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Leucine-rich repeat protein kinase 2 (LRRK2) is a multi-domain protein encompassing two of biology's most critical molecular switches, a kinase and a GTPase, and mutations in LRRK2 are key players in the pathogenesis of Parkinson's disease (PD). The availability of multiple structures (full-length and truncated) has opened doors to explore intra-domain cross-talk in LRRK2. A helix extending from the WD40 domain and stably docking onto the kinase domain is common in all available structures. This C-terminal (Ct) helix is a hub of phosphorylation and organelle-localization motifs and thus serves as a multi-functional protein : protein interaction module. To examine its intra-domain interactions, we have recombinantly expressed a stable Ct motif (residues 2480-2527) and used peptide arrays to identify specific binding sites. We have identified a potential interaction site between the Ct helix and a loop in the CORB domain (CORB loop) using a combination of Gaussian accelerated molecular dynamics simulations and peptide arrays. This Ct-Motif contains two auto-phosphorylation sites (T2483 and T2524), and T2524 is a 14-3-3 binding site. The Ct helix, CORB loop, and the CORB-kinase linker together form a part of a dynamic 'CAP' that regulates the N-lobe of the kinase domain. We hypothesize that in inactive, full-length LRRK2, the Ct-helix will also mediate interactions with the N-terminal armadillo, ankyrin, and LRR domains (NTDs) and that binding of Rab substrates, PD mutations, or kinase inhibitors will unleash the NTDs.

Conventional protein kinase C (cPKC) isozymes tune the signaling output of cells, with loss-of-function somatic mutations associated with cancer and gain-of-function germline mutations identified in neurodegeneration. PKC with impaired autoinhibition is removed from the cell by quality-control mechanisms to prevent the accumulation of aberrantly active enzyme. Here, we examine how a highly conserved residue in the C1A domain of cPKC isozymes permits quality-control degradation when mutated to histidine in cancer (PKCβ-R42H) and blocks down-regulation when mutated to proline in the neurodegenerative disease spinocerebellar ataxia (PKCγ-R41P). Using FRET-based biosensors, we determined that mutation of R42 to any residue, including lysine, resulted in reduced autoinhibition as indicated by higher basal activity and faster agonist-induced plasma membrane translocation. R42 is predicted to form a stabilizing salt bridge with E655 in the C-tail and mutation of E655, but not neighboring E657, also reduced autoinhibition. Western blot analysis revealed that whereas R42H had reduced stability, the R42P mutant was stable and insensitive to activator-induced ubiquitination and down-regulation, an effect previously observed by deletion of the entire C1A domain. Molecular dynamics (MD) simulations and analysis of stable regions of the domain using local spatial pattern (LSP) alignment suggested that P42 interacts with Q66 to impair mobility and conformation of one of the ligand-binding loops. Additional mutation of Q66 to the smaller asparagine (R42P/Q66N), to remove conformational constraints, restored degradation sensitivity. Our results unveil how disease-associated mutations of the same residue in the C1A domain can toggle between gain- or loss-of-function of PKC.

LRRK2 is a multi-domain protein with three catalytically inert N-terminal domains (NtDs) and four C-terminal domains, including a kinase and a GTPase domain. LRRK2 mutations are linked to Parkinson's Disease. Recent structures of LRRK2RCKW and a full-length inactive LRRK2 (fl-LRRK2INACT) monomer revealed that the kinase domain drives LRRK2 activation. The LRR domain and also an ordered LRR- COR linker, wrap around the C-lobe of the kinase domain and sterically block the substrate binding surface in fl-LRRK2INACT. Here we focus on the crosstalk between domains. Our biochemical studies of GTPase and kinase activities of fl-LRRK2 and LRRK2RCKW reveal how mutations influence this crosstalk differently depending on the domain borders investigated. Furthermore, we demonstrate that removing the NtDs leads to altered intramolecular regulation. To further investigate the crosstalk, we used Hydrogen-Deuterium exchange Mass Spectrometry (HDX-MS) to characterize the conformation of LRRK2RCKW   and Gaussian Accelerated Molecular Dynamics (GaMD) to create dynamic portraits of fl-LRRK2 and LRRK2RCKW. These models allowed us to investigate the dynamic changes in wild type and mutant LRRK2s. Our data show that the a3ROC helix, the Switch II motif in the ROC domain, and the LRR-ROC linker play crucial roles in mediating local and global conformational changes. We demonstrate how these regions are affected by other domains in fl-LRRK2 and LRRK2RCKW and show how unleashing of the NtDs as well as PD mutations lead to changes in conformation and dynamics of the ROC and kinase domains which ultimately impact kinase and GTPase activities. These allosteric sites are potential therapeutic targets.

It is investigated whether molecular-dynamics (MD) simulations can be used to enhance macromolecular crystallography (MX) studies. Historically, protein crystal structures have been described using a single set of atomic coordinates. Because conformational variation is important for protein function, researchers now often build models that contain multiple structures. Methods for building such models can fail, however, in regions where the crystallographic density is difficult to interpret, for example at the protein-solvent interface. To address this limitation, a set of MD-MX methods that combine MD simulations of protein crystals with conventional modeling and refinement tools have been developed. In an application to a cyclic adenosine monophosphate-dependent protein kinase at room temperature, the procedure improved the interpretation of ambiguous density, yielding an alternative water model and a revised protein model including multiple conformations. The revised model provides mechanistic insights into the catalytic and regulatory interactions of the enzyme. The same methods may be used in other MX studies to seek mechanistic insights.

