Crystal structure and allosteric activation of protein kinase C ßII.

Protein kinase C (PKC) isozymes are the paradigmatic effectors of lipid signaling. PKCs translocate to cell membranes and are allosterically activated upon binding of the lipid diacylglycerol to their C1A and C1B domains. The crystal structure of full-length protein kinase C ßII was determined at 4.0 Å, revealing the conformation of an unexpected intermediate in the activation pathway. Here, the kinase active site is accessible to substrate, yet the conformation of the active site corresponds to a low-activity state because the ATP-binding side chain of Phe629 of the conserved NFD motif is displaced. The C1B domain clamps the NFD helix in a low-activity conformation, which is reversed upon membrane binding. A low-resolution solution structure of the closed conformation of PKCßII was derived from small-angle X-ray scattering. Together, these results show how PKCßII is allosterically regulated in two steps, with the second step defining a novel protein kinase regulatory mechanism.

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.

PI(3,4,5)P3 Engagement Restricts Akt Activity to Cellular Membranes.

Protein kinase B/Akt regulates cellular metabolism, survival, and proliferation in response to hormones and growth factors. Hyperactivation of Akt is frequently observed in cancer, while Akt inactivation is associated with severe diabetes. Here, we investigated the molecular and cellular mechanisms that maintain Akt activity proportional to the activating stimulus. We show that binding of phosphatidylinositol-3,4,5-trisphosphate (PIP3) or PI(3,4)P2 to the PH domain allosterically activates Akt by promoting high-affinity substrate binding. Conversely, dissociation from PIP3 was rate limiting for Akt dephosphorylation, dependent on the presence of the PH domain. In cells, active Akt associated primarily with cellular membranes. In contrast, a transforming mutation that uncouples kinase activation from PIP3 resulted in the accumulation of hyperphosphorylated, active Akt in the cytosol. Our results suggest that intramolecular allosteric and cellular mechanisms cooperate to restrict Akt activity to cellular membranes, thereby enhancing the fidelity of Akt signaling and the specificity of downstream substrate phosphorylation.

Molecular basis for the recruitment of the Rab effector protein WDR44 by the GTPase Rab11.

The formation of complexes between Rab11 and its effectors regulates multiple aspects of membrane trafficking, including recycling and ciliogenesis. WD repeat-containing protein 44 (WDR44) is a structurally uncharacterized Rab11 effector that regulates ciliogenesis by competing with prociliogenesis factors for Rab11 binding. Here, we present a detailed biochemical and biophysical characterization of the WDR44-Rab11 complex and define specific residues mediating binding. Using AlphaFold2 modeling and hydrogen/deuterium exchange mass spectrometry, we generated a molecular model of the Rab11-WDR44 complex. The Rab11-binding domain of WDR44 interacts with switch I, switch II, and the interswitch region of Rab11. Extensive mutagenesis of evolutionarily conserved residues in WDR44 at the interface identified numerous complex-disrupting mutations. Using hydrogen/deuterium exchange mass spectrometry, we found that the dynamics of the WDR44-Rab11 interface are distinct from the Rab11 effector FIP3, with WDR44 forming a more extensive interface with the switch II helix of Rab11 compared with FIP3. The WDR44 interaction was specific to Rab11 over evolutionarily similar Rabs, with mutations defining the molecular basis of Rab11 specificity. Finally, WDR44 can be phosphorylated by Sgk3, with this leading to reorganization of the Rab11-binding surface on WDR44. Overall, our results provide molecular detail on how WDR44 interacts with Rab11 and how Rab11 can form distinct effector complexes that regulate membrane trafficking events.

Structure of autoinhibited Akt1 reveals mechanism of PIP3-mediated activation.

