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J. Biol. Chem., Vol. 283, Issue 32, 21909-21919, August 8, 2008

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Modulation of Lck Function through Multisite Docking to T Cell-specific Adapter Protein^*

Stine Granum¹, Thorny Cesilie Bie Andersen, Morten Sørlie, Marit Jørgensen^¶, Lise Koll, Tone Berge, Tor Lea^¶, Burkhard Fleckenstein^¶, Anne Spurkland^¶, and Vibeke Sundvold-Gjerstad

From the Department of Anatomy, Institute of Basic Medical Sciences, University of Oslo, Box 1105, Blindern, N-0317 Oslo, Norway, the Department of Chemistry, Biotechnology and Food Science, the Norwegian University of Life Sciences, 1432 As, Norway, and the ^¶Institute of Immunology, Rikshospitalet Medical Centre, 0027 Oslo, Norway

T cell-specific adapter protein (TSAd), encoded by the SH2D2A gene, interacts with Lck through its C terminus and thus modulates Lck activity. Here we mapped Lck phosphorylation and interaction sites on TSAd and evaluated their functional importance. The three C-terminal TSAd tyrosines Tyr²⁸⁰, Tyr²⁹⁰, and Tyr³⁰⁵ were phosphorylated by Lck and functioned as docking sites for the Lck Src homology 2 (SH2) domain. Binding affinities of the TSAd Tyr(P)²⁸⁰ and Tyr(P)²⁹⁰ phosphopeptides to the isolated Lck SH2 domain were similar to that observed for the Lck Tyr(P)⁵⁰⁵ phosphopeptide, whereas the TSAd Tyr(P)³⁰⁵ peptide displayed a 10-fold higher affinity. The proline-rich Lck SH3-binding site on TSAd as well as the Lck SH2 domain were required for efficient tyrosine phosphorylation of TSAd by Lck. Interaction sites on TSAd for both Lck SH2 and Lck SH3 were necessary for TSAd-mediated modulation of proximal TCR signaling events. We found that 20–30% of TSAd molecules are phosphorylated in activated T cells and that the proportion of TSAd to Lck molecules in such cells is 1:1. Therefore, in activated T cells, a considerable number of Lck molecules may potentially be engaged by TSAd. In conclusion, Lck binds to TSAd prolines and phosphorylates and interacts with the three C-terminal TSAd tyrosines. We propose that through multivalent interactions with Lck, TSAd diverts Lck from phosphorylating other substrates, thus modulating its functional activity through substrate competition.

Received for publication, February 1, 2008 , and in revised form, May 28, 2008.

^* This work was supported by the EMBIO (steering board for molecular life sciences at the University of Oslo), University of Oslo, the Norwegian Cancer Society, and the Norwegian Research Council, Medinnova, Novo Nordisk, Odd Fellow, and Anders Jahre Research foundations. 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 Table 1 and Fig. 1.

¹ To whom correspondence should be addressed. Tel.: 4722851154; Fax: 4722851278; E-mail: stine.granum{at}medisin.uio.no.

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