Specificity of T-cell protein tyrosine phosphatase toward phosphorylated synthetic peptides

Authors

  • Maria RUZZENE,

    1. Dipartimento di Chimica Biologica e Centro per lo Studio della Fisiologia Mitocondriale del Consiglio Nazionale delle Ricerche, Università di Padova, Italy
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  • Arianna DONELLA-DEANA,

    1. Dipartimento di Chimica Biologica e Centro per lo Studio della Fisiologia Mitocondriale del Consiglio Nazionale delle Ricerche, Università di Padova, Italy
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  • Oriano MARIN,

    1. Dipartimento di Chimica Biologica e Centro per lo Studio della Fisiologia Mitocondriale del Consiglio Nazionale delle Ricerche, Università di Padova, Italy
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  • John W. PERICH,

    1. Biochemistry and Molecular Biology Unit, School of Dental Science and School of Chemistry, University of Melbourne, Victoria, Australia
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  • Paolo RUZZA,

    1. Dipartimento di Chimica Organica e Centro di Studio sui Biopolimeri del Consiglio Nazionale delle Ricerche, Università di Padova, Italy
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  • Gianfranco BORIN,

    1. Dipartimento di Chimica Organica e Centro di Studio sui Biopolimeri del Consiglio Nazionale delle Ricerche, Università di Padova, Italy
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  • Andrea CALDERAN,

    1. Dipartimento di Chimica Organica e Centro di Studio sui Biopolimeri del Consiglio Nazionale delle Ricerche, Università di Padova, Italy
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  • Lorenzo A. PINNA

    Corresponding author
    1. Dipartimento di Chimica Biologica e Centro per lo Studio della Fisiologia Mitocondriale del Consiglio Nazionale delle Ricerche, Università di Padova, Italy
      Correspondence to L. A. Pinna, Dipartimento di Chimica Biologica, Via Trieste 75, I-35121 Padova, Italy
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Correspondence to L. A. Pinna, Dipartimento di Chimica Biologica, Via Trieste 75, I-35121 Padova, Italy

Abstract

The local specificity determinants for a T-cell protein tyrosine phosphatase (TC-PTP) have been inspected with the aid of a series of synthetic peptides, either enzymically or chemically phosphorylated, derived from the phosphoacceptor sites of phosphotyrosyl proteins. The truncated form of T-cell PTP, deprived of its C-terminal down-regulatory domain, readily dephosphorylates submicromolar concentrations of eptapeptides to eicosapeptides, reproducing the C-terminal down-regulatory site of pp60c-src (Tyr527), the phosphorylated loop IV of calmodulin and the main autophosphorylation site of two protein tyrosine kinases of the src family (Tyr416 of pp60c-src and Tyr412 of p51fgr). However, phosphopeptides of similar size, derived from phosphoacceptor tyrosyl sites of the abl and epidermal-growth-factor receptor protein tyrosine kinases, the phosphorylated loop III of calmodulin, and phosphoangiotensin II undergo either very slow or undetectable dephosphorylation, even if tested up to 1 μM concentration.

The replacement of either Ser-P or O-methylated phosphotyrosine for phosphotyrosine within suitable peptide substrates gives rise to totally inert derivatives. Moreover, amino acid substitutions around phosphotyrosine in the peptides src-(412–418), src-(414–418) and abl-(390–397) deeply influence the dephosphorylation efficiency. From these data and from a comparative analysis of efficient versus poor phosphopeptide substrates, it can be concluded that acidic residues located on the N-terminal side of phosphotyrosine, with special reference to position –3, play a crucial role in substrate recognition, while basic residues in the same positions act as negative determinants. In any event, the presence of at least two aminoacyl residues upstream of phosphotyrosine represents a necessary, albeit not sufficient, condition for detectable dephosphorylation to occur.

By replacing the truncated form of TC-PTP with the full length TC-PTP, the dephosphorylation efficiencies of all peptides tested are dramatically impaired. Such an effect is invariably accounted for by a substantial increase in Km values, accompanied by a more or less pronounced decrease in Vmax values. These data support the concept that the C-terminal regulatory domain of TC-PTP exerts its function primarily by altering the affinity of the enzyme toward its phosphotyrosyl targets.

Abbreviations
PTP

protein tyrosine phosphatase

TC-PTP

T-cell protein tyrosine phosphatase

TPK

tyrosine protein kinase

Fmoc

fluorenylmethoxycarbonyl

NMP

N-methylpyrrolidone

t-But

tert-butyl

Boc

tert-butyloxycarbonyl

DMAP

4-dimethylaminopyridine

RCML

reduced

MBP

myelin basic protein

EGF

epidermal growth factor

Yp

phosphotyrosine

Tp

phosphothreonine

Sp

phosphoserine

Enzyme
 

Protein-tyrosine-phosphate phosphohydrolase (EC 3.1.3.48)

Ancillary

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