Regulation of ZAP-70 Intracellular Localization: Visualization with the Green Fluorescent Protein

Sloan-Lancaster, Joanne; Zhang, Weiguo; Presley, John; Williams, Brandi L.; Abraham, Robert T.; Lippincott-Schwartz, Jennifer; Samelson, Lawrence E.

doi:10.1084/jem.186.10.1713

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Article| November 17 1997

Regulation of ZAP-70 Intracellular Localization: Visualization with the Green Fluorescent Protein

Joanne Sloan-Lancaster,

Joanne Sloan-Lancaster

From *the Section on Lymphocyte Signaling and ‡the Unit of Organelle Biology, Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892; and §The Department of Immunology, Mayo Clinic, Rochester, Minnesota 55905

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Weiguo Zhang,

Weiguo Zhang

From *the Section on Lymphocyte Signaling and ‡the Unit of Organelle Biology, Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892; and §The Department of Immunology, Mayo Clinic, Rochester, Minnesota 55905

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John Presley,

John Presley

From *the Section on Lymphocyte Signaling and ‡the Unit of Organelle Biology, Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892; and §The Department of Immunology, Mayo Clinic, Rochester, Minnesota 55905

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Brandi L. Williams,

Brandi L. Williams

From *the Section on Lymphocyte Signaling and ‡the Unit of Organelle Biology, Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892; and §The Department of Immunology, Mayo Clinic, Rochester, Minnesota 55905

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Robert T. Abraham,

Robert T. Abraham

From *the Section on Lymphocyte Signaling and ‡the Unit of Organelle Biology, Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892; and §The Department of Immunology, Mayo Clinic, Rochester, Minnesota 55905

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Jennifer Lippincott-Schwartz,

Jennifer Lippincott-Schwartz

From *the Section on Lymphocyte Signaling and ‡the Unit of Organelle Biology, Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892; and §The Department of Immunology, Mayo Clinic, Rochester, Minnesota 55905

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Lawrence E. Samelson

From *the Section on Lymphocyte Signaling and ‡the Unit of Organelle Biology, Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892; and §The Department of Immunology, Mayo Clinic, Rochester, Minnesota 55905

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Author and Article Information

Joanne Sloan-Lancaster , Weiguo Zhang , John Presley , Brandi L. Williams , Robert T. Abraham , Jennifer Lippincott-Schwartz , Lawrence E. Samelson

From *the Section on Lymphocyte Signaling and ‡the Unit of Organelle Biology, Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892; and §The Department of Immunology, Mayo Clinic, Rochester, Minnesota 55905

Address correspondence to Lawrence E. Samelson, NICHD, CBMB, Bldg. 18T, Rm 101, Bethesda, MD 20892. Phone: 301-496-6368; FAX: 301-402-0078; E-mail: [email protected]

Dr. Sloan-Lancaster is a fellow of the Damon-Runyon/Walter Winchell Cancer Research Fund. Dr. Zhang is supported by the Leukemia Society of America. Dr. Abraham is supported by a National Institutes of Health grant GM47286.

1

Abbreviations used in this paper: cfb3, cytosolic fragment of erythrocyte band 3; GFP, green fluorescent protein; IF, immunofluorescence; IP, immunoprecipitation; IRP, iron regulatory protein; ITAM, immunoreceptor tyrosine-based activation motif; KD, kinase dead; PTK, protein tyrosine kinase; PV, pervanadate; NLS, nuclear localization signal; ROI, region of interest; RT, room temperature.

Received: April 01 1997

Revision Received: June 30 1997

Online ISSN: 1540-9538

Print ISSN: 0022-1007

1997

J Exp Med (1997) 186 (10): 1713–1724.

https://doi.org/10.1084/jem.186.10.1713

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.

1997

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