Activation Loop Phosphorylation Research Articles

Abstract 5782: Probing monomer - dimer and conformational transitions of PLK1 using catalytic and polo-box domain inhibitors

Abstract Polo Like Kinase 1 (PLK1), a sought-after target for drug discovery, is an essential mitotic kinase consisting of a catalytic domain (KD) and a polobox domain (PBD), the latter of which plays roles in substrate recognition, subcellular localization and further acting as an autoinhibitory regulatory domain. PLK1 can exist either in an inhibited closed conformation or an open active state allowing interaction with and phosphorylation of substrates. PLK1 has also been shown to be regulated by interchange between monomeric and dimeric forms through binding to the Bora protein and by activation loop phosphorylation. We have described PBD-binding molecules termed abbapolins that inhibit cellular phosphorylation of a PLK1 substrate and induce loss of intracellular PLK1. In addition, due to their engagement of the cryptic pocket (ligand induced) of the PBD, we proposed that abbapolins would interfere with formation of PLK1 dimers. Here we provide evidence further supporting the ability of abbapolins to block PLK1 cellular dimerization and that this is related to their mechanism of anti-proliferative activity. In a separate study, using a cellular thermal shift assay, KD inhibitors were shown to decrease soluble PLK1, indicating that catalytic-site binding results in less stable PLK1 suggestive of an open conformation. We have extended these observations using a sensitive readout of cellular target engagement employing a Microtag overexpressed PLK1. In this format, we observe heat-induced destabilization of PLK1 upon nanomolar engagement by two lead KD inhibitors, BI2536 and onvansertib, currently being clinically evaluated. Furthermore, cyclapolin 9, a low molecular weight kinase inhibitor that, unlike the other two KD inhibitors does not protrude from the active site, did not induce significant destabilization. We have also carried out experiments using a fluorescently labeled version of BI2536 to probe the conformational effects of PBD binding upon the KD. These data solidify the observations that relief of autoinhibited PLK1 is induced by certain KD binders and highlight the conformational perturbations from KD versus PBD binding. These observations have implications for the development of ATP-competitive PLK1 inhibitors because catalytic inhibitors may conversely promote PLK1 non-catalytic functions, which may explain their lack of clinical efficacy to date. Furthermore, the demonstration that the abbapolins are mechanistically unique in terms of blocking the dimerization of PLK through the PBD provides impetus for their development as next generation PLK1 inhibitors. Citation Format: Mourad Sanhaji, Monika Raab, George Merhej, Chintada Nageswara Rao, Ivan Babic, Elmar Nurmemmedov, Klaus Strebhardt, MIchael Wyatt, Campbell McInnes. Probing monomer - dimer and conformational transitions of PLK1 using catalytic and polo-box domain inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5782.

Molecularly imprinted nanoparticles reveal regulatory scaffolding features in Pyk2 tyrosine kinase.

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.

Autoregulation of the LIM kinases by their PDZ domain

LIM domain kinases (LIMK) are important regulators of actin cytoskeletal remodeling. These protein kinases phosphorylate the actin depolymerizing factor cofilin to suppress filament severing, and are key nodes between Rho GTPase cascades and actin. The two mammalian LIMKs, LIMK1 and LIMK2, contain consecutive LIM domains and a PDZ domain upstream of the C-terminal kinase domain. The roles of the N-terminal regions are not fully understood, and the function of the PDZ domain remains elusive. Here, we determine the 2.0 Å crystal structure of the PDZ domain of LIMK2 and reveal features not previously observed in PDZ domains including a core-facing arginine residue located at the second position of the ‘x-Φ-G-Φ’ motif, and that the expected peptide binding cleft is shallow and poorly conserved. We find a distal extended surface to be highly conserved, and when LIMK1 was ectopically expressed in yeast we find targeted mutagenesis of this surface decreases growth, implying increased LIMK activity. PDZ domain LIMK1 mutants expressed in yeast are hyperphosphorylated and show elevated activity in vitro. This surface in both LIMK1 and LIMK2 is critical for autoregulation independent of activation loop phosphorylation. Overall, our study demonstrates the functional importance of the PDZ domain to autoregulation of LIMKs.

