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

Abstract 861: Selective small-molecule kinase inhibitors of the c-Src:FAK complex

Abstract The c-Src protein-tyrosine kinase is the prototype of a large non-receptor kinase family involved in a variety of signaling pathways in virtually every cell type. In normal cells, c-Src controls cell-cell adhesion, cell-matrix adhesion, and cell motility by interacting with and phosphorylating focal adhesion and extracellular matrix (ECM) proteins. Focal adhesion kinase (FAK), a key component of focal adhesions, activates c-Src by binding to its SH2 and SH3 domains and disrupting their negative 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. Both c-Src and FAK are over-expressed and constitutively active in many tumor types, enhancing focal adhesion turnover, cell migration and metastatic potential. The goal of our project is to determine whether distinct conformational changes occur in the c-Src kinase domain as a function of binding to FAK that can be selectively targeted with small molecule inhibitors. Using the FRET-based Z’-Lyte in vitro kinase assay, we first identified assay conditions where recombinant, downregulated c-Src activation was dependent on a synthetic phosphopeptide based on the FAK SH3- and SH2-binding motifs for c-Src. We then screened a chemical library of about 600 kinase-biased inhibitors for compounds that preferentially inhibited FAK-peptide-dependent c-Src activation compared to c-Src alone. Remarkably, 13 compounds showed at least five-fold selectivity for the FAK peptide:Src complex in the primary screen. Several of these compounds are of the Type II kinase inhibitor class and may exploit unique conformations of c-Src that are induced by FAK peptide binding to the SH3 and SH2 region of c-Src. These compounds are currently being evaluated for their impact on cell growth, invasion and migration in both fibroblasts and epithelial tumor cell lines. Discovery and development of selective inhibitors for a unique FAK-induced c-Src conformation may provide a new approach to targeted therapy for metastatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 861. doi:1538-7445.AM2012-861</jats:p

Mitochondrial ClpX activates an essential metabolic enzyme through partial unfolding

ABSTRACTMitochondria can control the activity, quality, and lifetime of their proteins with their autonomous system of chaperones, but the signals that direct substrate-chaperone interaction and outcome 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 incorporation of cofactor. Here, we ask how unfolding by mtClpX directs activation. We identified sequence and structural features in ALAS that position mtClpX and provide a grip for acting on ALAS. Observation of ALAS undergoing remodeling by mtClpX revealed that unfolding was limited to a subdomain extending from the mtClpX-binding site to the active site. Unfolding along this path was required for mtClpX to gate cofactor access to the ALAS active site. This targeted unfolding contrasts with the global unfolding canonically executed by ClpX homologs and suggests how substrate-chaperone interactions can direct the outcome of remodeling.</jats:p

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

Abstract B190: Allosteric modulation of Src family kinases via SH3 domain displacement

Abstract The Src family of non-receptor protein-tyrosine kinases (SFKs) plays a crucial role in cancer progression by affecting multiple signaling pathways linked to tumor cell adhesion, invasion and survival. Recent work from our group and others has shown that individual Src-family members may have unique and in some cases opposing roles in the regulation of cell growth and differentiation. Discovery and development of SFK isoform-selective inhibitors and agonists is therefore essential for the most effective therapeutic targeting of this ubiquitous kinase family. Current SFK inhibitors target the ATP-binding site of the kinase domain or an allosteric pocket nearby. However, the highly conserved nature of the active site across the Src kinase family limits identification of isoform-selective compounds. One possible solution to this problem involves small molecule modulation of SFK activity via the regulatory SH3 and SH2 domains. SFK activity is tightly regulated by intramolecular interactions of the SH3 domain with the SH2:kinase linker and the SH2 domain with the phosphorylated C-terminal tail. Small molecules that either enhance or disrupt these interactions are anticipated to result in kinase inhibition or activation, respectively. Recently, we reported that binding of a short proline-rich peptide to the SH3 domains of several Src-family members is sufficient to induce kinase activation in vitro. This peptide, known as VSL12 (VSLARRPLPLP), was postulated to disrupt the regulatory SH3:linker interaction in the downregulated kinase. In the present study, we used VSL12 as probe to better understand the structural basis of kinase activation by SH3 displacement. We first compared the affinity and kinetics of the VSL12 peptide interaction with SFK SH3 domains vs. the near full-length kinase proteins using surface plasmon resonance. We found that VSL12 bound to the isolated SH3 domains of Src, Hck, Lyn and Fyn with similar micromolar potencies. Surprisingly, the KD values for VSL12 binding to the corresponding near-full-length kinases were in the low nanomolar range, suggesting that the peptide makes additional contacts with residues outside of the SH3 domain that result in the stable, active complex. This idea was tested further using differential scanning fluorimetry to explore the thermal stability of the protein:peptide complexes. Addition of VSL12 enhanced the thermal stability of a recombinant Src SH3-SH2-linker protein, consistent with increased molecular order. However, VSL12 decreased the thermal stability of near full-length c-Src, which may reflect conformational changes in the kinase domain that accompany kinase activation. Addition of the kinase domain inhibitor dasatinib restored thermal stability of the Src:VSL12 complex. Complexes of the Src SH3-SH2-linker protein with the VSL12 peptide readily formed crystals that diffract X-rays to 2.6 Å resolution, providing further support for the stabilization of the complex by VSL12 binding. Determining the structures of the Src SH3-SH2-linker protein and the near-full-length kinase in complex with VSL12 will provide fresh insight as to the mechanism of c-Src activation by SH3 domain displacement and aid in the design of potent and selective non-peptide allosteric modulators. Citation Format: Heather L. Rust, Jamie A. Moroco, John J. Alvarado, John J. Engen, Thomas E. Smithgall. Allosteric modulation of Src family kinases via SH3 domain displacement. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B190.</jats:p

