Differential sensitivity of Src-family kinases to activation by SH3 domain displacement

Abstract

Src-family kinases (SFKs) are non-receptor protein-tyrosine kinases involved in a variety of signaling pathways in virtually every cell type. The SFKs share a common negative regulatory mechanism that involves intramolecular interactions of the SH3 domain with the PPII helix formed by the SH2-kinase linker as well as the SH2 domain with a conserved phosphotyrosine residue in the C-terminal tail. Growing evidence suggests that individual SFKs may exhibit distinct activation mechanisms dictated by the relative strengths of these intramolecular interactions. To elucidate the role of the SH3:linker interaction in the regulation of individual SFKs, we used a synthetic SH3 domain-binding peptide (VSL12) to probe the sensitivity of downregulated c-Src, Hck, Lyn and Fyn to SH3-based activation in a kinetic kinase assay. All four SFKs responded to VSL12 binding with enhanced kinase activity, demonstrating a conserved role for SH3:linker interaction in the control of catalytic function. However, the sensitivity and extent of SH3-based activation varied over a wide range. In addition, autophosphorylation of the activation loops of c-Src and Hck did not override regulatory control by SH3:linker displacement, demonstrating that these modes of activation are independent. Our results show that despite the similarity of their downregulated conformations, individual Src-family members show diverse responses to activation by domain displacement which may reflect their adaptation to specific signaling environments in vivo.

Figure 1. Structural features of Src-family kinases.

A) Crystal structure of inactive c-Src (PDB: 2SRC) showing the intramolecular interactions necessary for downregulation of kinase activity. Shown are the SH3 domain (red), 3–2 connector (gray), SH2 domain (blue), SH2-kinase linker (orange), kinase domain (N-lobe, pink; C-lobe, light blue), and the C-terminal pTyr tail (cyan, pTyr527 side chain shown in green). The N-lobe αC-helix is shown in green. The SH3 domain interacts with the PPII helix formed by the linker, while the SH2 domain interacts with the pTyr tail. In the inactive state, the activation loop (purple) adopts a partially helical conformation and the autophosphorylation site (Tyr416) points inward towards the catalytic cleft. B) Sequence alignment of Src-family kinase SH3 domains and SH2-kinase linkers. The Src family can be divided into two subfamilies based on sequence homology as shown (A and B subgroups). Key hydrophobic residues that contribute to the binding surface are highlighted in bold and marked with an asterisk (their positions in the structure of the Src SH3 domain are modeled in Figure 2B). The conserved aspartate residues (Asp99 in c-Src) that contribute to VSL12 peptide binding are also bolded and marked with a †. SFK linker sequences are more diverse and display suboptimal residues at key positions that face the SH3 domain in the inactive state. The positions of linker residues that contact the SH3 domain in the inactive structure of c-Src are modeled in Figure 2B.

Figure 2. Interaction of the VSL12 peptide with the SH3 domain of c-Src.

A) Comparison of the sequence of the SH3-binding peptide VSL12 (top) with that of the c-Src SH2-kinase linker (bottom). Note that the VSL12 sequence is presented in the C- to N-terminal orientation relative to the linker. B) Comparison of c-Src SH3 domain interaction with the VSL12 peptide and the SH3-kinase linker. The NMR solution structure of the Src SH3 domain (red) with the VSL12 peptide (cyan) is modeled on the left (PDB: 1QWF) . The side chains of the SH3 domain residues that interact with VSL12 are shown in green (tyrosines 90, 92, 136 and Trp118), and interacting VSL12 side chains are shown in cyan (Pro12, Leu11, Pro9, and Leu8). The ionic contact between VSL12 Arg6 and SH3 Asp99 is also shown. The analogous interaction of the Src SH3 domain with the SH2-kinase linker from the inactive structure of c-Src is shown on the right (PDB: 2SRC) . Linker residue Pro250 contributes to SH3 interaction in the P₀ position of the linker PPII helix, while Gln253 occupies the P₊₃ position and is rotated away from the SH3 surface. The position of Lys257 is also shown; it does not contact Asp99 in this structure.