Topological analysis of protein residue networks (PRNs) is a common method that can help to understand the roles of individual residues. Here, we used protein kinase A as a study object and asked what already known functionally important residues can be detected by network analysis. Along several traditional approaches to weight edges in PRNs we used local spatial pattern (LSP) alignment that assigns high weights to edges only if CαCβ vectors for the corresponding residues retain their mutual positions and orientation. Our results show that even short molecular dynamic simulations of 10 to 20 ns can give convergent values for betweenness and degree centralities calculated from the LSP-based PRNs. Using these centralities, we were able to clearly distinguish a group of residues that are highly conserved in protein kinases and play important functional and regulatory roles. In comparison, traditional methods based on cross-correlation and linear mutual information were much less efficient for this particular task. These results call for reevaluation of the current methods to generate PRNs.

The Hedgehog (Hh) cascade is central to development, tissue homeostasis and cancer. A pivotal step in Hh signal transduction is the activation of glioma-associated (GLI) transcription factors by the atypical G protein-coupled receptor (GPCR) SMOOTHENED (SMO). How SMO activates GLI remains unclear. Here we show that SMO uses a decoy substrate sequence to physically block the active site of the cAMP-dependent protein kinase (PKA) catalytic subunit (PKA-C) and extinguish its enzymatic activity. As a result, GLI is released from phosphorylation-induced inhibition. Using a combination of in vitro, cellular and organismal models, we demonstrate that interfering with SMO-PKA pseudosubstrate interactions prevents Hh signal transduction. The mechanism uncovered echoes one used by the Wnt cascade, revealing an unexpected similarity in how these two essential developmental and cancer pathways signal intracellularly. More broadly, our findings define a mode of GPCR-PKA communication that may be harnessed by a range of membrane receptors and kinases.

Spinocerebellar ataxia type 14 (SCA14) is a neurodegenerative disease caused by germline variants in the diacylglycerol (DAG)/Ca2+-regulated protein kinase Cγ (PKCγ), leading to Purkinje cell degeneration and progressive cerebellar dysfunction. Most of the identified mutations cluster in the DAG-sensing C1 domains. Here, we found with a FRET-based activity reporter that SCA14-associated PKCγ mutations, including a previously undescribed variant, D115Y, enhanced the basal activity of the kinase by compromising its autoinhibition. Unlike other mutations in PKC that impair its autoinhibition but lead to its degradation, the C1 domain mutations protected PKCγ from such down-regulation. This enhanced basal signaling rewired the brain phosphoproteome, as revealed by phosphoproteomic analysis of cerebella from mice expressing a human SCA14-associated H101Y mutant PKCγ transgene. Mutations that induced a high basal activity in vitro were associated with earlier average age of onset in patients. Furthermore, the extent of disrupted autoinhibition, but not agonist-stimulated activity, correlated with disease severity. Molecular modeling indicated that almost all SCA14 variants not within the C1 domain were located at interfaces with the C1B domain, suggesting that mutations in and proximal to the C1B domain are a susceptibility for SCA14 because they uniquely enhance PKCγ basal activity while protecting the enzyme from down-regulation. These results provide insight into how PKCγ activation is modulated and how deregulation of the cerebellar phosphoproteome by SCA14-associated mutations affects disease progression.

Protein kinase A (PKA) is a holoenzyme consisting of a regulatory (R)-subunit dimer and two catalytic (C)-subunits. There are two major families of C-subunits, Cα and Cβ, and four functionally nonredundant R-subunits (RIα, RIβ, RIIα, RIIβ). In addition to binding to and being regulated by the R-subunits, the C-subunits are regulated by two tail regions that each wrap around the N- and C-lobes of the kinase core. Although the C-terminal (Ct-) tail is classified as an intrinsically disordered region (IDR), the N-terminal (Nt-) tail is dominated by a strong helix that is flanked by short IDRs. In contrast to the Ct-tail, which is a conserved and highly regulated feature of all PKA, PKG, and protein kinase C protein kinase group (AGC) kinases, the Nt-tail has evolved more recently and is highly variable in vertebrates. Surprisingly and in contrast to the kinase core and the Ct-tail, the entire Nt-tail is not conserved in nonmammalian PKAs. In particular, in humans, Cβ actually represents a large family of C-subunits that are highly variable in their Nt-tail and also expressed in a highly tissue-specific manner. Although we know so much about the Cα1-subunit, we know almost nothing about these Cβ isoforms wherein Cβ2 is highly expressed in lymphocytes, and Cβ3 and Cβ4 isoforms account for ∼50% of PKA signaling in brain. Based on recent disease mutations, the Cβ proteins appear to be functionally important and nonredundant with the Cα isoforms. Imaging in retina also supports nonredundant roles for Cβ as well as isoform-specific localization to mitochondria. This represents a new frontier in PKA signaling. SIGNIFICANCE STATEMENT: How tails and adjacent domains regulate each protein kinase is a fundamental challenge for the biological community. Here we highlight how the N- and C-terminal tails of PKA (Nt-tails/Ct-tails) affect the structure and regulate the function of the kinase core and show the combinatorial variations that are introduced into the Nt-tail of the Cα- and Cβ-subunits in contrast to the Ct-tail, which is conserved across the entire AGC subfamily of protein kinases.

The 2 major molecular switches in biology, kinases and GTPases, are both contained in the Parkinson disease-related leucine-rich repeat kinase 2 (LRRK2). Using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and molecular dynamics (MD) simulations, we generated a comprehensive dynamic allosteric portrait of the C-terminal domains of LRRK2 (LRRK2RCKW). We identified 2 helices that shield the kinase domain and regulate LRRK2 conformation and function. One helix in COR-B (COR-B Helix) tethers the COR-B domain to the αC helix of the kinase domain and faces its activation loop, while the C-terminal helix (Ct-Helix) extends from the WD40 domain and interacts with both kinase lobes. The Ct-Helix and the N-terminus of the COR-B Helix create a "cap" that regulates the N-lobe of the kinase domain. Our analyses reveal allosteric sites for pharmacological intervention and confirm the kinase domain as the central hub for conformational control.