The protein kinase Akt is one of the primary effectors of growth factor signaling in the cell. Akt responds specifically to the lipid second messengers phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P3] and phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2] via its PH domain, leading to phosphorylation of its activation loop and the hydrophobic motif of its kinase domain, which are critical for activity. We have now determined the crystal structure of Akt1, revealing an autoinhibitory interface between the PH and kinase domains that is often mutated in cancer and overgrowth disorders. This interface persists even after stoichiometric phosphorylation, thereby restricting maximum Akt activity to PI(3,4,5)P3- or PI(3,4)P2-containing membranes. Our work helps to resolve the roles of lipids and phosphorylation in the activation of Akt and has wide implications for the spatiotemporal control of Akt and potentially lipid-activated kinase signaling in general.

In vitro reconstitution of Sgk3 activation by phosphatidylinositol 3-phosphate.

Serum- and glucocorticoid-regulated kinase 3 (Sgk3) is a serine/threonine protein kinase activated by the phospholipid phosphatidylinositol 3-phosphate (PI3P) downstream of growth factor signaling via class I phosphatidylinositol 3-kinase (PI3K) signaling and by class III PI3K/Vps34-mediated PI3P production on endosomes. Upregulation of Sgk3 activity has recently been linked to a number of human cancers; however, the precise mechanism of activation of Sgk3 is unknown. Here, we use a wide range of cell biological, biochemical, and biophysical techniques, including hydrogen-deuterium exchange mass spectrometry, to investigate the mechanism of activation of Sgk3 by PI3P. We show that Sgk3 is regulated by a combination of phosphorylation and allosteric activation. We demonstrate that binding of Sgk3 to PI3P via its regulatory phox homology (PX) domain induces large conformational changes in Sgk3 associated with its activation and that the PI3P-binding pocket of the PX domain of Sgk3 is sequestered in its inactive conformation. Finally, we reconstitute Sgk3 activation via Vps34-mediated PI3P synthesis on phosphatidylinositol liposomes in vitro. In addition to identifying the mechanism of Sgk3 activation by PI3P, our findings open up potential therapeutic avenues in allosteric inhibitor development to target Sgk3 in cancer.

It Takes Two to Tango: Activation of Protein Kinase D by Dimerization.

The recent discovery and structure determination of a novel ubiquitin-like dimerization domain in protein kinase D (PKD) has significant implications for its activation. PKD is a serine/threonine kinase activated by the lipid second messenger diacylglycerol (DAG). It is an essential and highly conserved protein that is implicated in plasma membrane directed trafficking processes from the trans-Golgi network. However, many open questions surround its mechanism of activation, its localization, and its role in the biogenesis of cargo transport carriers. In reviewing this field, the focus is primarily on the mechanisms that control the activation of PKD at precise locations in the cell. In light of the new structural findings, the understanding of the mechanisms underlying PKD activation is critically evaluated, with particular emphasis on the role of dimerization in PKD autophosphorylation, and the provenance and recognition of the DAG that activates PKD.

Conformational sampling of membranes by Akt controls its activation and inactivation.

The protein kinase Akt controls myriad signaling processes in cells, ranging from growth and proliferation to differentiation and metabolism. Akt is activated by a combination of binding to the lipid second messenger PI(3,4,5)P3 and its subsequent phosphorylation by phosphoinositide-dependent kinase 1 and mechanistic target of rapamycin complex 2. The relative contributions of these mechanisms to Akt activity and signaling have hitherto not been understood. Here, we show that phosphorylation and activation by membrane binding are mutually interdependent. Moreover, the converse is also true: Akt is more rapidly dephosphorylated in the absence of PIP3, an autoinhibitory process driven by the interaction of its PH and kinase domains. We present biophysical evidence for the conformational changes in Akt that accompany its activation on membranes, show that Akt is robustly autoinhibited in the absence of PIP3 irrespective of its phosphorylation, and map the autoinhibitory PH-kinase interface. Finally, we present a model for the activation and inactivation of Akt by an ordered series of membrane binding, phosphorylation, dissociation, and dephosphorylation events.

A ubiquitin-like domain controls protein kinase D dimerization and activation by trans-autophosphorylation.

