Comparison of the Specificity of Bacterially Expressed Cytoplasmic Protein-Tyrosine Phosphatases SHP and SH-PTP2 Towards Synthetic Phosphopeptide Substrates

Authors

  • Ute Dechert,

    1. The Biomedical Research Centre and Department of Medicine, University of British Columbia, Vancouver, Canada
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  • Michael Affolter,

    1. The Biomedical Research Centre and Department of Medicine, University of British Columbia, Vancouver, Canada
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  • Kenneth W. Harder,

    1. The Biomedical Research Centre and Department of Medicine, University of British Columbia, Vancouver, Canada
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  • James Matthews,

    1. Howard Hughes Medical Institute and Department of Pathology, Washington University School of Medicine, St Louis MO, USA
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  • Philip Owen,

    1. The Biomedical Research Centre and Department of Medicine, University of British Columbia, Vancouver, Canada
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  • Ian Clark-Lewis,

    1. The Biomedical Research Centre and Department of Medicine, University of British Columbia, Vancouver, Canada
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  • Matthew L. Thomas,

    1. Howard Hughes Medical Institute and Department of Pathology, Washington University School of Medicine, St Louis MO, USA
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  • Ruedi Aebersold,

    1. The Biomedical Research Centre and Department of Medicine, University of British Columbia, Vancouver, Canada
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  • Frank R. Jirik

    Corresponding author
    1. The Biomedical Research Centre and Department of Medicine, University of British Columbia, Vancouver, Canada
      Correspondence to F. R. Jirik, Biomedical Research Centre, University of British Columbia, 2222 Health Sciences Mall, Vancouver, B. C., Canada V6T 1Z3
      Fax:+1 604 822 9710.
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Correspondence to F. R. Jirik, Biomedical Research Centre, University of British Columbia, 2222 Health Sciences Mall, Vancouver, B. C., Canada V6T 1Z3
Fax:+1 604 822 9710.

Abstract

SHP and SH-PTP2 are related cytoplasmic protein-tyrosine phosphatases having two tandem amino-terminal src homology 2 domains linked to a single catalytic domain. There is growing evidence that these two molecules may exhibit opposing effects within specific signaling pathways. However, the relative contributions of the src homology 2 domains or the catalytic domains to these opposing effects are not well known. To evaluate the potential contribution of the catalytic domains, we compared the substrate specificity of the two phosphatases. As seen previously, the catalytic activities of bacterially expressed SHP and SH-PTP2 were regulated by the presence of the linked src homology 2 domains. In addition, we characterized a cryptic thrombin cleavage site within the carboxy-terminus of SHP that led to a striking increase in the activity of the catalytic domain. Employing a panel of phosphopeptide substrates whose sequences were modeled after intracellular phosphorylation sites, both SHP and SH-PTP2 demonstrated a similar specificity pattern. Similar to SH-PTP2, SHP failed to elicit detectable phosphate release from several phosphopeptide substrates, while displaying catalytic efficiencies that ranged over ≈40–1.6×103 M−1 s−1 towards other substrates. In contrast, the PTP-1B phosphatase dephosphorylated all of the phosphopeptide substrates tested with approximately equal ease. The overall similarity demonstrated by the catalytic domains of SHP and SH-PTP2 suggested that differences in the in vivo behavior of these two molecules might not stem from differences in the substrate specificity of the catalytic domains, suggesting instead that the specificity of the src homology 2 domains is more important in this regard.

Abbreviations
CSF-1R

colony stimulating factor-1 receptor

GST

gluthathione S -transferase

P Np

p -nitrophenyl phosphate

PhMeSO2F

phenylmethanesulfonylfluoride

PDGFR

platelet-derived growth factor receptor

PTPase

protein-tyrosine phosphatase

SH2

src homology 2

Enzymes
 

Protein-tyrosine phosphatase (EC 3.1.3.48)

 

protein-tyrosine kinase (EC 2.7.1.112)

Ancillary

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