Many of the fundamental concepts of signal transduction and kinase activity are attributed to the discovery and crystallization of cAMP-dependent protein kinase, or protein kinase A. PKA is one of the best-studied kinases in human biology, with emphasis in biochemistry and biophysics, all the way to metabolism, hormone action, and gene expression regulation. It is surprising, however, that our understanding of PKA's role in disease is largely underappreciated. Although genetic mutations in the PKA holoenzyme are known to cause diseases such as Carney complex, Cushing syndrome, and acrodysostosis, the story largely stops there. With the recent explosion of genomic medicine, we can finally appreciate the broader role of the Gαs-PKA pathway in disease, with contributions from aberrant functioning G proteins and G protein-coupled receptors, as well as multiple alterations in other pathway components and negative regulators. Together, these represent a broad family of diseases we term the Gαs-PKA pathway signalopathies. The Gαs-PKA pathway signalopathies encompass diseases caused by germline, postzygotic, and somatic mutations in the Gαs-PKA pathway, with largely endocrine and neoplastic phenotypes. Here, we present a signaling-centric review of Gαs-PKA-driven pathophysiology and integrate computational and structural analysis to identify mutational themes commonly exploited by the Gαs-PKA pathway signalopathies. Major mutational themes include hotspot activating mutations in Gαs, encoded by GNAS, and mutations that destabilize the PKA holoenzyme. With this review, we hope to incite further study and ultimately the development of new therapeutic strategies in the treatment of a wide range of human diseases. SIGNIFICANCE STATEMENT: Little recognition is given to the causative role of Gαs-PKA pathway dysregulation in disease, with effects ranging from infectious disease, endocrine syndromes, and many cancers, yet these disparate diseases can all be understood by common genetic themes and biochemical signaling connections. By highlighting these common pathogenic mechanisms and bridging multiple disciplines, important progress can be made toward therapeutic advances in treating Gαs-PKA pathway-driven disease.

To explore how pathogenic mutations of the multidomain leucine-rich repeat kinase 2 (LRRK2) hijack its finely tuned activation process and drive Parkinson's disease (PD), we used a multitiered approach. Most mutations mimic Rab-mediated activation by "unleashing" kinase activity, and many, like the kinase inhibitor MLi-2, trap LRRK2 onto microtubules. Here we mimic activation by simply deleting the inhibitory N-terminal domains and then characterize conformational changes induced by MLi-2 and PD mutations. After confirming that LRRK2RCKW retains full kinase activity, we used hydrogen-deuterium exchange mass spectrometry to capture breathing dynamics in the presence and absence of MLi-2. Solvent-accessible regions throughout the entire protein are reduced by MLi-2 binding. With molecular dynamics simulations, we created a dynamic portrait of LRRK2RCKW and demonstrate the consequences of kinase domain mutations. Although all domains contribute to regulating kinase activity, the kinase domain, driven by the DYGψ motif, is the allosteric hub that drives LRRK2 regulation.

Familial mutations of the protein kinase A (PKA) R1α regulatory subunit lead to a generalized predisposition for a wide range of tumors, from pituitary adenomas to pancreatic and liver cancers, commonly referred to as Carney complex (CNC). CNC mutations are known to cause overactivation of PKA, but the molecular mechanisms underlying such kinase overactivity are not fully understood in the context of the canonical cAMP-dependent activation of PKA. Here, we show that oligomerization-induced sequestration of R1α from the catalytic subunit of PKA (C) is a viable mechanism of PKA activation that can explain the CNC phenotype. Our investigations focus on comparative analyses at the level of structure, unfolding, aggregation, and kinase inhibition profiles of wild-type (wt) PKA R1α, the A211D and G287W CNC mutants, as well as the cognate acrodysostosis type 1 (ACRDYS1) mutations A211T and G287E. The latter exhibit a phenotype opposite to CNC with suboptimal PKA activation compared with wt. Overall, our results show that CNC mutations not only perturb the classical cAMP-dependent allosteric activation pathway of PKA, but also amplify significantly more than the cognate ACRDYS1 mutations nonclassical and previously unappreciated activation pathways, such as oligomerization-induced losses of the PKA R1α inhibitory function.

The complex mTORC2 is accepted to be the kinase that controls the phosphorylation of the hydrophobic motif, a key regulatory switch for AGC kinases, although whether mTOR directly phosphorylates this motif remains controversial. Here, we identified an mTOR-mediated phosphorylation site that we termed the TOR interaction motif (TIM; F-x3-F-pT), which controls the phosphorylation of the hydrophobic motif of PKC and Akt and the activity of these kinases. The TIM is invariant in mTORC2-dependent AGC kinases, is evolutionarily conserved, and coevolved with mTORC2 components. Mutation of this motif in Akt1 and PKCβII abolished cellular kinase activity by impairing activation loop and hydrophobic motif phosphorylation. mTORC2 directly phosphorylated the PKC TIM in vitro, and this phosphorylation event was detected in mouse brain. Overexpression of PDK1 in mTORC2-deficient cells rescued hydrophobic motif phosphorylation of PKC and Akt by a mechanism dependent on their intrinsic catalytic activity, revealing that mTORC2 facilitates the PDK1 phosphorylation step, which, in turn, enables autophosphorylation. Structural analysis revealed that PKC homodimerization is driven by a TIM-containing helix, and biophysical proximity assays showed that newly synthesized, unphosphorylated PKC dimerizes in cells. Furthermore, disruption of the dimer interface by stapled peptides promoted hydrophobic motif phosphorylation. Our data support a model in which mTORC2 relieves nascent PKC dimerization through TIM phosphorylation, recruiting PDK1 to phosphorylate the activation loop and triggering intramolecular hydrophobic motif autophosphorylation. Identification of TIM phosphorylation and its role in the regulation of PKC provides the basis for AGC kinase regulation by mTORC2.

