Identification of substrates of human protein-tyrosine phosphatase PTPN22.
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, CD3epsilon, and valosin containing protein) and two known substrates of PEP, the mouse homolog of PTPN22 (Lck and Zap70). T cell antigen receptor (TCR) zeta 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 TCRzeta, 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 TCRzeta in vitro and in cells, and its substrate trap variant co-immunoprecipitated with TCRzeta when both were coexpressed in 293T cells, establishing TCRzeta as a direct substrate of PTPN22.
Wu
Jiansheng
J
Celera Genomics, South San Francisco, California 94080, USA. lakedaly@yahoo.com
Katrekar
Anjali
A
Honigberg
Lee A
LA
Smith
Ashley M
AM
Conn
Marion T
MT
Tang
Jie
J
Jeffery
Doug
D
Mortara
Kyle
K
Sampang
Jun
J
Williams
Steve R
SR
Buggy
Joseph
J
Clark
James M
JM
D000595:Amino Acid Sequence; D001665:Binding Sites; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D019169:Jurkat Cells; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000378:metabolism; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011392:Proline / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D054596:Protein Tyrosine Phosphatase, Non-Receptor Type 22; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry* / Q000378:metabolism; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013379:Substrate Specificity; D013601:T-Lymphocytes / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D014443:Tyrosine / Q000737:chemistry; D051746:ZAP-70 Protein-Tyrosine Kinase / Q000378:metabolism
D018076:DNA, Complementary; D008565:Membrane Proteins; D010455:Peptides; D011948:Receptors, Antigen, T-Cell; D011994:Recombinant Proteins; C054979:antigen T cell receptor, zeta chain; D014443:Tyrosine; D011392:Proline; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human; C516765:PTPN22 protein, human; D054596:Protein Tyrosine Phosphatase, Non-Receptor Type 22; D017027:Protein Tyrosine Phosphatases; C516766:Ptpn22 protein, mouse