Protein kinase D (PKD) is an essential Ser/Thr kinase in animals and controls a variety of diverse cellular functions, including vesicle trafficking and mitogenesis. PKD is activated by recruitment to membranes containing the lipid second messenger diacylglycerol (DAG) and subsequent phosphorylation of its activation loop. Here, we report the crystal structure of the PKD N terminus at 2.2 Å resolution containing a previously unannotated ubiquitin-like domain (ULD), which serves as a dimerization domain. A single point mutation in the dimerization interface of the ULD not only abrogated dimerization in cells but also prevented PKD activation loop phosphorylation upon DAG production. We further show that the kinase domain of PKD dimerizes in a concentration-dependent manner and autophosphorylates on a single residue in its activation loop. We also provide evidence that PKD is expressed at concentrations 2 orders of magnitude below the ULD dissociation constant in mammalian cells. We therefore propose a new model for PKD activation in which the production of DAG leads to the local accumulation of PKD at the membrane, which drives ULD-mediated dimerization and subsequent trans-autophosphorylation of the kinase domain.

Lipid-dependent Akt-ivity: where, when, and how.

Akt is an essential protein kinase activated downstream of phosphoinositide 3-kinase and frequently hyperactivated in cancer. Canonically, Akt is activated by phosphoinositide-dependent kinase 1 and mechanistic target of rapamycin complex 2, which phosphorylate it on two regulatory residues in its kinase domain upon targeting of Akt to the plasma membrane by PI(3,4,5)P3 Recent evidence, however, has shown that, in addition to phosphorylation, Akt activity is allosterically coupled to the engagement of PI(3,4,5)P3 or PI(3,4)P2 in cellular membranes. Furthermore, the active membrane-bound conformation of Akt is protected from dephosphorylation, and Akt inactivation by phosphatases is rate-limited by its dissociation. Thus, Akt activity is restricted to membranes containing either PI(3,4,5)P3 or PI(3,4)P2 While PI(3,4,5)P3 has long been associated with signaling at the plasma membrane, PI(3,4)P2 is gaining increasing traction as a signaling lipid and has been implicated in controlling Akt activity throughout the endomembrane system. This has clear implications for the phosphorylation of both freely diffusible substrates and those localized to discrete subcellular compartments.

Coiled-coils: The long and short of it.

Coiled-coils are found in proteins throughout all three kingdoms of life. Coiled-coil domains of some proteins are almost invariant in sequence and length, betraying a structural and functional role for amino acids along the entire length of the coiled-coil. Other coiled-coils are divergent in sequence, but conserved in length, thereby functioning as molecular spacers. In this capacity, coiled-coil proteins influence the architecture of organelles such as centrioles and the Golgi, as well as permit the tethering of transport vesicles. Specialized coiled-coils, such as those found in motor proteins, are capable of propagating conformational changes along their length that regulate cargo binding and motor processivity. Coiled-coil domains have also been identified in enzymes, where they function as molecular rulers, positioning catalytic activities at fixed distances. Finally, while coiled-coils have been extensively discussed for their potential to nucleate and scaffold large macromolecular complexes, structural evidence to substantiate this claim is relatively scarce.

Features critical for membrane binding revealed by DivIVA crystal structure.

DivIVA is a conserved protein in Gram-positive bacteria that localizes at the poles and division sites, presumably through direct sensing of membrane curvature. DivIVA functions as a scaffold and is vital for septum site selection during vegetative growth and chromosome anchoring during sporulation. DivIVA deletion causes filamentous growth in Bacillus subtilis, whereas overexpression causes hyphal branching in Streptomyces coelicolor. We have determined the crystal structure of the N-terminal (Nt) domain of DivIVA, and show that it forms a parallel coiled-coil. It is capped with two unique crossed and intertwined loops, exposing hydrophobic and positively charged residues that we show here are essential for membrane binding. An intragenic suppressor introducing a positive charge restores membrane binding after mutating the hydrophobic residues. We propose that the hydrophobic residues insert into the membrane and that the positively charged residues bind to the membrane surface. A low-resolution crystal structure of the C-terminal (Ct) domain displays a curved tetramer made from two parallel coiled-coils. The Nt and Ct parts were then merged into a model of the full length, 30 nm long DivIVA protein.

