Tania M. Palhano
Zanela
a,
Milad
Zangiabadi
b,
Yan
Zhao
b and
Eric S.
Underbakke
*a
aRoy J. Carver Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011, USA. E-mail: esu@iastate.edu
bDepartment of Chemistry, Iowa State University, Ames, Iowa 50011, USA
First published on 13th March 2024
Pyk2 is a multi-domain non-receptor tyrosine kinase that serves dual roles as a signaling enzyme and scaffold. Pyk2 activation involves a multi-stage cascade of conformational rearrangements and protein interactions initiated by autophosphorylation of a linker site. Linker phosphorylation recruits Src kinase, and Src-mediated phosphorylation of the Pyk2 activation loop confers full activation. The regulation and accessibility of the initial Pyk2 autophosphorylation site remains unclear. We employed peptide-binding molecularly imprinted nanoparticles (MINPs) to probe the regulatory conformations controlling Pyk2 activation. MINPs differentiating local structure and phosphorylation state revealed that the Pyk2 autophosphorylation site is protected in the autoinhibited state. Activity profiling of Pyk2 variants implicated FERM and linker residues responsible for constraining the autophosphorylation site. MINPs targeting each Src docking site disrupt the higher-order kinase interactions critical for activation complex maturation. Ultimately, MINPs targeting key regulatory motifs establish a useful toolkit for probing successive activational stages in the higher-order Pyk2 signaling complex.
Fig. 1 Tyrosine kinase regulatory features probed by MINPs (A) Domain organization of Pyk2, FAK, and Src kinase. Proline-rich regions (PRR) and phosphoacceptor tyrosines are annotated with linker autophosphorylation sites in bold. (B) Cartoon representation of peptide templated MINP preparation. Mixed micelles with functional monomers are cross-linked around peptide (yellow and blue) to afford a binding pocket with shape and functional group complementarity.8 (C) Schematic model for MINP-mediated inhibition of Pyk2 autophosphorylation. (D) Pyk2 FERM-kinase construct highlighting linker sequences used for MINP imprinting. |
Despite activation differences, key regulatory mechanisms are shared between Pyk2 and FAK. Both kinases are autoinhibited by engagement of the FERM domain with the kinase C-lobe.9,10 The FERM domain suppresses kinase activity by obstructing access to the kinase activation loop and substrate protein docking surface. The autoinhibitory conformation is relieved by stimuli such as integrin engagement for FAK or calcium signaling for Pyk2. Stimulated conformational changes expose the kinase active site and trigger the autophosphorylation of a key tyrosine (FAK Y397, Pyk2 Y402) in the FERM-kinase linker.1,5 The phospho-Y397 and a neighboring proline-rich region (PRR) establish a scaffold for signaling effectors like Src kinase.11,12 The engagement of the Src SH2 and SH3 domains with the FERM-kinase linker leads to Src-mediated phosphorylation of a pair of activation loop tyrosines. Phosphorylation of the activation loop ultimately enhances the catalytic activity, which in turn promotes the phosphorylation of downstream targets.
Although the mechanistic details of Pyk2 activation are obscured by limited high-resolution structural models, investigations dissecting Pyk2 regulation revealed key features.13–15 Pyk2 autophosphorylates residue Y402 independently of Src kinase activity. Autophosphorylation is sufficient for Src to dock and outcompete the dynamic FERM-kinase autoinhibitory interface to phosphorylate the Pyk2 activation loop tyrosines (Y579, Y580). These observations underscore the intrinsic dynamics of the Pyk2 autoinhibitory conformation and highlight the importance of the accessibility of linker residue Y402 for the maturation of the Pyk2–Src activation complex.
Despite the intrinsic capacity of Pyk2 for autophosphorylation, the regulatory mechanisms governing activation remain unclear. In FAK, the autophosphorylation site Y397 and surrounding residues are constrained in an antiparallel strand of a small FERM domain beta sheet.9 The neighboring proline-rich region also makes intramolecular contacts with the FERM domain.16 Indeed, perturbations to the FAK FERM domain beta sheet (e.g., K38A) impact autophosphorylation rate.17 To date, it is unknown whether the autophosphorylation site of Pyk2 is also constrained via secondary structure. Additionally, the regulatory contributions of the Src SH2 and SH3 domains in the Pyk2 activation complex are unclear.
To gain insight into the dynamics and signaling scaffolding of the Pyk2 autophosphorylation site, we employed chemical probes and biochemical characterizations, including peptide-binding molecularly imprinted nanoparticles (MINPs), mutagenesis, and activity profiling. MINPs are nanoparticle probes prepared from cross-linkable micelles containing template molecules such as peptides. The MINP micelles include diverse functional monomers presenting chemical moieties that provide specific interactions with template peptide features (Fig. 1B and Scheme S1, ESI†). Cross-linking of the micelles generates a solid particle with docked template peptide nestled into a stable pocket lined with interaction moieties. Template removal yields cavities (i.e., imprinted sites) with shapes and interaction networks complementary to the target peptide.18,19 The resultant “plastic antibodies” can bind peptides with high affinity (i.e., low nanomolar dissociation constants) and distinguish closely related amino acids such as leucine/isoleucine,18 aspartate/glutamate,20 and lysine/arginine.21 The capacity for exquisite specificity enables MINPs to inhibit specific post-translational modifications8,22 or probe the functional role of a specific peptide sequence.19 Hence, we investigated the mechanisms regulating Pyk2 activation by leveraging the distinctive features of MINPs to selectively target and probe specific Pyk2 regulatory features (Fig. 1C).
