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J. Biol. Chem., Vol. 281, Issue 16, 11002-11010, April 21, 2006

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Identification of Substrates of Human Protein-tyrosine Phosphatase PTPN22^*

From the Celera Genomics, South San Francisco, California 94080

Stimulation of mature T cells activates a downstream signaling cascade involving temporally and spatially regulated phosphorylation and dephosphorylation events mediated by protein-tyrosine kinases and phosphatases, respectively. PTPN22 (Lyp), a non-receptor protein-tyrosine phosphatase, is expressed exclusively in cells of hematopoietic origin, notably in T cells where it represses signaling through the T cell receptor. We used substrate trapping coupled with mass spectrometry-based peptide identification in an unbiased approach to identify physiological substrates of PTPN22. Several potential substrates were identified in lysates from pervanadate-stimulated Jurkat cells using PTPN22-D195A/C227S, an optimized substrate trap mutant of PTPN22. These included three novel PTPN22 substrates (Vav, CD3, and valosin containing protein) and two known substrates of PEP, the mouse homolog of PTPN22 (Lck and Zap70). T cell antigen receptor (TCR) was also identified as a potential substrate in Jurkat lysates by direct immunoblotting. In vitro experiments with purified recombinant proteins demonstrated that PTPN22-D195A/C227S interacted directly with activated Lck, Zap70, and TCR, confirming the initial substrate trap results. Native PTPN22 dephosphorylated Lck and Zap70 at their activating tyrosine residues Tyr-394 and Tyr-493, respectively, but not at the regulatory tyrosines Tyr-505 (Lck) or Tyr-319 (Zap70). Native PTPN22 also dephosphorylated TCR in vitro and in cells, and its substrate trap variant co-immunoprecipitated with TCR when both were coexpressed in 293T cells, establishing TCR as a direct substrate of PTPN22.

Received for publication, January 17, 2006

^* 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 To whom correspondence should be addressed: 615 John Muir Dr. D616, San Francisco, CA 94132. Tel.: 415-337-6873; Fax: 415-337-6873; E-mail: lakedaly{at}yahoo.com.

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