Protein kinase A (PKA) signaling is essential for numerous processes but the subcellular localization of specific PKA regulatory (R) and catalytic (C) subunits has yet to be explored comprehensively. Additionally, the localization of the Cβ subunit has never been spatially mapped in any tissue even though ∼50% of PKA signaling in neuronal tissues is thought to be mediated by Cβ. Here we used human retina with its highly specialized neurons as a window into PKA signaling in the brain and characterized localization of PKA Cα, Cβ, RIIα, and RIIβ subunits. We found that each subunit presented a distinct localization pattern. Cα and Cβ were localized in all cell layers (photoreceptors, interneurons, retinal ganglion cells), while RIIα and RIIβ were selectively enriched in photoreceptor cells where both showed distinct patterns of co-localization with Cα but not Cβ. Only Cα was observed in photoreceptor outer segments and at the base of the connecting cilium. Cβ in turn, was highly enriched in mitochondria and was especially prominent in the ellipsoid of cone cells. Further investigation of Cβ using RNA BaseScope technology showed that two Cβ splice variants (Cβ4 and Cβ4ab) likely code for the mitochondrial Cβ proteins. Overall, our data indicates that PKA Cα, Cβ, RIIα, and RIIβ subunits are differentially localized and are likely functionally non-redundant in the human retina. Furthermore, Cβ is potentially important for mitochondrial-associated neurodegenerative diseases previously linked to PKA dysfunction.

It is difficult to imagine where the signaling community would be today without the Protein Data Bank. This visionary resource, established in the 1970s, has been an essential partner for sharing information between academics and industry for over 3 decades. We describe here the history of our journey with the protein kinases using cAMP-dependent protein kinase as a prototype. We summarize what we have learned since the first structure, published in 1991, why our journey is still ongoing, and why it has been essential to share our structural information. For regulation of kinase activity, we focus on the cAMP-binding protein kinase regulatory subunits. By exploring full-length macromolecular complexes, we discovered not only allostery but also an essential motif originally attributed to crystal packing. Massive genomic data on disease mutations allows us to now revisit crystal packing as a treasure chest of possible protein:protein interfaces where the biological significance and disease relevance can be validated. It provides a new window into exploring dynamic intrinsically disordered regions that previously were deleted, ignored, or attributed to crystal packing. Merging of crystallography with cryo-electron microscopy, cryo-electron tomography, NMR, and millisecond molecular dynamics simulations is opening a new world for the signaling community where those structure coordinates, deposited in the Protein Data Bank, are just a starting point!

Protein kinase C (PKC) family members are multi-domain proteins whose function is exquisitely tuned by interdomain interactions that control the spatiotemporal dynamics of their signaling. Despite extensive mechanistic studies on this family of enzymes, no structure of a full-length enzyme that includes all domains has been solved. Here, we take into account the biochemical mechanisms that control autoinhibition, the properties of each individual domain, and previous structural studies to propose a unifying model for the general architecture of PKC family members. This model shows how the C2 domains of conventional and novel PKC isozymes, which have different topologies and different positions in the primary structure, can occupy the same position in the tertiary structure of the kinase. This common architecture of conventional and novel PKC isozymes provides a framework for understanding how disease-associated mutations impair PKC function.

The fidelity of intracellular signaling hinges on the organization of dynamic activity architectures. Spatial compartmentation was first proposed over 30 years ago to explain how diverse G protein-coupled receptors achieve specificity despite converging on a ubiquitous messenger, cyclic adenosine monophosphate (cAMP). However, the mechanisms responsible for spatially constraining this diffusible messenger remain elusive. Here, we reveal that the type I regulatory subunit of cAMP-dependent protein kinase (PKA), RIα, undergoes liquid-liquid phase separation (LLPS) as a function of cAMP signaling to form biomolecular condensates enriched in cAMP and PKA activity, critical for effective cAMP compartmentation. We further show that a PKA fusion oncoprotein associated with an atypical liver cancer potently blocks RIα LLPS and induces aberrant cAMP signaling. Loss of RIα LLPS in normal cells increases cell proliferation and induces cell transformation. Our work reveals LLPS as a principal organizer of signaling compartments and highlights the pathological consequences of dysregulating this activity architecture.

When the J-domain of the heat shock protein DnaJB1 is fused to the catalytic (C) subunit of cAMP-dependent protein kinase (PKA), replacing exon 1, this fusion protein, J-C subunit (J-C), becomes the driver of fibrolamellar hepatocellular carcinoma (FL-HCC). Here, we use cryo-electron microscopy (cryo-EM) to characterize J-C bound to RIIβ, the major PKA regulatory (R) subunit in liver, thus reporting the first cryo-EM structure of any PKA holoenzyme. We report several differences in both structure and dynamics that could not be captured by the conventional crystallography approaches used to obtain prior structures. Most striking is the asymmetry caused by the absence of the second cyclic nucleotide binding (CNB) domain and the J-domain in one of the RIIβ:J-C protomers. Using molecular dynamics (MD) simulations, we discovered that this asymmetry is already present in the wild-type (WT) RIIβ2C2 but had been masked in the previous crystal structure. This asymmetry may link to the intrinsic allosteric regulation of all PKA holoenzymes and could also explain why most disease mutations in PKA regulatory subunits are dominant negative. The cryo-EM structure, combined with small-angle X-ray scattering (SAXS), also allowed us to predict the general position of the Dimerization/Docking (D/D) domain, which is essential for localization and interacting with membrane-anchored A-Kinase-Anchoring Proteins (AKAPs). This position provides a multivalent mechanism for interaction of the RIIβ holoenzyme with membranes and would be perturbed in the oncogenic fusion protein. The J-domain also alters several biochemical properties of the RIIβ holoenzyme: It is easier to activate with cAMP, and the cooperativity is reduced. These results provide new insights into how the finely tuned allosteric PKA signaling network is disrupted by the oncogenic J-C subunit, ultimately leading to the development of FL-HCC.