Structural basis of a redox-dependent conformational switch that regulates the stress kinase p38α

Many functional aspects of the protein kinase p38α have been illustrated by more than three hundred structures determined in the presence of reducing agents. These structures correspond to free forms and complexes with activators, substrates, and inhibitors. Here we report the conformation of an oxidized state with an intramolecular disulfide bond between Cys119 and Cys162 that is conserved in vertebrates. The structure of the oxidized state does not affect the conformation of the catalytic site, but alters the docking groove by partially unwinding and displacing the short αD helix due to the movement of Cys119 towards Cys162. The transition between oxidized and reduced conformations provides a mechanism for fine-tuning p38α activity as a function of redox conditions, beyond its activation loop phosphorylation. Moreover, the conformational equilibrium between these redox forms reveals an unexplored cleft for p38α inhibitor design that we describe in detail.

Phosphorylation-dependent pseudokinase domain dimerization drives full-length MLKL oligomerization

The necroptosis pathway is a lytic, pro-inflammatory mode of cell death that is widely implicated in human disease, including renal, pulmonary, gut and skin inflammatory pathologies. The precise mechanism of the terminal steps in the pathway, where the RIPK3 kinase phosphorylates and triggers a conformation change and oligomerization of the terminal pathway effector, MLKL, are only emerging. Here, we structurally identify RIPK3-mediated phosphorylation of the human MLKL activation loop as a cue for MLKL pseudokinase domain dimerization. MLKL pseudokinase domain dimerization subsequently drives formation of elongated homotetramers. Negative stain electron microscopy and modelling support nucleation of the MLKL tetramer assembly by a central coiled coil formed by the extended, ~80 Å brace helix that connects the pseudokinase and executioner four-helix bundle domains. Mutational data assert MLKL tetramerization as an essential prerequisite step to enable the release and reorganization of four-helix bundle domains for membrane permeabilization and cell death.

Structure shows that the BIR2 domain of E3 ligase XIAP binds across the RIPK2 kinase dimer interface.

RIPK2 is an essential adaptor for NOD signalling and its kinase domain is a drug target for NOD-related diseases, such as inflammatory bowel disease. However, recent work indicates that the phosphorylation activity of RIPK2 is dispensable for signalling and that inhibitors of both RIPK2 activity and RIPK2 ubiquitination prevent the essential interaction between RIPK2 and the BIR2 domain of XIAP, the key RIPK2 ubiquitin E3 ligase. Moreover, XIAP BIR2 antagonists also block this interaction. To reveal the molecular mechanisms involved, we combined native mass spectrometry, NMR, and cryo-electron microscopy to determine the structure of the RIPK2 kinase BIR2 domain complex and validated the interface with in cellulo assays. The structure shows that BIR2 binds across the RIPK2 kinase antiparallel dimer and provides an explanation for both inhibitory mechanisms. It also highlights why phosphorylation of the kinase activation loop is dispensable for signalling while revealing the structural role of RIPK2-K209 residue in the RIPK2-XIAP BIR2 interaction. Our results clarify the features of the RIPK2 conformation essential for its role as a scaffold protein for ubiquitination.

Activation loop phosphorylation tunes conformational dynamics underlying Pyk2 tyrosine kinase activation

Pyk2 is a multidomain non-receptor tyrosine kinase that undergoes a multistage activation mechanism. Activation is instigated by conformational rearrangements relieving autoinhibitory FERM domain interactions. The kinase autophosphorylates a central linker residue to recruit Src kinase. Pyk2 and Src mutually phosphorylate activation loops to confer full activation. While the mechanisms of autoinhibition are established, the conformational dynamics associated with autophosphorylation and Src recruitment remain unclear. We employ hydrogen/deuterium exchange mass spectrometry and kinase activity profiling to map the conformational dynamics associated with substrate binding and Src-mediated activation loop phosphorylation. Nucleotide engagement stabilizes the autoinhibitory interface, while phosphorylation deprotects both FERM and kinase regulatory surfaces. Phosphorylation organizes active site motifs linking catalytic loop with activation segment. Dynamics of the activation segment anchor propagate to EF/G helices to prevent reversion of the autoinhibitory FERM interaction. We employ targeted mutagenesis to dissect how phosphorylation-induced conformational rearrangements elevate kinase activity above the basal autophosphorylation rate.

