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Characterization of the proto-oncogene pim-1: kinase activity and substrate recognition sequence
M Friedmann1, M S Nissen, D S Hoover
1Department of Biochemistry, Washington State University, Pullman 99164.
Abstract:
The human pim-1 proto-oncogene was expressed in Escherichia coli as a glutathione-S-transferase (GST)-fusion protein and the enzymatic properties of its kinase activity were characterized. Likewise, a Pim-1 mutant lacking intrinsic kinase activity was constructed by site-directed mutagenesis (Lys67 to Met) and expressed in E. coli. In vitro assays with the mutant Pim-1 kinase showed no contaminating kinase activity. The wild-type Pim-1 kinase-GST fusion protein showed a pH optimum of 7 to 7.5 and optimal activity was observed at either 10 mM MgCl2 or 5 mM MnCl2. Higher cation concentrations were inhibitory, as was the addition of NaCl to the assays. Previous work by this laboratory assaying several proteins and peptides showed histone H1 and the peptide Kemptide to be efficiently phosphorylated by recombinant Pim-1 kinase. Here we examine the substrate sequence specificity of Pim-1 kinase in detail. Comparison of different synthetic peptide substrates showed Pim-1 to have a strong substrate preference for the peptide Lys-Arg-Arg-Ala-Ser*-Gly-Pro with an almost sixfold higher specificity constant kcat/Km over that of the substrate Kemptide (Leu-Arg-Arg-Ala-Ser*-Leu-Gly). The presence of basic amino acid residues on the amino terminal side of the target Ser/Thr was shown to be essential for peptide substrate recognition. Furthermore, phosphopeptide analysis of calf thymus histone H1 phosphorylated in vitro by Pim-1 kinase resulted in fragments containing sequences similar to that of the preferred synthetic substrate peptide shown above. Therefore, under optimized in vitro conditions, the substrate recognition sequence for Pim-1 kinase is (Arg/Lys)3-X-Ser/Thr*-X', where X' is likely neither a basic nor a large hydrophobic residue.
Insights
The Pim-1 kinase, a proto-oncogene, prefers specific peptide sequences for phosphorylation, with basic residues near the target serine being crucial for recognition. This finding refines our understanding of Pim-1 kinase substrate specificity.
Area of Science:
- Molecular Biology
- Enzymology
- Oncogene Research
Background:
- The human pim-1 proto-oncogene encodes a serine/threonine kinase implicated in cell proliferation and cancer.
- Recombinant Pim-1 kinase has previously been shown to phosphorylate histone H1 and Kemptide.
Purpose of the Study:
- To characterize the enzymatic properties and substrate sequence specificity of the human Pim-1 kinase.
- To determine the optimal in vitro conditions for Pim-1 kinase activity.
- To elucidate the specific amino acid sequence recognized by Pim-1 kinase for phosphorylation.
Main Methods:
- Expression of wild-type and mutant human Pim-1 kinase as glutathione-S-transferase (GST)-fusion proteins in Escherichia coli.
- In vitro kinase assays to determine optimal pH, cation concentrations (MgCl2, MnCl2), and NaCl inhibition.
- Phosphorylation of synthetic peptide substrates and calf thymus histone H1 to analyze substrate sequence specificity using kcat/Km values and phosphopeptide analysis.
Main Results:
- Wild-type Pim-1 kinase-GST fusion protein exhibited optimal activity at pH 7-7.5, 10 mM MgCl2 or 5 mM MnCl2, with higher cation concentrations and NaCl being inhibitory.
- Pim-1 kinase demonstrated a strong preference for the peptide sequence Lys-Arg-Arg-Ala-Ser*-Gly-Pro, showing a sixfold higher specificity constant (kcat/Km) compared to Kemptide.
- Analysis confirmed the essential role of basic amino acid residues (Arg/Lys) on the amino-terminal side of the target serine/threonine for substrate recognition.
Conclusions:
- The optimal substrate recognition sequence for Pim-1 kinase under in vitro conditions is (Arg/Lys)3-X-Ser/Thr*-X', where X' is not a basic or large hydrophobic residue.
- Understanding Pim-1 kinase substrate specificity provides insights into its biological functions and potential as a therapeutic target in cancer.