Protein kinase A (PKA) holoenzyme, comprised of a cAMP-binding regulatory (R)-subunit dimer and 2 catalytic (C)-subunits, is the master switch for cAMP-mediated signaling. Of the 4 R-subunits (RIα, RIβ, RIIα, RIIβ), RIα is most essential for regulating PKA activity in cells. Our 2 RIα2C2 holoenzyme states, which show different conformations with and without ATP, reveal how ATP/Mg2+ functions as a negative orthosteric modulator. Biochemical studies demonstrate how the removal of ATP primes the holoenzyme for cAMP-mediated activation. The opposing competition between ATP/cAMP is unique to RIα. In RIIβ, ATP serves as a substrate and facilitates cAMP-activation. The isoform-specific RI-holoenzyme dimer interface mediated by N3A-N3A' motifs defines multidomain cross-talk and an allosteric network that creates competing roles for ATP and cAMP. Comparisons to the RIIβ holoenzyme demonstrate isoform-specific holoenzyme interfaces and highlights distinct allosteric mechanisms for activation in addition to the structural diversity of the isoforms.

A dense interplay between structure and dynamics underlies the working of proteins, especially enzymes. Protein kinases are molecular switches that are optimized for their regulation rather than catalytic turnover rates. Using long-simulations dynamic allostery analysis, this study describes an exploration of the dynamic kinase:peptide complex. We have used protein kinase A (PKA) as a model system as a generic prototype of the protein kinase superfamily of signaling enzymes. Our results explain the role of dynamic coupling of active-site residues that must work in coherence to provide for a successful activation or inhibition response from the kinase. Amino acid networks-based community analysis allows us to ponder the conformational entropy of the kinase:nucleotide:peptide ternary complex. We use a combination of 7 peptides that include 3 types of PKA-binding partners: Substrates, products, and inhibitors. The substrate peptides provide for dynamic insights into the enzyme:substrate complex, while the product phospho-peptide allows for accessing modes of enzyme:product release. Mapping of allosteric communities onto the PKA structure allows us to locate the more unvarying and flexible dynamic regions of the kinase. These distributions, when correlated with the structural elements of the kinase core, allow for a detailed exploration of key dynamics-based signatures that could affect peptide recognition and binding at the kinase active site. These studies provide a unique dynamic allostery-based perspective to kinase:peptide complexes that have previously been explored only in a structural or thermodynamic context.

Leucine-rich repeat kinase 2 (LRRK2) is a large multidomain protein, and LRRK2 mutants are recognized risk factors for Parkinson's disease (PD). Although the precise mechanisms that control LRRK2 regulation and function are unclear, the importance of the kinase domain is strongly implicated, since 2 of the 5 most common familial LRRK2 mutations (G2019S and I2020T) are localized to the conserved DFGψ motif in the kinase core, and kinase inhibitors are under development. Combining the concept of regulatory (R) and catalytic (C) spines with kinetic and cell-based assays, we discovered a major regulatory mechanism embedded within the kinase domain and show that the DFG motif serves as a conformational switch that drives LRRK2 activation. LRRK2 is quite unusual in that the highly conserved Phe in the DFGψ motif, which is 1 of the 4 R-spine residues, is replaced with tyrosine (DY2018GI). A Y2018F mutation creates a hyperactive phenotype similar to the familial mutation G2019S. The hydroxyl moiety of Y2018 thus serves as a "brake" that stabilizes an inactive conformation; simply removing it destroys a key hydrogen-bonding node. Y2018F, like the pathogenic mutant I2020T, spontaneously forms LRRK2-decorated microtubules in cells, while the wild type and G2019S require kinase inhibitors to form filaments. We also explored 3 different mechanisms that create kinase-dead pseudokinases, including D2017A, which further emphasizes the highly synergistic role of key hydrophobic and hydrophilic/charged residues in the assembly of active LRRK2. We thus hypothesize that LRRK2 harbors a classical protein kinase switch mechanism that drives the dynamic activation of full-length LRRK2.

The intricacies of allosteric regulation of protein kinases continue to engage the research community. Allostery, or control from a distance, is seen as a fundamental biomolecular mechanism for proteins. From the traditional methods of conformational selection and induced fit, the field has grown to include the role of protein motions in defining a dynamics-based allosteric approach. Harnessing of these continuous motions in the protein to exert allosteric effects can be defined by a "violin" model that focuses on distributions of protein vibrations as opposed to concerted pathways. According to this model, binding of an allosteric modifier causes global redistribution of dynamics in the protein kinase domain that leads to changes in its catalytic properties. This model is consistent with the "entropy-driven allostery" mechanism proposed by Cooper and Dryden in 1984 and does not require, but does not exclude, any major structural changes. We provide an overview of practical implementation of the violin model and how it stands amidst the other known models of protein allostery. Protein kinases have been described as the biomolecules of interest. © 2019 IUBMB Life, 71(6):685-696, 2019.

Eukaryotic protein kinases (EPKs) regulate almost every biological process and have evolved to be dynamic molecular switches; this is in stark contrast to metabolic enzymes, which have evolved to be efficient catalysts. In particular, the highly conserved active site of every EPK is dynamically and transiently assembled by a process that is highly regulated and unique for every protein kinase. We review here the essential features of the kinase core, focusing on the conserved motifs and residues that are embedded in every kinase. We explore, in particular, how the hydrophobic core architecture specifically drives the dynamic assembly of the regulatory spine and consequently the organization of the active site where the γ-phosphate of ATP is positioned by a convergence of conserved motifs including a conserved regulatory triad for transfer to a protein substrate. In conclusion, we show how the flanking N- and C-terminal tails often classified as intrinsically disordered regions, as well as flanking domains, contribute in a highly kinase-specific manner to the regulation of the conserved kinase core. Understanding this process as well as how one kinase activates another remains as two of the big challenges for the kinase signaling community. © 2019 IUBMB Life, 71(6):672-684, 2019.

