PTPH1 is a predominant protein-tyrosine phosphatase capable of interacting with and dephosphorylating the T cell receptor zeta subunit.
Protein-tyrosine phosphatases (PTPases) play key roles in regulating tyrosine phosphorylation levels in cells, yet the identity of their substrates remains limited. We report here on the identification of PTPases capable of dephosphorylating the phosphorylated immune tyrosine-based activation motifs present in the T cell receptor zeta subunit. To characterize these PTPases, we purified enzyme activities directed against the phosphorylated T cell receptor zeta subunit by a combination of anion and cation chromatography procedures. A novel ELISA-based PTPase assay was developed to rapidly screen protein fractions for enzyme activity following the various chromatography steps. We present data that SHP-1 and PTPH1 are present in highly enriched protein fractions that exhibit PTPase activities toward a tyrosine-phosphorylated TCR zeta substrate (specific activity ranging from 0.23 to 40 pmol/min/microg). We also used a protein-tyrosine phosphatase substrate-trapping library comprising the catalytic domains of 47 distinct protein-tyrosine phosphatases, representing almost all the tyrosine phosphatases identified in the human genome. PTPH1 was the predominant phosphatase capable of complexing phospho-zeta. Subsequent transfection assays indicated that SHP-1 and PTPH1 are the two principal PTPases capable of regulating the phosphorylation state of the TCR zeta ITAMs, with PTPH1 directly dephosphorylating zeta. This is the first reported demonstration that PTPH1 is a candidate PTPase capable of interacting with and dephosphorylating TCR zeta.
Sozio
Margaret S
MS
Center for Immunology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9093, USA.
Mathis
Meredith A
MA
Young
Jennifer A
JA
Wälchli
Sebastien
S
Pitcher
Lisa A
LA
Wrage
Philip C
PC
Bartók
Beatrix
B
Campbell
Amanda
A
Watts
Julian D
JD
Aebersold
Ruedi
R
Hooft van Huijsduijnen
Rob
R
van Oers
Nicolai S C
NS
D001665:Binding Sites; D002384:Catalysis; D002852:Chromatography, Ion Exchange / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D019169:Jurkat Cells; D008565:Membrane Proteins / Q000378:metabolism*; D010766:Phosphorylation; D054589:Protein Tyrosine Phosphatase, Non-Receptor Type 3; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism
D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D011948:Receptors, Antigen, T-Cell; C054979:antigen T cell receptor, zeta chain; D014443:Tyrosine; C516656:PTPN3 protein, human; C509249:PTPN6 protein, human; D054589:Protein Tyrosine Phosphatase, Non-Receptor Type 3; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases