Sequential dephosphorylation of a multiply phosphorylated insulin receptor peptide by protein tyrosine phosphatases.
The question of whether protein tyrosine phosphatases (PTPases) dephosphorylate a multiply phosphorylated peptide in a random or ordered manner was investigated using the synthetic triphosphotyrosyl peptide TRDIY(P)ETDY(P)Y(P)RK, corresponding to the major sites of autophosphorylation of the insulin receptor, as a substrate for four purified PTPases. All four enzymes dephosphorylated the triphospho peptide to produce diphospho, monophospho, and nonphosphorylated forms. Partially dephosphorylated peptides were separated by reverse-phase HPLC, and the di- and monophospho peptides were collected and analyzed by solid-phase sequencing to determine the order of dephosphorylation of the three sites by each of the PTPases. The quantitative analysis of the signals for derivatives of tyrosine and phosphotyrosine generated at positions 5, 9, and 10 of the peptide showed that the low molecular weight human placental PTPase 1B preferentially dephosphorylated the two phosphotyrosines at positions 9 and 10 whereas the integral membrane enzyme CD45 (from human spleen) and the bacterially expressed rat LAR preferentially dephosphorylated the phosphotyrosine at position 5. A second low molecular weight enzyme, termed TCPTPase, did not display any specificity for a particular phosphotyrosyl residue. These results demonstrate that different PTPases exhibit a characteristic pattern of dephosphorylation of the triphospho peptide model substrate, raising the possibility that features in the primary structure surrounding the dephosphorylation site may contribute to substrate specificity.
Ramachandran
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C
Department of Biochemistry, Merck Frosst Centre for Therapeutic Research, Kirkland, Quebec, Canada.
Aebersold
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R
Tonks
N K
NK
Pot
D A
DA
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010446:Peptide Fragments; D019000:Phosphotyrosine; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D011972:Receptor, Insulin / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D013997:Time Factors; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D010446:Peptide Fragments; D019000:Phosphotyrosine; D014443:Tyrosine; D011972:Receptor, Insulin; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases