same membrane is shown (bottom).
Mentions: The ZAP-70 GFP fusion protein was constructed in a mammalian expression vector fusing the GFP coding sequence to the COOH terminus of ZAP-70 kinase (Fig. 1 a). To assess the enzymatic activity of the chimeric protein, Cos 7 cells were transfected with ZAP-70 GFP and Lck F505, left untreated, or stimulated with PV, and an in vitro kinase assay was performed on the anti–ZAP-70 IPs (Fig. 1 b, top). Enzymatic activity was measured by detection of autophosphorylation and phosphorylation of cfb3, a substrate for ZAP-70. ZAP-70 GFP showed significant kinase activity, which was increased upon cellular stimulation, indicating that the fusion protein retained the enzymatic properties of the native ZAP-70 molecule (8). An anti–ZAP-70 immunoblot of the membrane demonstrated the amounts of protein in the lanes and confirmed that the antigenic properties of ZAP-70 had been retained (Fig. 1 b, bottom). Anti-GFP antiserum also identified this single species (data not shown), whereas an irrelevant mAb failed to IP the chimeric protein (Fig. 1 b).

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