PKD autoinhibition in trans regulates activation loop autophosphorylation in cis

Phosphorylation is a ubiquitous mechanism by which signals are transduced in cells. Protein kinases, enzymes that catalyze the phosphotransfer reaction are, themselves, often regulated by phosphorylation. Paradoxically, however, a substantial fraction of more than 500 human protein kinases are capable of catalyzing their own activation loop phosphorylation. Commonly, these kinases perform this autophosphorylation reaction in trans, whereby transient dimerization leads to the mutual phosphorylation of the activation loop of the opposing protomer. In this study, we demonstrate that protein kinase D (PKD) is regulated by the inverse mechanism of dimerization-mediated trans-autoinhibition, followed by activation loop autophosphorylation in cis. We show that PKD forms a stable face-to-face homodimer that is incapable of either autophosphorylation or substrate phosphorylation. Dissociation of this trans-autoinhibited dimer results in activation loop autophosphorylation, which occurs exclusively in cis. Phosphorylation serves to increase PKD activity and prevent trans-autoinhibition, thereby switching PKD on. Our findings not only reveal the mechanism of PKD regulation but also have profound implications for the regulation of many other eukaryotic kinases.

Divergent kinase WNG1 is regulated by phosphorylation of an atypical activation sub-domain.

Apicomplexan parasites like Toxoplasma gondii grow and replicate within a specialized organelle called the parasitophorous vacuole. The vacuole is decorated with parasite proteins that integrate into the membrane after trafficking through the parasite secretory system as soluble, chaperoned complexes. A regulator of this process is an atypical protein kinase called WNG1. Phosphorylation by WNG1 appears to serve as a switch for membrane integration. However, like its substrates, WNG1 is secreted from the parasite dense granules, and its activity must, therefore, be tightly regulated until the correct membrane is encountered. Here, we demonstrate that, while another member of the WNG family can adopt multiple multimeric states, WNG1 is monomeric and therefore not regulated by multimerization. Instead, we identify two phosphosites on WNG1 that are required for its kinase activity. Using a combination of in vitro biochemistry and structural modeling, we identify basic residues that are also required for WNG1 activity and appear to recognize the activating phosphosites. Among these coordinating residues are the ‘HRD’ Arg, which recognizes activation loop phosphorylation in canonical kinases. WNG1, however, is not phosphorylated on its activation loop, but rather on atypical phosphosites on its C-lobe. We propose a simple model in which WNG1 is activated by increasing ATP concentration above a critical threshold once the kinase traffics to the parasitophorous vacuole.

Human RIPK3 C-lobe phosphorylation is essential for necroptotic signaling

Necroptosis is a caspase-independent, pro-inflammatory mode of programmed cell death which relies on the activation of the terminal effector, MLKL, by the upstream protein kinase RIPK3. To mediate necroptosis, RIPK3 must stably interact with, and phosphorylate the pseudokinase domain of MLKL, although the precise molecular cues that provoke RIPK3 necroptotic signaling are incompletely understood. The recent finding that RIPK3 S227 phosphorylation and the occurrence of a stable RIPK3:MLKL complex in human cells prior to exposure to a necroptosis stimulus raises the possibility that additional, as-yet-unidentified phosphorylation events activate RIPK3 upon initiation of necroptosis signaling. Here, we sought to identify phosphorylation sites of RIPK3 and dissect their regulatory functions. Phosphoproteomics identified 21 phosphorylation sites in HT29 cells overexpressing human RIPK3. By comparing cells expressing wild-type and kinase-inactive D142N RIPK3, autophosphorylation sites and substrates of other cellular kinases were distinguished. Of these 21 phosphosites, mutational analyses identified only pT224 and pS227 as crucial, synergistic sites for stable interaction with MLKL to promote necroptosis, while the recently reported activation loop phosphorylation at S164/T165 negatively regulate the kinase activity of RIPK3. Despite being able to phosphorylate MLKL to a similar or higher extent than wild-type RIPK3, mutation of T224, S227, or the RHIM in RIPK3 attenuated necroptosis. This finding highlights the stable recruitment of human MLKL by RIPK3 to the necrosome as an essential checkpoint in necroptosis signaling, which is independent from and precedes the phosphorylation of MLKL.

Conformations of p90 Ribosomal S6 Kinase (RSK) Activation

Protein kinase have evolved to be dynamic macromolecular switches that alternate between inactive and active conformations. Although most kinase are typically phosphorylated and activated by other kinases, relatively little is known about the detailed mechanistic steps required for one kinase to activate another. The p90 ribosomal S6 kinases (RSKs) are a family of serine/threonine kinases that lie downstream of the Ras‐MAPK pathway and regulate cell proliferation, cell survival, cell growth, and cell motility. RSK is an interesting model system to study kinase activation because it contains two distinct kinase domains, an N‐terminal kinase (NTK), and a C‐terminal kinase (CTK) in the same polypeptide chain. The NTK is a member of the AGC kinase superfamily, and the CTK is a member of the CAMK family. A flexible linker bridges the NTK and CTK, and embedded within the linker is the highly conserved tail of AGC kinases (AGC tail) which contains the conserved turn and hydrophobic motifs (HF). RSK has a complex activation mechanism that includes sequential phosphorylation events and requires two additional kinases: extracellular signal‐regulated kinase (ERK2) and 3‐phosphoinositide‐dependent protein kinase 1 (PDK1). ERK2 phosphorylates the CTK and the turn motif. The phosphorylated CTK is then able to phosphorylate the linker at the HF, this enables PDK1 to bind the linker and then phosphorylate and activate the NTK2. The precise structural details of RSK kinase activation are not fully understood and a more rigorous biochemical characterization could provide valuable insight into how kinase complexes assemble and how the linker facilitates multiple binding and phosphorylation events.To gain a better understanding of the FL RSK2‐ERK2 signaling complex we have used cryoEM and hydrogen deuterium exchange mass spectrometry (HDX) to gain insight into the overall shape and solvent accessibility of the overall complex. We have determined a low‐resolution density map of the RSK2‐ERK2 complex, revealing a compact shape. Additionally, all three kinase domains are relatively well protected from solvent, with the exception of the activation loops of the CTK, and the aC helix of the NTK. While the tails of RSK2 and linker (AGC tail) are highly solvent exposed. We hypothesize that the linker region needs such solvent accessibility in order to enable the various interactions and phosphorylation events necessary for RSK activation. We have also utilized peptide array binding experiments to determine the residues within the linker that are necessary to enable ERK2 phosphorylation of the turn motif, CTK phosphorylation of the HF, and PDK1 binding and subsequent phosphorylation of the NTK activation loop. ERK2 appears to bind to the linker and NTK in a multivalent fashion via the C‐lobe (300‐320) and the N‐lobe. The CTK seems to primarily interact with HF in the linker. And PDK1 binds to both the HF and a conserved FDX1‐2Y/F motif in the linker.

The mechanism of activation of MEK1 by B-Raf and KSR1

MEK1 interactions with B-Raf and KSR1 are key steps in Ras/Raf/MEK/ERK signaling. Despite this, vital mechanistic details of how these execute signal transduction are still enigmatic. Among these is why, despite B-Raf and KSR1 kinase domains similarity, the B-Raf/MEK1 and KSR1/MEK1 complexes have distinct contributions to MEK1 activation, and broadly, what is KSR1’s role. Our molecular dynamics simulations clarify these still unresolved ambiguities. Our results reveal that the proline-rich (P-rich) loop of MEK1 plays a decisive role in MEK1 activation loop (A-loop) phosphorylation. In the inactive B-Raf/MEK1 heterodimer, the collapsed A-loop of B-Raf interacts with the P-rich loop and A-loop of MEK1, minimizing MEK1 A-loop fluctuation and preventing it from phosphorylation. In the active B-Raf/MEK1 heterodimer, the P-rich loop moves in concert with the A-loop of B-Raf as it extends. This reduces the number of residues interacting with MEK1 A-loop, allowing increased A-loop fluctuation, and bringing Ser222 closer to ATP for phosphorylation. B-Raf αG-helix Arg662 promotes MEK1 activation by orienting Ser218 towards ATP. In KSR1/MEK1, the KSR1 αG-helix has Ala826 in place of B-Raf Arg662. This difference results in much fewer interactions between KSR1 αG-helix and MEK1 A-loop, thus a more flexible A-loop. We postulate that if KSR1 were to adopt an active configuration with an extended A-loop as seen in other protein kinases, then the MEK1 P-rich loop would extend in a similar manner, as seen in the active B-Raf/MEK1 heterodimer. This would result in highly flexible MEK1 A-loop, and KSR1 functioning as an active, B-Raf-like, kinase.

HDL promotes adiponectin gene expression via the CAMKK/CAMKIV pathway.