A critical evaluation of protein kinase regulation by activation loop autophosphorylation.

Phosphorylation of proteins is a ubiquitous mechanism of regulating their function, localization, or activity. Protein kinases, enzymes that use ATP to phosphorylate protein substrates are, therefore, powerful signal transducers in eukaryotic cells. The mechanism of phosphoryl-transfer is universally conserved among protein kinases, which necessitates the tight regulation of kinase activity for the orchestration of cellular processes with high spatial and temporal fidelity. In response to a stimulus, many kinases enhance their own activity by autophosphorylating a conserved amino acid in their activation loop, but precisely how this reaction is performed is controversial. Classically, kinases that autophosphorylate their activation loop are thought to perform the reaction in trans, mediated by transient dimerization of their kinase domains. However, motivated by the recently discovered regulation mechanism of activation loop cis-autophosphorylation by a kinase that is autoinhibited in trans, we here review the various mechanisms of autoregulation that have been proposed. We provide a framework for critically evaluating biochemical, kinetic, and structural evidence for protein kinase dimerization and autophosphorylation, and share some thoughts on the implications of these mechanisms within physiological signaling networks.

The membrane surface as a platform that organizes cellular and biochemical processes.

Membranes are essential for life. They act as semi-permeable boundaries that define cells and organelles. In addition, their surfaces actively participate in biochemical reaction networks, where they confine proteins, align reaction partners, and directly control enzymatic activities. Membrane-localized reactions shape cellular membranes, define the identity of organelles, compartmentalize biochemical processes, and can even be the source of signaling gradients that originate at the plasma membrane and reach into the cytoplasm and nucleus. The membrane surface is, therefore, an essential platform upon which myriad cellular processes are scaffolded. In this review, we summarize our current understanding of the biophysics and biochemistry of membrane-localized reactions with particular focus on insights derived from reconstituted and cellular systems. We discuss how the interplay of cellular factors results in their self-organization, condensation, assembly, and activity, and the emergent properties derived from them.

Structure and regulation of the myotonic dystrophy kinase-related Cdc42-binding kinase.

Protein kinases of the dystonia myotonica protein kinase (DMPK) family are critical regulators of actomyosin contractility in cells. The DMPK kinase MRCK1 is required for the activation of myosin, leading to the development of cortical tension, apical constriction, and early gastrulation. Here, we present the structure, conformation, and membrane-binding properties of Caenorhabditis elegans MRCK1. MRCK1 forms a homodimer with N-terminal kinase domains, a parallel coiled coil of 55 nm, and a C-terminal tripartite module of C1, pleckstrin homology (PH), and citron homology (CNH) domains. We report the high-resolution structure of the membrane-binding C1-PH-CNH module of MRCK1 and, using high-throughput and conventional liposome-binding assays, determine its binding to specific phospholipids. We further characterize the interaction of the C-terminal CRIB motif with Cdc42. The length of the coiled-coil domain of DMPK kinases is remarkably conserved over millions of years of evolution, suggesting that they may function as molecular rulers to position kinase activity at a fixed distance from the membrane.

ATP-competitive and allosteric inhibitors induce differential conformational changes at the autoinhibitory interface of Akt1.

Akt is a master regulator of pro-growth signaling in the cell. Akt is activated by phosphoinositides that disrupt the autoinhibitory interface between the kinase and pleckstrin homology (PH) domains and then is phosphorylated at T308 and S473. Akt hyperactivation is oncogenic, which has spurred development of potent and selective inhibitors as therapeutics. Using hydrogen deuterium exchange mass spectrometry (HDX-MS), we interrogated the conformational changes upon binding Akt ATP-competitive and allosteric inhibitors. We compared inhibitors against three different states of Akt1. The allosteric inhibitor caused substantive conformational changes and restricts membrane binding. ATP-competitive inhibitors caused extensive allosteric conformational changes, altering the autoinhibitory interface and leading to increased membrane binding, suggesting that the PH domain is more accessible for membrane binding. This work provides unique insight into the autoinhibitory conformation of the PH and kinase domain and conformational changes induced by Akt inhibitors and has important implications for the design of Akt targeted therapeutics.

