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J. Biol. Chem., Vol. 280, Issue 23, 21949-21954, June 10, 2005

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Dynamic Regulation of Tec Kinase Localization in Membrane-proximal Vesicles of a T Cell Clone Revealed by Total Internal Reflection Fluorescence and Confocal Microscopy^*

Lawrence P. Kane and Simon C. Watkins¶

From the Immunology and ^¶Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261

Tec family tyrosine kinases are key regulators of lymphocyte activation and effector function. Several Tec family kinases (Tec, Itk, Rlk/Txk) are expressed in T cells, but it is still not clear to what degree these are redundant or have unique functions. We recently demonstrated that Tec alone, among the Tec kinase family members examined, can induce nuclear factor of activated T cell-dependent transcription. This unique functional characteristic correlated with a unique pattern of subcellular localization, as Tec (but not other family members) was found in small vesicles, the appearance of which requires signaling through the T cell receptor for antigen. Here we report on our studies of these Tec-containing structures in live T cells, using total internal reflection fluorescence microscopy. With this technique, we showed that, in live T cells, the Tec vesicles are located at the plasma membrane, the vesicles are unique to Tec (and not the related kinase Itk), and their formation and maintenance require T cell receptor signaling through Src family kinases and PI 3-kinase. Finally, we have imaged isolated T cell membranes by confocal microscopy, confirming the membrane-proximal location of Tec vesicles, as well as demonstrating overlap of these vesicles with the tyrosine kinase Lck, the Tec substrate PLC-1, and the early endosomal antigen 1 marker EEA1.

Received for publication, November 15, 2004 , and in revised form, April 1, 2005.

^* This work was supported by startup funds from the University of Pittsburgh School of Medicine (to L. P. K.) and by support from the National Institutes of Health (P01CA73743). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Movies 1–5.

To whom correspondence should be addressed: Dept. of Immunology, BST E-1056, University of Pittsburgh, Pittsburgh, PA 15261. E-mail: lkane{at}pitt.edu.

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