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. 2003 Dec 9;100(25):14707-12.
doi: 10.1073/pnas.2534493100. Epub 2003 Dec 1.

Determination of the substrate-docking site of protein tyrosine kinase C-terminal Src kinase

Sungsoo Lee  1 , Xiaofeng Lin, Nguyen Hai Nam, Keykavous Parang, Gongqin Sun
Affiliations

Determination of the substrate-docking site of protein tyrosine kinase C-terminal Src kinase

Sungsoo Lee et al. Proc Natl Acad Sci U S A. .

Abstract

Protein tyrosine kinases (PTK) are key enzymes of mammalian signal transduction. For the fidelity of signal transduction, each PTK phosphorylates only one or a few proteins on specific Tyr residues. Substrate specificity is thought to be mediated by PTK-substrate docking interactions and recognition of the phosphorylation site sequence by the kinase active site. However, a substrate-docking site has not been determined on any PTK. C-terminal Src kinase (Csk) is a PTK that specifically phosphorylates Src family kinases on a C-terminal Tyr. In this study, by sequence alignment and site-specific mutagenesis, we located a substrate-docking site on Csk. Mutations in the docking site disabled Csk to phosphorylate, regulate, and complex with Src but only moderately affected its general kinase activity. A peptide mimicking the docking site potently inhibited (IC50 = 21 microM) Csk phosphorylation of Src but only moderately inhibited (IC50 = 422 microM) its general kinase activity. Determination of the substrate-docking site provides the structural basis of substrate specificity in Csk and a model for understanding substrate specificity in other PTKs.

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Figures

Fig. 1.
Fig. 1.
Alignment of the amino acid sequences of Csk, Chk, Src, and Hck in the peptide-binding lobe. Residues that are uniquely conserved in the Csk family are highlighted blue (polar or charged) or red (hydrophobic or Gly).
Fig. 2.
Fig. 2.
Ala scanning mutagenesis to identify the substrate-docking site on Csk. (A) Ala scanning of polar or charged residues that are uniquely conserved in the Csk family. The activity of the Ala mutants toward an artificial and a physiological substrate are determined. (B) Structure of the Csk peptide-binding lobe. Residues that are potentially part of the substrate-docking site, the catalytic loop, and the activation loop are indicated. (C) Ala scanning of residues in or near the identified substrate-docking site.
Fig. 3.
Fig. 3.
Effect of DM and QM on Csk kinase function. The details and rationale of the mutations are described in the text. (A) Effect of docking site mutations on Csk phosphorylation of kdSrc. (B) Effect of docking site mutations Csk's ability to inactivate Src.
Fig. 4.
Fig. 4.
Pull-down assay to determine the interaction of Csk and mutants with kdSrc. (A) Purified GST, GST-Csk, GST-DM, and GST-QM. (B and C) kdSrc pull-down assay with various Csk variants. Each of the purified GST or fusion proteins (100 pmol) was incubated with purified kdSrc (200 pmol) and precipitated with glutathione-agarose. The proteins retained by the beads were analyzed by SDS/PAGE and Coomassie blue staining.
Fig. 5.
Fig. 5.
Inhibition of Csk activity by peptide P2 that mimics the substrate-docking site. The Ki of P2 inhibition of Csk is 21 μM toward kdSrc but 422 μM toward polyE4Y.
Fig. 6.
Fig. 6.
Structures of Csk catalytic domain and the substrate-docking site. (A) Ribbon structure of Csk with identified residues in the substrate-docking site shown in a ball-and-stick model. Several loop structures relevant to peptide substrate binding and catalysis are indicated by arrows. (B) Surface structure of Csk (yellow) and the substrate-docking site (colored by electrostatic potential). The active site cleft is indicated.
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