Figure 3. Surface plasmon resonance (SPR) analysis of SH3 interactions with the VSL12 peptide.

SPR was used to evaluate VSL12 peptide binding kinetics and affinity for the isolated SH3 domains of c-Src, Hck, Fyn and Lyn as indicated. The biotinylated peptide was immobilized to 80 Response Units (RU) on the surface of a streptavidin (SA) biosensor chip. The recombinant purified Src SH3 domain proteins were flowed past the peptide over the concentration ranges shown. Association was measured for 180 s, followed by a 300 s dissociation phase. Each panel shows a representative sensorgram, with the double-referenced binding data (black traces) fit to a 1∶1 Langmuir binding model (red trace). Kinetic constants derived from this experiment are presented in Table 1.

Figure 4. Linear relationship between SFK-YEEI activity and kinase protein input.

A) Representative time course of ADP production for six concentrations of Src-YEEI. At higher kinase concentrations, the reaction rates plateau as the fluorescence reading reaches saturation. The linear portion of each curve was fit by linear regression analysis to provide the slope, which corresponds to the rate of the reaction in pmol ADP produced/min. B) Reaction rates for each SFK-YEEI protein are plotted against input kinase concentration. Curves were best-fit by linear regression analysis (dotted lines) and used to estimate the specific activity for each kinase (inset).

Figure 5. Differential sensitivity of individual SFK-YEEI proteins to activation by SH3 domain displacement.

Each of the SFK-YEEI proteins shown was assayed in the presence of VSL12 over a range of concentrations (0.1 to 300 µM). ATP and substrate concentrations were set to the K_m for each kinase, and input kinase concentrations were set to achieve a basal reaction velocity of 1 pmol ADP produced/min. A) Each of the kinases is activated by VSL12 in a concentration-dependent manner. Plots of reaction velocity vs. VSL12 concentration were best-fit by the Michaelis-Menten equation, allowing for the determination of the V_max. Each data point was assayed in triplicate and is shown as the mean ±S.E. B) Comparison of basal rate (left) and V_max (right) for each kinase in the presence of VSL12. Bars heights correspond to the mean values from triplicate experiments ±S.E.

Figure 6. Src-YEEI and Hck-YEEI retain sensitivity to activation by SH3 domain displacement after autophosphorylation.

Src-YEEI (A) and Hck-YEEI (B) were incubated in the absence or presence of ATP (at the K_m) for 3 hours at 25°C to induce autophosphorylation of Tyr416 in the activation loop. The autophosphorylated kinases were then assayed for responsiveness to the SH3-binding peptide, VSL12. C) Autophosphorylation of Src-YEEI and Hck-YEEI was measured in response to the VSL12 peptide by running the kinase reactions in the absence of the substrate peptide. For all three panels, the basal reaction velocity (no VSL12) was subtracted from the rate at each VSL12 concentration, and the resulting linear reaction velocities were plotted as a function of VSL12 concentration as shown. Each data point was assayed in triplicate and the average value is plotted ±S.E.

Figure 7. SFK linker mutants display higher basal kinase activity than their wild-type counterparts and are refractory to activation by VSL12.

A) Sequences of the wild-type (WT) linkers of Src and Hck are shown. Residues involved in intramolecular engagement of the SH3 domain are highlighted in bold, and are replaced with alanines in the respective Src-3A and Hck-2A mutants as shown. B) Reaction velocities for equivalent amounts (125 ng/well) of Src-YEEI and Hck-YEEI with wild-type vs. mutant linkers were determined using the ADP Quest assay. Results are shown as the mean velocity for three replicate determinations ±S.E. C) Each of the SFK-YEEI proteins shown was assayed in the presence of VSL12 over a range of concentrations (0 to 300 µM). ATP and substrate concentrations were set to the K_m for each wild-type kinase, and input kinase concentrations were set to achieve a basal reaction velocity of 1 pmol ADP produced/min. Plots of reaction velocity vs. VSL12 concentration were best-fit by the Michaelis-Menten equation for the wild-type kinases, indicative of saturable activation kinetics by VSL12. Each data point was assayed in triplicate and is plotted as the mean velocity ±S.E.