The mammalian target of rapamycin complex 1 (mTORC1) regulates cell growth, metabolism, and autophagy. Extensive research has focused on pathways that activate mTORC1 like growth factors and amino acids; however, much less is known about signaling cues that directly inhibit mTORC1 activity. Here, we report that G-protein coupled receptors (GPCRs) paired to Gαs proteins increase cyclic adenosine 3'5' monophosphate (cAMP) to activate protein kinase A (PKA) and inhibit mTORC1. Mechanistically, PKA phosphorylates the mTORC1 component Raptor on Ser 791, leading to decreased mTORC1 activity. Consistently, in cells where Raptor Ser 791 is mutated to Ala, mTORC1 activity is partially rescued even after PKA activation. Gαs-coupled GPCRs stimulation leads to inhibition of mTORC1 in multiple cell lines and mouse tissues. Our results uncover a signaling pathway that directly inhibits mTORC1, and suggest that GPCRs paired to Gαs proteins may be potential therapeutic targets for human diseases with hyperactivated mTORC1.

Since publication of the crystal structure of protein kinase (PK)A three decades ago, a structural portrait of the conserved kinase core has been drawn. The next challenge is to elucidate structures of full-length kinases and to address the intrinsically disordered regions (IDRs) that typically flank the core as well as the small linear motifs (SLiMs) that are embedded within the IDRs. It is increasingly apparent that unstructured regions integrate the kinase catalytic chassis into multienzyme-based regulatory networks. The extracellular signal-regulated kinase-ribosomal S6 PK-phosphoinositide-dependent kinase (ERK-RSK-PDK) complex is an excellent example to demonstrate how IDRs and SLiMs govern communication between four different kinase catalytic cores to mediate activation and how in molecular terms these promote the formation of kinase heterodimers in a context dependent fashion.

Protein kinases are dynamic molecular switches that sample multiple conformational states. The regulatory subunit of PKA harbors two cAMP-binding domains [cyclic nucleotide-binding (CNB) domains] that oscillate between inactive and active conformations dependent on cAMP binding. The cooperative binding of cAMP to the CNB domains activates an allosteric interaction network that enables PKA to progress from the inactive to active conformation, unleashing the activity of the catalytic subunit. Despite its importance in the regulation of many biological processes, the molecular mechanism responsible for the observed cooperativity during the activation of PKA remains unclear. Here, we use optical tweezers to probe the folding cooperativity and energetics of domain communication between the cAMP-binding domains in the apo state and bound to the catalytic subunit. Our study provides direct evidence of a switch in the folding-energy landscape of the two CNB domains from energetically independent in the apo state to highly cooperative and energetically coupled in the presence of the catalytic subunit. Moreover, we show that destabilizing mutational effects in one CNB domain efficiently propagate to the other and decrease the folding cooperativity between them. Taken together, our results provide a thermodynamic foundation for the conformational plasticity that enables protein kinases to adapt and respond to signaling molecules.

The full gene sequence encoding for the Trypanosoma equiperdum ortholog of the cAMP-dependent protein kinase (PKA) regulatory (R) subunits was cloned. A poly-His tagged construct was generated [TeqR-like(His)8], and the protein was expressed in bacteria and purified to homogeneity. The size of the purified TeqR-like(His)8 was determined to be ∼57,000 Da by molecular exclusion chromatography indicating that the parasite protein is a monomer. Limited proteolysis with various proteases showed that the T. equiperdum R-like protein possesses a hinge region very susceptible to proteolysis. The recombinant TeqR-like(His)8 did not bind either [3H] cAMP or [3H] cGMP up to concentrations of 0.40 and 0.65 μM, respectively, and neither the parasite protein nor its proteolytically generated carboxy-terminal large fragments were capable of binding to a cAMP-Sepharose affinity column. Bioinformatics analyses predicted that the carboxy-terminal region of the trypanosomal R-like protein appears to fold similarly to the analogous region of all known PKA R subunits. However, the protein amino-terminal portion seems to be unrelated and shows homology with proteins that contained Leu-rich repeats, a folding motif that is particularly appropriate for protein-protein interactions. In addition, the three-dimensional structure of the T. equiperdum protein was modeled using the crystal structure of the bovine PKA RIα subunit as template. Molecular docking experiments predicted critical changes in the environment of the two putative nucleotide binding clefts of the parasite protein, and the resulting binding energy differences support the lack of cyclic nucleotide binding in the trypanosomal R-like protein.

Tyrosine kinases are enzymes playing a critical role in cellular signaling. Molecular dynamics umbrella sampling potential of mean force computations are used to quantify the impact of activating and inactivating mutations of c-Src kinase. The potential of mean force computations predict that a specific double mutant can stabilize c-Src kinase into an active-like conformation while disabling the binding of ATP in the catalytic active site. The active-like conformational equilibrium of this catalytically dead kinase is affected by a hydrophobic unit that connects to the hydrophobic spine network via the C-helix. The αC-helix plays a crucial role in integrating the hydrophobic residues, making it a hub for allosteric regulation of kinase activity and the active conformation. The computational free-energy landscapes reported here illustrate novel design principles focusing on the important role of the hydrophobic spines. The relative stability of the spines could be exploited in future efforts to artificially engineer active-like but catalytically dead forms of protein kinases.