Adiponectin (APN) is an adipokine that protects against diabetes and atherosclerosis. High-density lipoprotein (HDL) mediates reverse cholesterol transport, which also protects against atherosclerosis. In this process, the human homolog of the B class type I scavenger receptor (SR-BI/CLA-1) facilitates the cellular uptake of cholesterol from HDL. The level of circulating APN is positively correlated with the serum level of HDL-cholesterol. In this study, we investigated whether HDL stimulates the gene expression of APN through the Ca2+/calmodulin (CaM)-dependent protein kinase IV (CaMKIV) cascade. APN expression was examined using real-time PCR and western blot analysis in 3T3-L1 cells incubated with HDL. CaMKIV activity was assessed by the detection of activation loop phosphorylation (at Thr196 residue), and the effect of the constitutively active form, CaMKIVc, on APN promoter activity was investigated. Our results showed that HDL stimulated APN gene expression via hSR-BI/CLA-1. Furthermore, we explored the signaling pathways by which HDL stimulated APN expression in 3T3-L1 cells. The stimulation of APN gene expression by HDL appears to be mediated by CaMKK, as STO-609, a specific inhibitor of CaMKK2, prevents this effect. We revealed that CaMKIVc increased APN gene transcriptional activity, and the CaMKIV-dominant negative mutant blocked the effect of HDL on APN promoter activity. Finally, knockdown of hSR-BI/CLA-1 also canceled the effect of HDL on APN gene expression. These results suggest that HDL has an important role to improve the function of adipocytes by activating hSR-BI/CLA-1, and CaMKK/CaMKIV pathway is conceivable as one of the signaling pathways of this activation mechanism.

Activation loop phosphorylation of a non-RD receptor kinase initiates plant innate immune signaling.

Receptor kinases (RKs) are fundamental for extracellular sensing and regulate development and stress responses across kingdoms. In plants, leucine-rich repeat receptor kinases (LRR-RKs) are primarily peptide receptors that regulate responses to myriad internal and external stimuli. Phosphorylation of LRR-RK cytoplasmic domains is among the earliest responses following ligand perception, and reciprocal transphosphorylation between a receptor and its coreceptor is thought to activate the receptor complex. Originally proposed based on characterization of the brassinosteroid receptor, the prevalence of complex activation via reciprocal transphosphorylation across the plant RK family has not been tested. Using the LRR-RK ELONGATION FACTOR TU RECEPTOR (EFR) as a model, we set out to understand the steps critical for activating RK complexes. While the EFR cytoplasmic domain is an active protein kinase in vitro and is phosphorylated in a ligand-dependent manner in vivo, catalytically deficient EFR variants are functional in antibacterial immunity. These results reveal a noncatalytic role for EFR in triggering immune signaling and indicate that reciprocal transphoshorylation is not a ubiquitous requirement for LRR-RK complex activation. Rather, our analysis of EFR along with a detailed survey of the literature suggests a distinction between LRR-RKs with RD- versus non-RD protein kinase domains. Based on newly identified phosphorylation sites that regulate the activation state of the EFR complex in vivo, we propose that LRR-RK complexes containing a non-RD protein kinase may be regulated by phosphorylation-dependent conformational changes of the ligand-binding receptor, which could initiate signaling either allosterically or through driving the dissociation of negative regulators of the complex.

Maternal Ethanol Exposure Acutely Elevates Src Family Kinase Activity in the Fetal Cortex

Fetal alcohol syndrome (FAS) is characterized by disrupted fetal brain development and postnatal cognitive impairment. The targets of alcohol are diverse, and it is not clear whether there are common underlying molecular mechanisms producing these disruptions. Prior work established that acute ethanol exposure causes a transient increase in tyrosine phosphorylation of multiple proteins in cultured embryonic cortical cells. In this study, we show that a similar tyrosine phosphorylation transient occurs in the fetal brain after maternal dosing with ethanol. Using phospho-specific antibodies and immunohistochemistry, we mapped regions of highest tyrosine phosphorylation in the fetal cerebral cortex and found that areas of dendritic and axonal growth showed elevated tyrosine phosphorylation 10 min after maternal ethanol exposure. These were also areas of Src expression and Src family kinase (SFK) activation loop phosphorylation (pY416) expression. Importantly, maternal pretreatment with the SFK inhibitor dasatinib completely prevents both the pY416 increase and the tyrosine phosphorylation response. The phosphorylation response was observed in the perisomatic region and neurites of immature migrating and differentiating primary neurons. Importantly, the initial phosphotyrosine transient (~ 30 min) targets both Src and Dab1, two critical elements in Reelin signaling, a pathway required for normal cortical development. This initial phosphorylation response is followed by sustained reduction in Ser3 phosphorylation of n-cofilin, a critical actin severing protein and an identified downstream effector of Reelin signaling. This biochemical disruption is associated with sustained reduction of F-actin content and disrupted Golgi apparatus morphology in developing cortical neurons. The finding outlines a model in which the initial activation of SFKs by ethanol has the potential to disrupt multiple developmentally important signaling systems for several hours after maternal exposure.