Abstract 2377: The AML linked Src family kinase Fgr is uncoupled from SH2 and SH3 domain regulation and drives oncogenic transformation

Abstract Fgr is a member of the Src family of non-receptor protein-tyrosine kinases, which are over-expressed and constitutively active in many human cancers. Fgr expression is restricted to myeloid hematopoietic cells and strongly upregulated in a subset of primary human AML bone marrow samples, suggesting that Fgr kinase activity may contribute to AML pathogenesis. To investigate the oncogenic potential of Fgr, we expressed the wild-type kinase in Rat-2 fibroblasts and scored transformation in soft-agar colony-forming assays. Remarkably, Fgr expression resulted in strong transforming activity, suggesting that over-expression of the kinase is sufficient to overcome regulatory control by its SH2 and SH3 domains. To test this idea, we mutated the negative regulatory tail tyrosine of Fgr. Phosphorylation of the homologous tyrosines in c-Src, Hck and other Src-family members by the regulatory kinase Csk causes intramolecular engagement of the SH2 domain to suppress kinase activity. Substitution of the Fgr tail tyrosine with phenylalanine did not further enhance the transforming or kinase activity of Fgr in Rat-2 cells, suggesting that its kinase domain is uncoupled from regulation by its non-catalytic SH3-SH2 region. To explore the regulatory mechanism further, we expressed near-full-length Fgr in insect cells, purified it to homogeneity, and confirmed that it was singly phosphorylated on its negative regulatory tail (and not the activation loop). Hydrogen-deuterium exchange mass spectrometry demonstrated that the SH3 and SH2 domains are protected from deuterium uptake as observed previously for recombinant, downregulated Hck, suggesting that they are packed against the back of the kinase domain. We then tested recombinant Fgr for sensitivity to activation in vitro using peptide ligands for the SH3 and SH2 domains, as well as a peptide that engages both domains simultaneously. Fgr kinase activity was unaffected by these peptides, providing further evidence that the kinase domain is not allosterically coupled to SH3 and SH2 control. In contrast, Hck and c-Src were both stimulated by these peptides, consistent with a domain displacement mechanism of activation described previously. Control experiments using surface plasmon resonance spectroscopy confirmed that the SH3 and SH2 domains of both Hck and Fgr bind to each of the peptide ligands with similar kinetics and affinity. Taken together, our data show that Fgr is a unique Src family member in that its kinase domain is not subject to regulatory domain control despite evidence for intramolecular interactions with the SH3 and SH2 domains. As a consequence, simple over-expression of Fgr is sufficient to induce transformation of rodent fibroblasts, unlike Hck or other Src family members. By extension, over-expression of Fgr may contribute to AML development and selective targeting of its kinase activity may be of therapeutic benefit. Citation Format: Kexin Shen, Heather R. Dorman, Haibin Shi, Ravi K. Patel, Jamie A. Moroco, John R. Engen, Thomas E. Smithgall. The AML linked Src family kinase Fgr is uncoupled from SH2 and SH3 domain regulation and drives oncogenic transformation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2377. doi:10.1158/1538-7445.AM2017-2377</jats:p

K_m Values for ATP and Peptide Substrate for Near-Full-Length SFKs.

<p>The K_m values for ATP and the substrate peptide, YIYGSFK, were determined for each SFK-YEEI protein using the ADP Quest assay as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105629#s4" target="_blank">Materials and Methods</a>. ATP experiments were performed three times for each kinase and substrate experiments were performed four times for each kinase, except for Src-YEEI, where ATP experiments were performed twice and substrate experiments were performed three times. Mean values are shown for each kinetic constant ±S.E.</p