Structural Basis for the Recognition of c-Src by Its Inactivator Csk

Nicholas M. Levinson, Markus A. Seeliger, Philip A. Cole, and John Kuriyan

Cell, Vol 134, 124-134, 11 July 2008 (local copy)

Abstract / Figures from the paper / Supplementary Information


The catalytic activity of the Src family of tyrosine kinases is suppressed by phosphorylation on a tyrosine residue located near the C-terminus (Tyr 527 in c-Src), which is catalyzed by C-terminal Src Kinase (Csk). Given the promiscuity of most tyrosine kinases, it is remarkable that the C-terminal tails of the Src family kinases are the only known targets of Csk. We have determined the crystal structure of a complex between the kinase domains of Csk and c-Src at 2.9 Å resolution, revealing that interactions between these kinases position the C-terminal tail of c-Src at the edge of the active site of Csk. Csk cannot phosphorylate substrates that lack this docking mechanism because the conventional substrate binding site, used by most tyrosine kinases to recognize substrates, is destabilized in Csk by a deletion in the activation loop.

Illustrations from the paper.

Click on the small image to get a bigger one.

Figure 1. Binding of Csk to c-Src measured by surface plasmon resonance (A) Constructs of Csk and c-Src used in this paper. (B) Representative data from the surface plasmon resonance experiments. The sensorgrams obtained with different concentrations of CskKD are shown on the left. The equilibrium response at each concentration was fitted to a single-site binding model, shown on the right, to derive the equilibrium dissociation constant. (C) Equilibrium dissociation constants for several different constructs of Csk and c-Src at 150 mM NaCl. Values shown are the mean and standard deviation from at least two separate titrations.

Figure 2. Structure of the complex between the kinase domains of Csk and c-Src (A) The kinase domains of c-Src and Csk are depicted in their relative orientations in the structure, but separated so that the Csk:c-Src interface, highlighted as dots, is visible. The Csk:c-Src complex is oriented such that the kinase domain of Csk is shown in the standard view (active site facing the viewer). The orientation of the kinase domain of c-Src in the complex is related to the standard view (on the left) by a rotation of ~90° about the vertical axis and ~120° about an axis perpendicular to the page. (B) Overview of the CskKD:c-SrcKD complex. The approximate path of the activation loop, which is disordered between residues 335 and 349, is indicated by a dotted red line. The region of the c-Src that interacts with Csk (residues 504-523), and the c-Src tail (residues 524-533), are highlighted in orange and magenta, respectively. The sidechain of the C-terminal residue of c-Src (Leu 533) is shown as spheres, as is the Cα atom of Tyr 527.

Figure 3. The Csk:c-Src interface probed by mutagenesis (A-C) Details of the Csk:c-Src interface are shown. The coloring scheme is the same as in Figure 2. Residues important for the binding of Csk to c-Src (see below) are indicated. (D) The effects of mutations on the binding of Csk to c-Src as measured by surface plasmon resonance. On the left, the binding of mutants of CskFL (25 μM) to c-SrcKD is shown. On the right the binding of wt CskFL (10 μM) to mutants of c-SrcKD, immobilized on different surfaces, is shown. Values are the means and standard deviations from three series of injections. (E) The effects of mutations in the Csk:c-Src interface on the ability of Csk to phosphorylate c-Src, measured by phosphorimaging, are shown.

Figure 4. The conformation of the c-Src tail (A) On the left the kinase domain of Csk from the CskKD:c-SrcKD structure is depicted with only the C-terminal region of c-Src (the αH/αI loop, helices I and I' and the c-Src tail) shown. The canonical binding site for peptide substrates is illustrated by the substrate peptide from the structure of the insulin receptor kinase domain (IRK), shown in yellow (Hubbard, 1997). On the right, the structure of protein kinase A, in complex with an inhibitory peptide that binds in the docking groove, is depicted for comparison (Knighton et al., 1991). (B) An enlarged view of the C-terminal portion of c-Src in the CskKD:c-SrcKD structure. Perpendicular aromatic:aromatic interactions that stabilize this conformation of the c-Src tail are indicated with sticks and dots. (C) In one of the complexes in the CskSH2KD:c-SrcKD structure the tail of c-Src binds in the active site of Csk. The substrate peptide from the IRK ternary complex, aligned on the kinase domain of Csk, is shown in yellow. A difference electron density map, contoured at 2 standard deviations over the mean (2δ), is shown in red. The map is calculated with coefficients (|Fo|-|Fc|)eiα(c), where |Fo| are the observed structure factor amplitudes and |Fc| and αc) are amplitudes and phases calculated from a model lacking the c-Src tail. See Figure S5 for a more comprehensive view of the electron density map that demonstrates the significance of the features shown here.

Figure 5. Activation loop anchoring in tyrosine kinases (A) The structure of the insulin receptor kinase domain (IRK) bound to a substrate peptide is shown. The panel on the right shows an enlarged view depicting the two anchor points and the loops to which they form hydrogen bonds (shown as dashed lines). (B) The activation loops from twelve structures of active tyrosine kinases (see Experimental Procedures). The structures were aligned on the catalytic loops, but only the activation loops are shown. (C) Hydrophobic interactions couple anchor point 2 to the peptide binding site. The structure of IRK is shown. Residues of the substrate peptide that interact with the hydrophobic residues are indicated.

Figure 6. Activation loop anchoring and the activity of Csk and c-Src towards peptide substrates A) On the left a schematic diagram is shown that highlights the characteristic features of the anchoring mechanism. An alignment of the activation loop sequences of several tyrosine kinases is shown on the right. (B) The kinase activity of constructs of Csk containing insertions in the activation loop is shown relative to wt Csk. The constructs are referred to as CtoS-1 (for Csk to Src), CtoS-2, CtoS-3, and CtoS-4 depending on whether 1, 2, 3, or 4 residues respectively were inserted. The mean value and standard deviation of the relative rates from three experiments are shown. (C) The relative kinase activity of mutants of c-Src. These constructs of the kinase domain either had the hydrophobic latch mutated to alanine alone (IA) or in combination with the deletion of the variable-length loop (IA delta). The graphs show the mean and standard deviation from two experiments.

Figure 7. The inactive assembled state of c-Src is incompatible with the Csk:c-Src complex (A) The crystal structure of inactive assembled c-Src (pdb code 2SRC) is aligned on the C-terminal lobe of c-Src from our CskKD:c-SrcKD structure. Clashes between the tail of inactive c-Src and the C-lobe of Csk are highlighted in black. (B) Csk cannot bind tail-phosphorylated c-Src as measured by surface plasmon resonance. A construct of c-Src that contains the SH2, SH3 and kinase domains (Src3D) was phosphorylated by Csk on Tyr 527 (Src3D pY527) prior to immobilization on the sensor surface. The binding of CskFL (10 μM) to the surface was then measured. The graphs show the mean and standard deviation of two experiments.

Supplementary Information

Supplementary Information