Methods

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

Chemicals and Reagents. All reagents used for bacterial culture and protein expression were purchased from Fisher. Chromatographic resins, glutathione-agarose, iminodiacetic acid-agarose, and Sephadex G25 were purchased from Sigma. DNA primers were synthesized by Integrated DNA Technologies (Coralville, IA). [γ-³²P]ATP (6,000 Ci mol^–1; 1 Ci = 37 GBq) was purchased from Perkin-Elmer. Peptides were synthesized by solid phase synthesis, purified by HPLC, and confirmed by electrospray mass spectrometry.

Recombinant Kinase and Substrate Expression and Site-Specific Mutagenesis. Wild type human Csk was expressed in E. coli (DH5α) by using pGEX-Csk-st plasmid (22, 23). Site-specific mutants were generated by using QuikChange (Stratagene). The entire coding regions of the mutant plasmids were sequenced to confirm that the correct mutations were incorporated. The GST–Csk fusion proteins were purified by glutathione affinity chromatography and stored in 50 mM Tris-Cl, pH 8.0, at –20°C in 30% glycerol. The chicken Src mutant devoid of kinase activity (kdSrc) was coexpressed with GroEL and GroES chaperone in BL21(DE3) as described (18). kdSrc was purified by a Ni²⁺-iminodiacetic acid agarose column as described (24). Lys295Met mutation abolishes the Src kinase activity but does not affect its ability to serve as a substrate for Csk (18, 25). Protein concentrations were determined by the Bradford method and A₂₈₀ in the 6 M urea. Purity of the purified enzymes was examined by SDS/PAGE and Coomassie blue staining.

Kinase Activity Assays. Kinase activity of Csk and mutants was determined by using polyE₄Y or kdSrc and [γ-³²P]ATP (600 dpm pmol^–1) as the substrates as described (26). Briefly, phosphorylation reactions were performed in 50-μl volumes at 30°Cinthe protein kinase assay buffer: 50 mM N-(2-hydroxyethyl)piperazine-N′-3-propanesulfonic acid (pH 8.0) containing 5% glycerol, 0.005% Triton X-100, and 0.05% 2-mercaptoethanol. The standard assay used 3 nM WT Csk, 12 mM MgCl₂, 0.2 mM ATP, and 1 mg ml^–1 polyE₄Y or 10 μM kdSrc. After a 10-min reaction time, 35 μl of the reaction mixture was spotted onto Whatman filter paper squares (2 × 1 cm), which were washed in 5% trichloroacetic acid at 65°C three times for 20 min each. The radioactivity incorporated into polyE₄Y or kdSrc was determined by liquid scintillation counting. Assays were performed in duplicate, and each assay was repeated at least twice with reproducible results.

To determine the catalytic parameters of Csk by using kdSrc as a substrate, 0.71–7.1 μM kdSrc was used as the variable substrate. The assays were performed as described above. The reaction minus Csk was used as background controls. The background was 4Y by kdSrc was detected under these conditions. The K_m and k_cat values were determined by using double reciprocal plot.

To determine the inhibition of Csk activity by peptides, Csk activity in the presence of various concentrations of the peptide was determined. The kinase activity as a function of the peptide concentration was fitted into a curve-fitting program (labfit)to determine the IC₅₀.

Csk Inactivation of Src. The ability of Csk and mutants to inactivate Src was determined as described (14). Appropriate level of Src activity was incubated with Csk or Csk mutants in the presence of 0.1 mM ATP and 12 mM MgCl₂ at 30°C for 10 min. At the end of the incubation, 0.4 mg/ml RCM-lysozyme and [γ-³²P]ATP was added. Because RCM-lysozyme was preferentially phosphorylated by Src, the residual Src activity after the initial incubation could be accurately determined without removing Csk from the incubation.

Pull-Down Assay. To determine the interaction between Csk and kdSrc, pull-down assays were performed. Purified GST, GST-Csk, or GST-mutant Csk fusion proteins (100 pmol) were incubated with 200 pmol of kdSrc in 50 μl of kinase assay buffer without ATP and MgCl₂ at 30°C for 15 min with gentle agitation. Glutathione-agarose bead suspension (20 μl) was added into each incubation and incubated for another 15 min. The incubation mixtures were passed through a small column to collect the beads, which were then washed three times with 100 μl of kinase assay buffer each. The proteins associated with the beads were analyzed by SDS/PAGE.