Activation of the essential kinase PDK1 by phosphoinositide-driven trans-autophosphorylation.

3-phosphoinositide-dependent kinase 1 (PDK1) is an essential serine/threonine protein kinase, which plays a crucial role in cell growth and proliferation. It is often referred to as a 'master' kinase due to its ability to activate at least 23 downstream protein kinases implicated in various signaling pathways. In this study, we have elucidated the mechanism of phosphoinositide-driven PDK1 auto-activation. We show that PDK1 trans-autophosphorylation is mediated by a PIP3-mediated face-to-face dimer. We report regulatory motifs in the kinase-PH interdomain linker that allosterically activate PDK1 autophosphorylation via a linker-swapped dimer mechanism. Finally, we show that PDK1 is autoinhibited by its PH domain and that positive cooperativity of PIP3 binding drives switch-like activation of PDK1. These results imply that the PDK1-mediated activation of effector kinases, including Akt, PKC, Sgk, S6K and RSK, many of whom are not directly regulated by phosphoinositides, is also likely to be dependent on PIP3 or PI(3,4)P2.

Structures of three MORN repeat proteins and a re-evaluation of the proposed lipid-binding properties of MORN repeats.

MORN (Membrane Occupation and Recognition Nexus) repeat proteins have a wide taxonomic distribution, being found in both prokaryotes and eukaryotes. Despite this ubiquity, they remain poorly characterised at both a structural and a functional level compared to other common repeats. In functional terms, they are often assumed to be lipid-binding modules that mediate membrane targeting. We addressed this putative activity by focusing on a protein composed solely of MORN repeats-Trypanosoma brucei MORN1. Surprisingly, no evidence for binding to membranes or lipid vesicles by TbMORN1 could be obtained either in vivo or in vitro. Conversely, TbMORN1 did interact with individual phospholipids. High- and low-resolution structures of the MORN1 protein from Trypanosoma brucei and homologous proteins from the parasites Toxoplasma gondii and Plasmodium falciparum were obtained using a combination of macromolecular crystallography, small-angle X-ray scattering, and electron microscopy. This enabled a first structure-based definition of the MORN repeat itself. Furthermore, all three structures dimerised via their C-termini in an antiparallel configuration. The dimers could form extended or V-shaped quaternary structures depending on the presence of specific interface residues. This work provides a new perspective on MORN repeats, showing that they are protein-protein interaction modules capable of mediating both dimerisation and oligomerisation.

Mutations in MAST1 Cause Mega-Corpus-Callosum Syndrome with Cerebellar Hypoplasia and Cortical Malformations.

Corpus callosum malformations are associated with a broad range of neurodevelopmental diseases. We report that de novo mutations in MAST1 cause mega-corpus-callosum syndrome with cerebellar hypoplasia and cortical malformations (MCC-CH-CM) in the absence of megalencephaly. We show that MAST1 is a microtubule-associated protein that is predominantly expressed in post-mitotic neurons and is present in both dendritic and axonal compartments. We further show that Mast1 null animals are phenotypically normal, whereas the deletion of a single amino acid (L278del) recapitulates the distinct neurological phenotype observed in patients. In animals harboring Mast1 microdeletions, we find that the PI3K/AKT3/mTOR pathway is unperturbed, whereas Mast2 and Mast3 levels are diminished, indicative of a dominant-negative mode of action. Finally, we report that de novo MAST1 substitutions are present in patients with autism and microcephaly, raising the prospect that mutations in this gene give rise to a spectrum of neurodevelopmental diseases.