Ultimately, our study provides new insights into Pyk2 regulation by revealing that the Y402 site is conformationally constrained in the autoinhibited state due to the formation of a short beta-sheet with the FERM domain. Disruption of this regulatory substructure results in trans autophosphorylation of the Y402 site, allowing Src docking and subsequent Src-mediated activation loop phosphorylation of Pyk2. Our findings clarify mechanisms of Pyk2 regulation and activation while demonstrating that MINPs can serve as effective conformational probes and chemical tools to investigate protein–protein interactions.
In this study, we sought to explore the correlation between MINP inhibitor potency and the accessibility of the Y402 site in the Pyk2 basal state. We first examined an AlphaFold-derived model of the autoinhibitory conformation of Pyk224,25 (Fig. 2A). The AlphaFold model of Pyk2 recapitulated a beta strand sequestering the autophosphorylation site to the FERM domain as observed in the autoinhibited FAK model derived by X-ray crystallography.9 However, the Pyk2 Y402 beta strand exhibited an inverted (i.e., parallel) orientation and a very low confidence score in AlphaFold modeling. Our efforts to generate new models of autoinhibited Pyk2 using different versions of AlphaFold on a local server failed to reproduce the antiparallel, FERM-engaged beta strand observed in FAK. The apparent modeling ambiguity further motivated our investigations into autophosphorylation site accessibility in Pyk2.
Comparisons of Pyk2 AlphaFold models and the reported FAK structure enabled us to predict residues putatively responsible for stabilizing the beta sheet and sequestering the autophosphorylation site. We assessed residue-specific contributions to the stabilization of the putative interaction between the FERM domain and the FERM-kinase linker by testing a disruptive variant E404P, predicted to enhance the autophosphorylation rate. Surprisingly, we found that E404P Pyk2 variant exhibits an autophosphorylation rate similar to WT Pyk2 FERM-kinase (Fig. 2B and Fig. S1, ESI†). We considered two possible explanations for this observation. Pyk2 regulation may differ from FAK, and Y402 sequestration does not limit basal autophosphorylation. Alternatively, the local structure constraining Y402 may be stabilized by multiple cooperative interactions and/or the E404P substitution is insufficient to disrupt and liberate the linker. Importantly, kinase specificity for the local autophosphorylation motif remained a factor, and further residue substitutions risked perturbating recognition by the kinase active site.
To further interrogate the regulatory role of autophosphorylation site sequestration, we tested the inhibitory effects of site-specific MINPs. We assessed whether the E404P variant would present a more accessible binding site for MINP(Y402) and thus enhance inhibitory potency (Fig. 2C and Fig. S2, ESI†). In vitro kinase assays of the WT and E404P Pyk2 FERM-kinase revealed that MINP(Y402) inhibited the autophosphorylation of the E404P Pyk2 variant far more effectively than WT Pyk2. Specifically, at a Pyk2:MINP ratio of 1:6, we observed near-complete inhibition of autophosphorylation of the E404P variant of Pyk2, while the wild-type (WT) Pyk2 phosphorylation was inhibited by only 30%. A non-imprinted nanoparticle (NINP), prepared without the template, exhibited negligible inhibition. The enhanced inhibitory potency of MINP(Y402) towards the E404P Pyk2 variant supports the hypothesis that the Y402 site and surrounding residues are constrained in the Pyk2 basal state. Thus, despite the sequence mismatch generated by the E404P Pyk2 mutation, the local structural perturbation in the Pyk2 FERM-kinase linker affords a better binding site for MINP(Y402). Based on the observed differential impact of MINP inhibition between the linker targets, we inferred that MINPs can differentiate between local polypeptide accessibility and/or conformations. Notably, the putative β-strand conformation would be uniquely resistant to MINP recognition due to its fully extended torsion angles that differ significantly from the free, flexible peptide template imprinted on the MINP. This observation suggests that the MINP templating strategy can be leveraged to differentiate local polypeptide conformation or accessibility.
The canonical mechanism for full activation of autophosphorylated Pyk2 involves Src docking and subsequent activation loop phosphorylation at Pyk2 residues Y579 and Y580.14,15,26 Given the pronounced increase in Pyk2 autophosphorylation upon disruption of the putative FERM:linker interface, we tested whether a K60 variant also impacted the phosphorylation status of the activation loop. We monitored site-specific phosphorylation in WT and K60P Pyk2 FERM-kinase. Blotting with site-specific antibodies revealed that the autophosphorylation target of the K60P variant was primarily the FERM-kinase linker residue Y402 (Fig. 3B and Fig. S4, ESI†). Like WT Pyk2, the K60P variant exhibited negligible autophosphorylation of its own activation loop.
