DIVERSITY IN SRC-FAMILY KINASE ACTIVATION MECHANISMS: IMPLICATIONS FOR SELECTIVE INHIBITOR DISCOVERY

The Src kinase family encompasses eight non-receptor protein tyrosine kinases in mammals that regulate signaling pathways in virtually every cell type. Src-family kinases (SFKs) share a common regulatory mechanism that requires two intramolecular interactions to maintain the inactive state. These involve binding of the SH3 domain and a PPII helix in the SH2-kinase linker and interaction of the SH2 domain and a phosphotyrosine residue in the C-terminal tail. To compare the activation dynamics of the individual SFKs, a synthetic SFK SH3 domain-binding peptide (VSL12) was used to probe the sensitivity of SFKs to SH3-based activation. Surface plasmon resonance was used to confirm equivalent binding of the VSL12 peptide to the SH3 domains and near-full-length kinases. SFKs were tested with VSL12 in a kinetic kinase assay to measure the changes in the rate of activity induced by SH3:linker displacement. All SFKs tested were susceptible to activation, but to varying degrees. Further, autophosphorylation of the c-Src and Hck activation loops prior to VSL12-induced activation revealed that c-Src can achieve a higher level of activation if primed before SH3 domain displacement. These results suggest that distinct activation thresholds may exist for individual SFKs. To apply these findings in an inhibitor discovery setting, the interaction of c-Src with focal adhesion kinase (FAK) was studied. FAK activates c-Src by binding to its SH2 and SH3 domains and disrupting their regulatory influence on the kinase domain. The c-Src:FAK complex plays a major role in the migration and invasion of both normal and cancer cells, making it an attractive target for drug discovery. To test the idea that FAK binding induces allosteric changes in the kinase domain active site, a screening assay was developed to target these changes with small molecule inhibitors. Assay conditions were identified where c-Src activity was dependent on a phosphopeptide encompassing the FAK SH3- and SH2-binding motifs for c-Src. A focused library of kinase-biased inhibitors was screened to identify compounds displaying selectivity for the c-Src:pFAK peptide complex. An aminopyrimidinyl carbamate, WH-4-124-2, was discovered that showed five-fold selectivity for the complex. Molecular docking studies of this inhibitor on the kinase domain of Lck bound to imatinib suggest that WH-4-124-2 is a “Type II” kinase inhibitor that prefers the unphosphorylated, “DFG-out” conformation of the kinase. Selective inhibitors of a specific FAK-induced c-Src conformation may provide a unique approach to selective targeting of this key cancer cell signaling pathway

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

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. © 2014 Moroco et al

Mitochondrial ClpX activates an essential biosynthetic enzyme through partial unfolding

© 2020, eLife Sciences Publications Ltd. All rights reserved. Mitochondria control the activity, quality, and lifetime of their proteins with an autonomous system of chaperones, but the signals that direct substrate-chaperone interactions and outcomes are poorly understood. We previously discovered that the mitochondrial AAA+ protein unfoldase ClpX (mtClpX) activates the initiating enzyme for heme biosynthesis, 5-aminolevulinic acid synthase (ALAS), by promoting cofactor incorporation. Here, we ask how mtClpX accomplishes this activation. Using S. cerevisiae proteins, we identified sequence and structural features within ALAS that position mtClpX and provide it with a grip for acting on ALAS. Observation of ALAS undergoing remodeling by mtClpX revealed that unfolding is limited to a region extending from the mtClpX-binding site to the active site. Unfolding along this path is required for mtClpX to gate cofactor binding to ALAS. This targeted unfolding contrasts with the global unfolding canonically executed by ClpX homologs and provides insight into how substrate-chaperone interactions direct the outcome of remodeling

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

<p>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.</p

Structural features of Src-family kinases.

<p>A) Crystal structure of inactive c-Src (PDB: 2SRC) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105629#pone.0105629-Xu1" target="_blank">[8]</a> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105629#pone-0105629-g002" target="_blank">Figure 2B</a>). 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105629#pone-0105629-g002" target="_blank">Figure 2B</a>.</p

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

<p>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).</p

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

<p>A) Comparison of the sequence of the SH3-binding peptide VSL12 (<i>top</i>) with that of the c-Src SH2-kinase linker (<i>bottom</i>). 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) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105629#pone.0105629-Feng1" target="_blank">[21]</a>. 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) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105629#pone.0105629-Xu1" target="_blank">[8]</a>. 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.</p

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

<p>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.</p

Binding Affinities of SFK SH3 Domains for the VSL12 Peptide as Measured by SPR.

<p>Analyses were performed with biotinylated VSL12 peptide bound to a streptavidin biosensor chip as described under Materials and Methods. Each protein was flowed past the chip surface over the concentration ranges indicated in the footnotes. Duplicate runs were performed for each concentration. A control cycle of buffer only was subtracted from all concentrations of reference-subtracted curves. To calculate the kinetic K_D, interaction data were curve-fit using a 1∶1 Langmuir model, with binding constants and chi-squared values calculated using the BIAevaluation software. To calculate the steady state K_D, the analyte response at equilibrium was plotted against the analyte concentration, and resulting curves were fit with the steady state model in the BIAevaluation software.</p>a<p>31.25, 62.5, 125, 250, 500, 1000 nM.</p>b<p>31.25, 62.5, 125, 250 nM.</p>c<p>31.25, 62.5, 125, 250, 500 nM.</p>d<p>SH2 domains tested: Src, Hck, Fyn and Lyn; kinase domains tested, Src and Hck. ND, binding not detected with 1 µM protein input.</p