cAMP-dependent protein kinase (PKAc) is a pivotal signaling protein in eukaryotic cells. PKAc has two well-characterized regulatory subunit proteins, RI and RII (each having α and β isoforms), which keep the PKAc catalytic subunit in a catalytically inactive state until activation by cAMP. Previous reports showed that the RIα regulatory subunit is phosphorylated by cGMP-dependent protein kinase (PKG) in vitro, whereupon phosphorylated RIα no longer inhibits PKAc at normal (1:1) stoichiometric ratios. However, the significance of this phosphorylation as a mechanism for activating type I PKA holoenzymes has not been fully explored, especially in cellular systems. In this study, we further examined the potential of RIα phosphorylation to regulate physiologically relevant "desensitization" of PKAc activity. First, the serine 101 site of RIα was validated as a target of PKGIα phosphorylation both in vitro and in cells. Analysis of a phosphomimetic substitution in RIα (S101E) showed that modification of this site increases PKAc activity in vitro and in cells, even without cAMP stimulation. Numerous techniques were used to show that although Ser101 variants of RIα can bind PKAc, the modified linker region of the S101E mutant has a significantly reduced affinity for the PKAc active site. These findings suggest that RIα phosphorylation may be a novel mechanism to circumvent the requirement of cAMP stimulus to activate type I PKA in cells. We have thus proposed a model to explain how PKG phosphorylation of RIα creates a "sensitized intermediate" state that is in effect primed to trigger PKAc activity.

Fibrous dysplasia (FD) is a disease caused by postzygotic activating mutations of GNAS (R201C and R201H) that encode the α-subunit of the Gs stimulatory protein. FD is characterized by the development of areas of abnormal fibroosseous tissue in the bones, resulting in skeletal deformities, fractures, and pain. Despite the well-defined genetic alterations underlying FD, whether GNAS activation is sufficient for FD initiation and the molecular and cellular consequences of GNAS mutations remains largely unresolved, and there are no currently available targeted therapeutic options for FD. Here, we have developed a conditional tetracycline (Tet)-inducible animal model expressing the GαsR201C in the skeletal stem cell (SSC) lineage (Tet-GαsR201C/Prrx1-Cre/LSL-rtTA-IRES-GFP mice), which develops typical FD bone lesions in both embryos and adult mice in less than 2 weeks following doxycycline (Dox) administration. Conditional GαsR201C expression promoted PKA activation and proliferation of SSCs along the osteogenic lineage but halted their differentiation to mature osteoblasts. Rather, as is seen clinically, areas of woven bone admixed with fibrous tissue were formed. GαsR201C caused the concomitant expression of receptor activator of nuclear factor kappa-B ligand (Rankl) that led to marked osteoclastogenesis and bone resorption. GαsR201C expression ablation by Dox withdrawal resulted in FD-like lesion regression, supporting the rationale for Gαs-targeted drugs to attempt FD cure. This model, which develops FD-like lesions that can form rapidly and revert on cessation of mutant Gαs expression, provides an opportunity to identify the molecular mechanism underlying FD initiation and progression and accelerate the development of new treatment options.

Eukaryotic protein kinases (EPKs) constitute a class of allosteric switches that mediate a myriad of signaling events. It has been postulated that EPKs' active and inactive states depend on the structural architecture of their hydrophobic cores, organized around two highly conserved spines: C-spine and R-spine. How the spines orchestrate the transition of the enzyme between catalytically uncommitted and committed states remains elusive. Using relaxation dispersion nuclear magnetic resonance spectroscopy, we found that the hydrophobic core of the catalytic subunit of protein kinase A, a prototypical and ubiquitous EPK, moves synchronously to poise the C subunit for catalysis in response to binding adenosine 5'-triphosphate. In addition to completing the C-spine, the adenine ring fuses the β structures of the N-lobe and the C-lobe. Additional residues that bridge the two spines (I150 and V104) are revealed as part of the correlated hydrophobic network; their importance was validated by mutagenesis, which led to inactivation. Because the hydrophobic architecture of the catalytic core is conserved throughout the EPK superfamily, the present study suggests a universal mechanism for dynamically driven allosteric activation of kinases mediated by coordinated signal transmission through ordered motifs in their hydrophobic cores.

Close-range electrostatic interactions that form salt bridges are key components of protein stability. Here we investigate the role of these charged interactions in modulating the allosteric activation of protein kinase A (PKA) via computational and experimental mutational studies of a conserved basic patch located in the regulatory subunit's B/C helix. Molecular dynamics simulations evidenced the presence of an extended network of fluctuating salt bridges spanning the helix and connecting the two cAMP binding domains in its extremities. Distinct changes in the flexibility and conformational free energy landscape induced by the separate mutations of Arg239 and Arg241 suggested alteration of cAMP-induced allosteric activation and were verified through in vitro fluorescence polarization assays. These observations suggest a mechanical aspect to the allosteric transition of PKA, with Arg239 and Arg241 acting in competition to promote the transition between the two protein functional states. The simulations also provide a molecular explanation for the essential role of Arg241 in allowing cooperative activation, by evidencing the existence of a stable interdomain salt bridge with Asp267. Our integrated approach points to the role of salt bridges not only in protein stability but also in promoting conformational transition and function.

The expertise of protein kinases lies in their dynamic structure, wherein they are able to modulate cellular signaling by their phosphotransferase activity. Only a few hundreds of protein kinases regulate key processes in human cells, and protein kinases play a pivotal role in health and disease. The present study dwells on understanding the working of the protein kinase-molecular switch as an allosteric network of "communities" composed of congruently dynamic residues that make up the protein kinase core. Girvan-Newman algorithm-based community maps of the kinase domain of cAMP-dependent protein kinase A allow for a molecular explanation for the role of protein conformational entropy in its catalytic cycle. The community map of a mutant, Y204A, is analyzed vis-à-vis the wild-type protein to study the perturbations in its dynamic profile such that it interferes with transfer of the γ-phosphate to a protein substrate. Conventional biochemical measurements are used to ascertain the effect of these dynamic perturbations on the kinetic profiles of both proteins. These studies pave the way for understanding how mutations far from the kinase active site can alter its dynamic properties and catalytic function even when major structural perturbations are not obvious from static crystal structures.

