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Intramolecular interactions in protein tyrosine phosphatase RPTPmu: kinetic evidence
A R Aricescu1, T A Fulga, V Cismasiu
1Department of Enzymology, Institute of Biochemistry, Splaiul Independentei 296, 77700 Bucharest, Romania.
Abstract:
The receptor-like protein tyrosine phosphatase RPTPmu contains three intracellular domains: the juxtamembrane (JM) and two phosphatase domains (D1 and D2). D1 is catalytically active in vitro. The functional roles of JM and D2 are still unclear. To find out whether and how they modulate the phosphatase activity of D1, we compared the enzymatic characteristics of two constructs, containing a truncated JM and either D1 or both phosphatase domains. p-Nitrophenyl phosphate and two peptide substrates were efficiently dephosphorylated by both constructs. The specificity constant of D1 alone was up to 50% higher. D2 induces (a) decreased K(m) values for peptide substrates, (b) decreased catalytic efficiency for these substrates, (c) shifting of the optimal pH to slightly lower values, and (d) looser binding of competitive inhibitors. These data suggest that the phosphatase activity of D1 is negatively modulated and its ligand binding capacity is sensibly modified by domain D2, having possible functional significance.
Insights
The juxtamembrane (JM) and D2 domains of RPTPmu negatively modulate phosphatase D1 activity. Domain D2 alters substrate binding and inhibitor interactions, suggesting functional significance in RPTPmu signaling.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Signaling
Background:
- Receptor-like protein tyrosine phosphatase RPTPmu has three intracellular domains: juxtamembrane (JM), and two phosphatase domains (D1 and D2).
- Domain D1 exhibits in vitro catalytic activity, while the functions of JM and D2 remain largely unknown.
- Understanding the modulation of D1 activity by other domains is crucial for elucidating RPTPmu's biological roles.
Purpose of the Study:
- To investigate whether and how the JM and D2 domains influence the phosphatase activity of D1.
- To characterize the enzymatic properties of RPTPmu constructs with different domain combinations.
Main Methods:
- Enzymatic assays using p-nitrophenyl phosphate and peptide substrates.
- Comparison of kinetic parameters (Km, catalytic efficiency) and pH optima.
- Assessment of competitive inhibitor binding.
Main Results:
- Both constructs (D1 alone and D1+D2) efficiently dephosphorylated tested substrates.
- RPTPmu D1 alone showed a higher specificity constant.
- Domain D2 decreased Km values and catalytic efficiency for peptide substrates, shifted optimal pH lower, and weakened competitive inhibitor binding.
Conclusions:
- RPTPmu's D2 domain negatively modulates the catalytic activity of D1.
- D2 significantly alters substrate binding and ligand interactions, indicating a crucial role in regulating RPTPmu function.
- These findings suggest functional implications for D2 in RPTPmu-mediated signaling pathways.