Probing protein-tyrosine phosphatase substrate specificity using a phosphotyrosine-containing phage library.
Protein tyrosine phosphatases (PTPs) play important, highly dynamic roles in signaling. Currently about 90 different PTP genes have been described. The enzymes are highly regulated at all levels of expression, and it is becoming increasingly clear that substrate specificity of the PTP catalytic domains proper contributes considerably to PTP functionality. To investigate PTP substrate selectivity, we have set up a procedure to generate phage libraries that presents randomized, phosphotyrosine-containing peptides. Phages that expressed suitable substrates were selected by immobilized, substrate-trapping GST-PTP fusion proteins. After multiple rounds of selection, positive clones were confirmed by SPOT analysis, dephosphorylation by wild-type enzyme, and Km determinations. We have identified distinct consensus substrate motifs for PTP1B, Sap-1, PTP-beta, SHP1, and SHP2. Our results confirm substrate specificity for individual PTPs at the peptide level. Such consensus sequences may be useful both for identifying potential PTP substrates and for the development of peptidomimetic inhibitors.
Wälchli
Sébastien
S
Serono Pharmaceutical Research Institute, CH-1228 Geneva, Switzerland.
Espanel
Xavier
X
Harrenga
Axel
A
Rossi
Mario
M
Cesareni
Gianni
G
Hooft van Huijsduijnen
Rob
R
D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000235:genetics*; D002213:Capsid / Q000737:chemistry; D020134:Catalytic Domain; D017931:DNA Primers; D015723:Gene Library; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D019000:Phosphotyrosine / Q000378:metabolism*; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D017931:DNA Primers; D010446:Peptide Fragments; D011994:Recombinant Proteins; D019000:Phosphotyrosine; D017027:Protein Tyrosine Phosphatases