Protein kinase A (PKA) plays critical roles in neuronal function that are mediated by different regulatory (R) subunits. Deficiency in either the RIβ or the RIIβ subunit results in distinct neuronal phenotypes. Although RIβ contributes to synaptic plasticity, it is the least studied isoform. Using isoform-specific antibodies, we generated high-resolution large-scale immunohistochemical mosaic images of mouse brain that provided global views of several brain regions, including the hippocampus and cerebellum. The isoforms concentrate in discrete brain regions, and we were able to zoom-in to show distinct patterns of subcellular localization. RIβ is enriched in dendrites and co-localizes with MAP2, whereas RIIβ is concentrated in axons. Using correlated light and electron microscopy, we confirmed the mitochondrial and nuclear localization of RIβ in cultured neurons. To show the functional significance of nuclear localization, we demonstrated that downregulation of RIβ, but not of RIIβ, decreased CREB phosphorylation. Our study reveals how PKA isoform specificity is defined by precise localization.

Most disease-related mutations that impair cAMP protein kinase A (PKA) signaling are present within the regulatory (R) PKA RI alpha-subunit (RIα). Although mutations in the PRKAR1A gene are linked to Carney complex (CNC) disease and, more recently, to acrodysostosis-1 (ACRDYS1), the two diseases show contrasting phenotypes. While CNC mutations cause increased PKA activity, ACRDYS1 mutations result in decreased PKA activity and cAMP resistant holoenzymes. Mapping the ACRDYS1 disease mutations reveals their localization to the second of two tandem cAMP-binding (CNB) domains (CNB-B), and here, we characterize a recurrent deletion mutant where the last 14 residues are missing. The crystal structure of a monomeric form of this mutant (RIα92-365) bound to the catalytic (C)-subunit reveals the dysfunctional regions of the RIα subunit. Beyond the missing residues, the entire capping motif is disordered (residues 357-379) and explains the disrupted cAMP binding. Moreover, the effects of the mutation extend far beyond the CNB-B domain and include the active site and N-lobe of the C-subunit, which is in a partially open conformation with the C-tail disordered. A key residue that contributes to this crosstalk, D267, is altered in our structure, and we confirmed its functional importance by mutagenesis. In particular, the D267 interaction with Arg241, a residue shown earlier to be important for allosteric regulation, is disrupted, thereby strengthening the interaction of D267 with the C-subunit residue Arg194 at the R:C interface. We see here how the switch between active (cAMP-bound) and inactive (holoenzyme) conformations is perturbed and how the dynamically controlled crosstalk between the helical domains of the two CNB domains is necessary for the functional regulation of PKA activity.

Scaffolding proteins organize the information flow from activated G protein-coupled receptors (GPCRs) to intracellular effector cascades both spatially and temporally. By this means, signaling scaffolds, such as A-kinase anchoring proteins (AKAPs), compartmentalize kinase activity and ensure substrate selectivity. Using a phosphoproteomics approach we identified a physical and functional connection between protein kinase A (PKA) and Gpr161 (an orphan GPCR) signaling. We show that Gpr161 functions as a selective high-affinity AKAP for type I PKA regulatory subunits (RI). Using cell-based reporters to map protein-protein interactions, we discovered that RI binds directly and selectively to a hydrophobic protein-protein interaction interface in the cytoplasmic carboxyl-terminal tail of Gpr161. Furthermore, our data demonstrate that a binary complex between Gpr161 and RI promotes the compartmentalization of Gpr161 to the plasma membrane. Moreover, we show that Gpr161, functioning as an AKAP, recruits PKA RI to primary cilia in zebrafish embryos. We also show that Gpr161 is a target of PKA phosphorylation, and that mutation of the PKA phosphorylation site affects ciliary receptor localization. Thus, we propose that Gpr161 is itself an AKAP and that the cAMP-sensing Gpr161:PKA complex acts as cilium-compartmentalized signalosome, a concept that now needs to be considered in the analyzing, interpreting, and pharmaceutical targeting of PKA-associated functions.

Obesity and metabolic syndrome reflect the dysregulation of molecular pathways that control energy homeostasis. Here, we show that the p75 neurotrophin receptor (p75(NTR)) controls energy expenditure in obese mice on a high-fat diet (HFD). Despite no changes in food intake, p75(NTR)-null mice were protected from HFD-induced obesity and remained lean as a result of increased energy expenditure without developing insulin resistance or liver steatosis. p75(NTR) directly interacts with the catalytic subunit of protein kinase A (PKA) and regulates cAMP signaling in adipocytes, leading to decreased lipolysis and thermogenesis. Adipocyte-specific depletion of p75(NTR) or transplantation of p75(NTR)-null white adipose tissue (WAT) into wild-type mice fed a HFD protected against weight gain and insulin resistance. Our results reveal that signaling from p75(NTR) to cAMP/PKA regulates energy balance and suggest that non-CNS neurotrophin receptor signaling could be a target for treating obesity and the metabolic syndrome.

Protein kinases have very dynamic structures and their functionality strongly depends on their dynamic state. Active kinases reveal a dynamic pattern with residues clustering into semirigid communities that move in μs-ms timescale. Previously detected hydrophobic spines serve as connectors between communities. Communities do not follow the traditional subdomain structure of the kinase core or its secondary structure elements. Instead they are organized around main functional units. Integration of the communities depends on the assembly of the hydrophobic spine and phosphorylation of the activation loop. Single mutations can significantly disrupt the dynamic infrastructure and thereby interfere with long-distance allosteric signaling that propagates throughout the whole molecule. Dynamics is proposed to be the underlying mechanism for allosteric regulation in protein kinases.