MAP4K4 expression in cardiomyocytes: multiple isoforms, multiple phosphorylations and interactions with striatins.

The Ser/Thr kinase MAP4K4, like other GCKIV kinases, has N-terminal kinase and C-terminal citron homology (CNH) domains. MAP4K4 can activate c-Jun N-terminal kinases (JNKs), and studies in the heart suggest it links oxidative stress to JNKs and heart failure. In other systems, MAP4K4 is regulated in striatin-interacting phosphatase and kinase (STRIPAK) complexes, in which one of three striatins tethers PP2A adjacent to a kinase to keep it dephosphorylated and inactive. Our aim was to understand how MAP4K4 is regulated in cardiomyocytes. The rat MAP4K4 gene was not properly defined. We identified the first coding exon of the rat gene using 5′-RACE, we cloned the full-length sequence and confirmed alternative-splicing of MAP4K4 in rat cardiomyocytes. We identified an additional α-helix C-terminal to the kinase domain important for kinase activity. In further studies, FLAG-MAP4K4 was expressed in HEK293 cells or cardiomyocytes. The Ser/Thr protein phosphatase inhibitor calyculin A (CalA) induced MAP4K4 hyperphosphorylation, with phosphorylation of the activation loop and extensive phosphorylation of the linker between the kinase and CNH domains. This required kinase activity. MAP4K4 associated with myosin in untreated cardiomyocytes, and this was lost with CalA-treatment. FLAG-MAP4K4 associated with all three striatins in cardiomyocytes, indicative of regulation within STRIPAK complexes and consistent with activation by CalA. Computational analysis suggested the interaction was direct and mediated via coiled-coil domains. Surprisingly, FLAG-MAP4K4 inhibited JNK activation by H2O2 in cardiomyocytes and increased myofibrillar organisation. Our data identify MAP4K4 as a STRIPAK-regulated kinase in cardiomyocytes, and suggest it regulates the cytoskeleton rather than activates JNKs.

Isoform‐Selective Regulation of Akt1 and Akt2 by Interdomain Interaction

The serine/threonine protein kinase PKB/Akt is a key signal transducer in a wide range of cellular responses and a prominent therapeutic target. The three highly homologous isoforms of Akt regulate critical and distinct physiological functions in cell growth, proliferation, and metabolism. However, the mechanisms that determine isoform-specific Akt signaling remain largely unclear. Here we examined the mechanisms by which Akt1 and Akt2, the most broadly expressed isoforms, are differentially activated upon growth factor stimulation. Using a newly developed FRET-based reporter of Akt2 activation (ReAktion2) along with a previously developed reporter of Akt1 activation (ReAktion), we found that growth factor triggers a conformational change in both Akt isoforms at the plasma membrane, which is critically dependent on activation-loop phosphorylation. Interestingly, the allosteric pan-Akt inhibitor, MK-2206, appears to selectively induce a conformational change in Akt2, but not Akt1 at the plasma membrane. This prompts us to further examine whether inter-domain interactions differ between Akt1 and Akt2. Our preliminary results suggest Akt1 activation enhances the interaction between its PH and linker-catalytic domains, which is dependent on phosphorylation of T308 within the catalytic domain, whereas this induced interdomain interaction is absent in Akt2. Furthermore, the different interdomain interaction of Akt isoforms may lead to different membrane departure, as single cell analysis of plasma membrane translocation revealed that Akt2 accumulates at the plasma membrane to a greater extent than Akt1. Taken together, our results suggest that distinct modes of interactions between the PH and linker-catalytic domains modulate the isoform-specific activation of Akt.

Allosteric Kinase Inhibitors Reshape MEK1 Kinase Activity Conformations in Cells and In Silico.

