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J. Biol. Chem., Vol. 283, Issue 39, 26409-26422, September 26, 2008

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Lck-dependent Fyn Activation Requires C Terminus-dependent Targeting of Kinase-active Lck to Lipid Rafts^*

Dominik Filipp¹, Behrouz Moemeni¹, Alessandra Ferzoco, Kirishanthy Kathirkamathamby, Jenny Zhang, Ondej Ballek, Dominique Davidson^¶, André Veillette^¶, and Michael Julius²

From the Sunnybrook Research Institute and the Department of Immunology, University of Toronto, Toronto, Ontario M4N 3M5, Canada, Laboratory of Immunobiology, Institute of Molecular Genetics AS CR, Videnska 1083, 142 20 Prague 4, Czech Republic, ^¶Laboratory of Molecular Oncology, Clinical Research Institute of Montréal, Montréal, Quebec H2W 1R7, Canada, the Department of Medicine, University of Montréal, Quebec H3A 1A1, Canada, and the Department of Medicine, McGill University, Montréal, Quebec H3T 1J4, Canada

Mechanisms regulating the activation and delivery of function of Lck and Fyn are central to the generation of the most proximal signaling events emanating from the T cell antigen receptor (TcR) complex. Recent results demonstrate that lipid rafts (LR) segregate Lck and Fyn and play a fundamental role in the temporal and spatial coordination of their activation. Specifically, TcR-CD4 co-aggregation-induced Lck activation outside LR results in Lck translocation to LR where the activation of LR-resident Fyn ensues. Here we report a structure-function analysis toward characterizing the mechanism supporting Lck partitioning to LR and its capacity to activate co-localized Fyn. Using NIH 3T3 cells ectopically expressing FynT, we demonstrate that only LR-associated, kinase-active ^Y505FLck reciprocally co-immunoprecipitates with and activates Fyn. Mutational analyses revealed a profound reduction in the formation of Lck-Fyn complexes and Fyn activation, using kinase domain mutants K273R and Y394F of ^Y505FLck, both of which have profoundly compromised kinase activity. The only kinase-active Lck mutants tested that revealed impaired physical and enzymatic engagement with Fyn were those involving truncation of the C-terminal sequence YQPQP. Remarkably, sequential truncation of YQPQP resulted in an increasing reduction of kinase-active Lck partitioning to LR, in both fibroblasts and T cells. This in turn correlated with an ablation of the capacity of these truncates to enhance TcR-mediated interleukin-2 production. Thus, Lck-dependent Fyn activation is predicated by proximity-mediated transphosphorylation of the Fyn kinase domain, and targeting kinase-active Lck to LR is dependent on the C-terminal sequence QPQP.

Received for publication, December 20, 2007 , and in revised form, July 18, 2008.

^* This work was supported by Grant FRN9735 from the Canadian Institutes of Health Research. 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.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed: Sunnybrook Research Institute, Rm. A3-33, 2075 Bayview Ave., Toronto, Ontario M4N 3M5, Canada. Fax: 416-480-4351; E-mail: michael.julius{at}sri.utoronto.ca.

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