A switch in nucleotide affinity governs activation of the Src and Tec family kinases

Abstract

The Tec kinases, closely related to Src family kinases, are essential for lymphocyte function in the adaptive immune system. Whilst the Src and Abl kinases are regulated by tail phosphorylation and N-terminal myristoylation respectively, the Tec kinases are notable for the absence of either regulatory element. We have found that the inactive conformations of the Tec kinase Itk and Src preferentially bind ADP over ATP, stabilising both proteins. We demonstrate that Itk adopts the same conformation as Src and that the autoinhibited conformation of Src is independent of its C-terminal tail. Allosteric activation of both Itk and Src depends critically on the disruption of a conserved hydrophobic stack that accompanies regulatory domain displacement. We show that a conformational switch permits the exchange of ADP for ATP, leading to efficient autophosphorylation and full activation. In summary, we propose a universal mechanism for the activation and autoinhibition of the Src and Tec kinases.

Figure 1

Autoinhibited Itk and Src kinases preferentially bind ADP. (a) Schematic of constructs evaluated in this study. Itk^32K, Src^32K, and Src^32KΔC contain the conserved module of SH3, SH2, and kinase (^32K) domains. Src^32K was truncated by 10 amino acids to remove its C-terminal tail (inclusive of the Y527 phosphorylation site), generating a construct we refer to as Src^32KΔC (ΔC-tail). (b) Competition nucleotide displacement assay for Itk^32K. 40 μM Itk^32K was incubated with 40 μM Mant-ADP until equilibrium was established. Unlabelled ADP or AMPPNP was titrated into the reaction and fluorescence anisotropy monitored. Comparison of the equilibrium inhibition constants (K _i) gives an indication of the relative strength of nucleotide binding. AMPPNP binds to Itk^32K with 42-fold weaker affinity. (c) Competition nucleotide displacement assay for Src^32K. 40 μM tail-phosphorylated Src^32K (pY527) or doubly phosphorylated Src^32K pY416 (pY527) was incubated with 40 μM Mant-ADP until equilibrium was established. Unlabelled ADP or AMPPNP was titrated into the reaction and fluorescence anisotropy monitored. Solid lines correspond to Src^32K, dashed lines to Src^32K pY416. AMPPNP binds to Src^32K with 20-fold weaker affinity than ADP. Activation loop phosphorylation (dashed lines) does not affect the nucleotide-binding properties of Src^32K. (d) Omit map showing clear electron density for ADP and a single magnesium ion in the nucleotide-binding pocket of Src. The magnesium is coordinated by Asn391, Asp404, and the α- and β- phosphates of ADP. The β-phosphate is directly coordinated by the guanidinium side chain of Arg388. (e) Thermodynamic cycle used for molecular dynamics calculations. The binding processes of ADP or ATP moving from bulk water to the bound state within Src kinase is computationally expensive to calculate. Instead we make use of a thermodynamic cycle. Because free energy is a state function, we can determine the relative binding free energy from the vertical arrows, where we convert ADP to ATP both when in solution and when bound to Src kinase.

Figure 2

Itk adopts a Src-like autoinhibited conformation. (a) Competition nucleotide displacement assay for Src^32KΔC. 40 μM Src^32KΔC was incubated with 40 μM Mant-ADP until equilibrium was established. Unlabelled ADP or AMPPNP was titrated into the reaction and fluorescence anisotropy monitored. AMPPNP binds to Src^32K with 23-fold weaker affinity than ADP. (b) Small angle X-ray scattering curves for Itk^32K (cyan), Src^32K (magenta), Src^32KΔC (blue), and Src^32K R175L (green). (c) Pair distribution functions for Itk^32K (cyan), Src^32K (magenta), Src^32KΔC (blue), and Src^32K R175L (green) indicate that the particles exhibit almost identical radii of gyration and maximum dimensions. (d) Table of physical parameters obtained from the scattering curves for Itk^32K, Src^32K, Src^32KΔC, and Src^32K R175L. (e)Ab initio model of Itk^32K and docking of autoinhibited Src (2SRC.pdb) into the molecular envelope. Two orientations of the envelope are shown for clarity. (f) Ab initio molecular envelope for Src^32KΔC illustrates the fit of the crystal structure of autoinhibited Src.