Mutations at different stages of the mitogen-activated protein kinase (MAPK) signaling pathway lead to aberrant activation of the involved protein kinase entities. These oncogenic modifications alter signal propagation which converge on the gatekeeper kinases MEK1/2, transmitting the input signal to ERK1/2. Thus, targeted MEK inhibition causes qualitative alterations of carcinogenic MAPK signals. Phosphorylation of the MEK1 activation loop at the positions S218 and S222 by RAF kinases triggers the conformational alignment of MEK’s catalytic pocket to enable ATP-binding and substrate phosphorylation. We have extended a kinase conformation (KinCon) biosensor platform to record MEK1 activity dynamics. In addition to MEK phosphorylation by BRAF, the integration of the phosphorylation-mimetic mutations S218D/S222D triggered opening of the kinase. Structural rearrangement may involve the flexibility of the N terminal MEK1 A-helix. Application of the allosterically acting MEK inhibitors (MEKi) trametinib, cobimentinib, refametinib, and selumetinib converted activated MEK1 KinCon reporters back into a more closed inactive conformation. We confirmed MEK1 KinCon activity dynamics upon drug engagement using the patient-derived melanoma cell line A2058, which harbors the V600E hotspot BRAF mutation. In order to confirm biosensor dynamics, we simulated structure dynamics of MEK1 kinase in the presence and absence of mutations and/or MEKi binding. We observed increased dynamics for the S218D/S222D double mutant particularly in the region of the distal A-helix and alpha-C helix. These data underline that MEK1 KinCon biosensors have the potential to be subjected to MEKi efficacy validations in an intact cell setting.

The influence of delay in mononuclear cell isolation on acute myeloid leukemia phosphorylation profiles

Mass-spectrometry (MS) based phosphoproteomics is increasingly used to explore aberrant cellular signaling and kinase driver activity, aiming to improve kinase inhibitor (KI) treatment selection in malignancies. Phosphorylation is a dynamic, highly regulated post-translational modification that may be affected by variation in pre-analytical sample handling, hampering the translational value of phosphoproteomics-based analyses. Here, we investigate the effect of delay in mononuclear cell isolation on acute myeloid leukemia (AML) phosphorylation profiles. We performed MS on immuno-precipitated phosphotyrosine (pY)-containing peptides isolated from AML samples after seven pre-defined delays before sample processing (direct processing, thirty minutes, one hour, two hours, three hours, four hours and 24 h delay). Up to four hours, pY phosphoproteomics profiles show limited variation. However, in samples processed with a delay of 24 h, we observed significant change in these phosphorylation profiles, with differential phosphorylation of 22 pY phosphopeptides (p < 0.01). This includes increased phosphorylation of pY phosphopeptides of JNK and p38 kinases indicative of stress response activation. Based on these results, we conclude that processing of AML samples should be standardized at all times and should occur within four hours after sample collection. SignificanceOur study provides a practical time-frame in which fresh peripheral blood samples from acute myeloid patients should be processed for phosphoproteomics, in order to warrant correct interpretation of in vivo biology. We show that up to four hours of delayed processing after sample collection, pY phosphoproteomic profiles remain stable. Extended delays are associated with perturbation of phosphorylation profiles. After a delay of 24 h, JNK activation loop phosphorylation is markedly increased and may serve as a biomarker for delayed processing.These findings are relevant for biomedical acute myeloid leukemia research, as phosphoproteomic techniques are of particular interest to investigate aberrant kinase signaling in relation to disease emergence and kinase inhibitor response. With these data, we aim to contribute to reproducible research with meaningful outcomes.

Post-Translational Modifications of the Energy Guardian AMP-Activated Protein Kinase.

Physical exercise elicits physiological metabolic perturbations such as energetic and oxidative stress; however, a diverse range of cellular processes are stimulated in response to combat these challenges and maintain cellular energy homeostasis. AMP-activated protein kinase (AMPK) is a highly conserved enzyme that acts as a metabolic fuel sensor and is central to this adaptive response to exercise. The complexity of AMPK’s role in modulating a range of cellular signalling cascades is well documented, yet aside from its well-characterised regulation by activation loop phosphorylation, AMPK is further subject to a multitude of additional regulatory stimuli. Therefore, in this review we comprehensively outline current knowledge around the post-translational modifications of AMPK, including novel phosphorylation sites, as well as underappreciated roles for ubiquitination, sumoylation, acetylation, methylation and oxidation. We provide insight into the physiological ramifications of these AMPK modifications, which not only affect its activity, but also subcellular localisation, nutrient interactions and protein stability. Lastly, we highlight the current knowledge gaps in this area of AMPK research and provide perspectives on how the field can apply greater rigour to the characterisation of novel AMPK regulatory modifications.