The lymphoid protein tyrosine phosphatase Lyp interacts with the adaptor molecule Grb2 and functions as a negative regulator of T-cell activation.
OBJECTIVE
Following activation of T cells, phosphorylation of tyrosine residues occurs through a complex signaling process involving protein tyrosine kinases, phosphatases, and a variety of adapter molecules including Grb2. We have attempted to identify new signaling molecules that are important for the activation response.


METHODS
Using a protein interaction screening protocol based on phage display, T-cell signaling components that associate with the adapter molecule, Grb2, the lymphoid-specific tyrosine phosphatase Lyp was identified. Using transcriptional reporter assays, the role of Lyp in T-cell activation was studied by overexpression of wild-type or catalytically inactive mutants of Lyp.


RESULTS
A GST fusion containing the C-terminal SH3 domain of Grb2 bound to the nucleotide exchange factor Sos or Grb2-associated binder 2 (Gab2). In contrast, the N-terminal SH3-containing fusion bound to the protein tyrosine phosphatase Lyp. Grb2 was co-immunoprecipitated with Lyp in 293T cells overexpressing both proteins. Using Northern blot analysis, Lyp was found to be expressed predominantly in hematopoietic tissue, including spleen, lymph node, thymus, peripheral blood leukocytes, bone marrow, and fetal liver. Two human T-cell lines, Jurkat and HuT78, expressed both Lyp mRNA and protein. Overexpression of wild-type Lyp or a catalytically inactive, substrate-trapping mutant (D195A) in Jurkat cells inhibited transcriptional activity initiated by anti-CD3 and anti-CD28 antibodies. In contrast, two other catalytically inactive mutants (R233M or C227S) had no effect.


CONCLUSIONS
These data demonstrate a novel interaction between the phosphatase Lyp and the adaptor Grb2 and are consistent with a negative regulatory role for Lyp in T-cell signaling.
Hill
Ronald J
RJ
Research Department, Sugen, Inc., South San Francisco, CA 94080, USA. ron-hill@sugen.com
Zozulya
Sergey
S
Lu
Ying-Lin
YL
Ward
Kevin
K
Gishizky
Mikhail
M
Jallal
Bahija
B
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D015152:Blotting, Northern; D002460:Cell Line; D051380:GRB2 Adaptor Protein; D015870:Gene Expression; D005982:Glutathione Transferase / Q000235:genetics; D006706:Homeostasis; D006801:Humans; D007163:Immunosorbent Techniques; D019169:Jurkat Cells; D008213:Lymphocyte Activation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009928:Organ Specificity; D010446:Peptide Fragments / Q000235:genetics; D019151:Peptide Library; D016133:Polymerase Chain Reaction; D054596:Protein Tyrosine Phosphatase, Non-Receptor Type 22; D017027:Protein Tyrosine Phosphatases / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000276:immunology*; D014162:Transfection
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D010446:Peptide Fragments; D019151:Peptide Library; D011506:Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; C516765:PTPN22 protein, human; D054596:Protein Tyrosine Phosphatase, Non-Receptor Type 22; D017027:Protein Tyrosine Phosphatases