NMR analysis of regioselectivity in dephosphorylation of a triphosphotyrosyl dodecapeptide autophosphorylation site of the insulin receptor by a catalytic fragment of LAR phosphotyrosine phosphatase.
An autophosphorylation site in the activated insulin receptor tyrosine kinase domain has three tyrosines phosphorylated when fully activated. To begin to examine recognition of triphosphotyrosyl sites by protein tyrosine phosphatases in possible control of signal transduction a triphosphotyrosyl dodecapeptide TRDIpYETDpYpYRK corresponding to residues 1,142-1,153 of the insulin receptor was prepared and incubated with the 40-kDa catalytic domain of the human PTPase LAR. To assess regioselectivity of recognition, the three diphosphotyrosyl regioisomers, and the three monophosphotyrosyl regioisomers were prepared and assayed. All seven peptides were PTPase substrates. To identify any preferences in dephosphorylation at pY5, pY9, or pY10, 1H-NMR analyses were conducted during enzyme incubations and distinguishing fingerprint regions determined for each of the seven phosphotyrosyl peptides. LAR PTPase shows strong preference for dephosphorylation first at pY5 (at tri-, di-, and monophosphotyrosyl levels). Initially this regioselectivity gives the Y5(pY9)(pY10) diphospho regioisomer, followed by equal dephosphorylation at pY9 or pY10 to give the corresponding monophosphoryl species on the way to fully dephosphorylated product. The NMR methodology is applicable to other peptides with multiple sites of phosphorylation that undergo attack by any phosphatase.
Lee
J P
JP
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Cho
H
H
Bannwarth
W
W
Kitas
E A
EA
Walsh
C T
CT
D000595:Amino Acid Sequence; D002384:Catalysis; D006023:Glycoproteins / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D011972:Receptor, Insulin / Q000378:metabolism*; D054630:Receptor-Like Protein Tyrosine Phosphatases, Class 4; D011956:Receptors, Cell Surface; D014443:Tyrosine / Q000378:metabolism*
D006023:Glycoproteins; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D014443:Tyrosine; D011972:Receptor, Insulin; C516841:PTPRA protein, human; D017027:Protein Tyrosine Phosphatases; D054630:Receptor-Like Protein Tyrosine Phosphatases, Class 4