Figure 3

An allosteric switch triggers nucleotide exchange. (a) Hydrophobic stack conservation in Src family kinases. The table illustrates the amino acid identity at each of the three positions in the hydrophobic stack in addition to the invariant tryptophan (Trp260 in Src) for all Src family kinase members. The positions are presented according to their N- to C-terminal order in the primary sequences. Where it was not possible to unambiguously determine the identity of the amino acid contributed by the SH2-kinase variable linker (V3), the residue is denoted by a question mark. (b) Small angle X-ray scattering of Itk^32K L351A (black). The scattering curve for wild type Itk^32K (cyan) is overlaid for comparison. Calculation of the pair distribution function for Itk^32K L351A shows an increase in the radius of gyration from 2.63 nm to 2.88 nm and an increase in D_max from 8.3 to 9.2 nm compared to wild type Itk^32K. (c) Rigid body modelling of Itk^32K L351A and fit to ab initio molecular envelope. Displacement of both the SH3 and SH2 domains is required for a good fit to the experimental scattering curve. (d) Determination of the binding constants of Itk^32K L351A for nucleotides by fluorescence anisotropy. Non-phosphorylated Itk^32K L351A binds ADP with similar affinity (21 μM) to wild type Itk^32K (20 μM) and binds ATP (32 μM) with slightly weaker, but comparable affinity, to ADP. (e) Determination of the thermal stabilities and relative binding affinities of non-phosphorylated Src^32KΔC L255A for nucleotides by differential scanning fluorimetry. Src^32KΔC L255A binds ATP with higher affinity (100 μM) than ADP (310 μM), while ATP also confers an additional 1.3 °C of thermal stability over ADP.

Figure 4

Nucleotide exchange permits trans-autophosphorylation. (a) Itk^32K autophosphorylation. Itk^32K and Itk^32K L351A were mixed with Mg²⁺ and ATP and allowed to autophosphorylate over time. Autophosphorylation was monitored with a specific antibody against the phosphorylated activation loop (pTyr511) and the results quantitated from the chemiluminescent signal. Itk^32K autophosphorylates on Tyr511 very slowly compared to Itk^32K L351A, which has a half time for maximal autophosphorylation of ~93 min. Under the same conditions, Itk^32K did not achieve maximal autophosphorylation. Full-length blots can be found in Supplementary Fig. 8a-b. (b) Src^32K autophosphorylation. Src^32K, Src^32KΔC, and Src^32K L255A were mixed with Mg²⁺ and ATP and allowed to autophosphorylate over time. Autophosphorylation was monitored with a specific antibody against the phosphorylated activation loop (pTyr416) and the results quantitated from the chemiluminescent signal. Src^32K autophosphorylates on Tyr416 with a half time for maximal autophosphorylation of ~70 min. Under the same conditions, Src^32KΔC, which lacks its regulatory C-terminal tail phosphorylated itself on Tyr416 approximately 1.8-fold faster. Src^32KΔC L255A, lacking its C-terminal tail and mutated in its hydrophobic stack, phosphorylates almost 6 times faster than Src^32KΔC and more than 10 times faster than Src^32K. Full-length blots can be found in Supplementary Fig. 8c-e. (c) Kinase activity assay of Itk^32K against an exogenous Src substrate (Cdk1 aa 6-20). Itk^32K is a very poor kinase against the peptide substrate, whereas Itk^32K L351A readily phosphorylates it. (d) Kinase activity assay of Src^32K against an exogenous Src substrate (Cdk1 aa 6-20). Removal of the C-terminal tail (Src^32KΔC) results in a modest activation of Src^32K compared with the dramatic activation seen with the hydrophobic stack mutant (Src^32KΔC L255A).

Figure 5

Model for the allosteric activation of Src and Tec family kinases. Src and Tec kinases adopt a closed, autoinhibited conformation in the cytosol of unstimulated cells stabilized by ADP. Activation of receptor tyrosine kinases in the membrane leads to the phosphorylation of motifs in their cytoplasmic domains and the consequent recruitment of phosphotyrosine-binding SH2-domain containing proteins, including Src and Tec kinases. Engagement of SH2 and SH3 binding motifs in the receptor or adaptor proteins leads to activation of these kinases. Displacement of the SH3 domain from contacts with the N-terminal lobe of the kinase domain disrupts a hydrophobic stack that stabilizes the inactive state, leading to a conformational change that drives the exchange of ADP for ATP, trans-autoactivation, and downstream substrate phosphorylation. Additional regulatory adaptations restrict Itk activity to PIP₃-containing membranes on account of its regulatory PH domain, while the C-terminal tail of Src prevents spurious activation at the membrane. (yellow, αC helix; orange, hydrophobic stack; blue, activation loop; red, ADP; green, ATP; Φ, hydrophobic amino acid).