Our results show that variants designed to disrupt FERM-kinase linker sequestration can increase MINP access or promote autophosphorylation. We surmised that the disruptive variants increase conformational accessibility of the linker autophosphorylation site. However, perturbing remote FERM:linker contacts could also destabilize the autoinhibitory interface between FERM F2 and kinase C-lobe subdomains (Fig. 2A).9,10 Indeed, previous studies established that disruption of the Pyk2 FERM F2:kinase C-lobe interface leads to increased autophosphorylation.15 Hence, we sought to test whether the K60P mutation disrupts the global autoinhibitory conformation of Pyk2. We employed global hydrogen/deuterium exchange mass spectrometry (HDX-MS) to monitor the general uptake of deuterium throughout the intact protein. Our results revealed negligible differences in global exchange between WT and K60P Pyk2 FERM-kinase (Fig. 3C). In contrast, targeted disruption of the FERM F2:kinase C-lobe autoinhibitory interface has been shown to increase global H/D exchange by 20–40 deuterons.10 Taken together, we conclude that point mutations in the FERM domain (e.g., K60P Pyk2) disrupt main chain H-bonding and ion pairing, liberating the linker segment harboring autophosphorylation site Y402. Interestingly, when challenged to predict the architecture of the K60P variant of Pyk2, AlphaFold consistently generates models with a disengaged, conformationally flexible FERM-kinase linker (Fig. 3D). Integrating MINP and variant activity with HDX-MS, we propose that linker liberation can enhance autophosphorylation without perturbing the dynamics of the autoinhibitory FERM-kinase conformation.
Interested in targeted strategies to interfere with specific protein–protein signaling interactions,29 we investigated the potential of MINPs to selectively target Pyk2 docking sites and disrupt the formation of the Pyk2–Src activation complex. Specifically, we hypothesized that MINPs imprinted with either phospho-Y402 sequence or PRR sequence (Fig. 4C) could outcompete the tandem Src motifs for Pyk2 binding. For this purpose, we assessed the inhibition of Src-mediated phosphorylation of Pyk2 activation loop tyrosines (Y579/Y580) in the presence of MINP(pY402) and MINP(PRR) alone or in combination. First, WT Pyk2 FERM-kinase was preincubated with ATP to allow for basal autophosphorylation to fully phosphorylate the Y402 site. Phospho-Y402 Pyk2 was pre-incubated with MINP or NINP followed by addition of Src. The impact of MINPs on Src-mediate Pyk2 activation loop phosphorylation was monitored using site-specific activation loop anti-phosphotyrosine (pY579/Y580) antibodies. Our results reveal that blocking either the Src SH3 or SH2 domain binding site in Pyk2 leads to substantial inhibition of Src-mediated phosphorylation of Pyk2 activation loop tyrosines (Fig. 4E and Fig. S7, ESI†). To control for the possibility that MINPs interfere with Src directly rather than blocking the targeted Pyk2 motifs, we tested Src activity in the presence of MINPs and NINP. Intrinsic Src kinase activity was unaffected by MINPs (Fig. S8, ESI†). Notably, despite similarly high binding affinities (Fig. 4C and Fig. S9, ESI†), MINP(pY402) exhibited more potent inhibition of Src activity than MINP(PRR). The combination of MINP(pY402) and MINP(PRR), however, resulted in further inhibition of Src-mediated phosphorylation of Pyk2 activation loop tyrosines (Fig. 4E). Thus, motif-specific MINP inhibition suggests that Src SH2 recognition of Pyk2 phosho-Y402 plays a critical role in nucleating the Src-Pyk2 activation complex. Nevertheless, the significant inhibition by MINP(PRR) reveals that blocking access to the Pyk2 PRR is sufficient for disruption of Src-mediated Pyk2 activation. Our results demonstrate that MINPs targeting either Src-binding motifs in Pyk2 can successfully outcompete Src domains and inhibit Src-mediated Pyk2 activation loop phosphorylation.
We also note an intriguing contrast between the activities of MINP(Y402), targeting Pyk2 autophosphorylation, and MINP(pY402), targeting Src SH2 docking via the same site modified by phosphorylation. MINP(Y402) inhibition is muted by the apparent inaccessibility of the unphosphorylated Y402 region (Fig. 2C). MINP(pY402), however, readily recognized the phosphotyrosine linker motif and potently inhibited Src activity. Given the role of ion pairing in stabilizing the sequestration of the Y402 region (Fig. 3), we speculate that Y402 phosphorylation prevents engagement with the FERM β-sheet due to local conformational changes associated with the additional negative charge. The increased accessibility of the linker upon Y402 phosphorylation may be important for Src SH2 docking.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d3cb00228d |
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