Biology subjects

Leonard, T. A.

Publications and source records attributed to Leonard, T. A..

10 recordsLinked to original sources

bioRxiv2026

Single-molecule reaction chambers reveal intramolecular protein kinase activation

Many protein kinases, ubiquitous signal transducers across all kingdoms of life, are activated by phosphorylation of one or more conserved residues in their activation loops. This reaction is widely considered to be intermolecular (trans) in nature, dependent on the transient dimerization of two kinase domains. However, since the substrate, product and reaction chemistry are identical, bulk solution kinase assays cannot distinguish between intra- and inter-molecular reactions. Discriminating between these two mechanisms is crucial because they impose different requirements on the cell for acute, high-fidelity signal transduction. Using single-molecule reaction chambers fabricated from DNA origami, we show that the paradigmatic insulin receptor kinase (IRK) undergoes intramolecular (cis) autophosphorylation with kinetics indistinguishable from those in free solution. Furthermore, we demonstrate that the bacterial serine/threonine kinase protein kinase B (PknB) undergoes intramolecular autophosphorylation, allosterically stimulated by intermolecular (trans) interactions. Our findings establish that intramolecular activation loop autophosphorylation is not only possible, but likely an ancient and conserved feature of protein kinase signaling. More broadly, our work provides new tools for single-molecule enzymology.

biochemistry↗

bioRxiv2026

Dimerization-dependent cis-autophosphorylation activates the UPR and ISR kinases

Maintaining cellular homeostasis requires the ability to detect and respond to various forms of stress. Protein kinases of the unfolded protein response (UPR) monitor protein folding in the endoplasmic reticulum, while the integrated stress response (ISR) kinases sense viral infection, nutrient deprivation, mitochondrial oxidative stress and proteotoxic stress. However, the precise molecular mechanism by which these kinases are activated is still unknown. In this study, we show that the ISR kinases PERK, HRI, and Gcn2, as well as the UPR kinase Ire1, undergo dimerization-dependent cis-autophosphorylation of their activation loops. The back-to-back dimerization of the kinase domain allosterically activates each kinase. We derive a simple mathematical model for dimerization-dependent cis-autophosphorylation, which we use to obtain kinetic parameters of PERK autophosphorylation. We show that dimerization promotes not only activation loop phosphorylation of PERK, but also phosphorylation of its substrate, eIF2. In cells, kinase domain dimerization is necessary and sufficient for the activation of PERK. In summary, we propose a model in which a dimer is the minimal functional unit of all UPR and ISR kinases.

biochemistry↗

bioRxiv2025

A complex of MAST1 and 14-3-3 regulates Tau phosphorylation in the developing cortex

The MAST family of serine/threonine kinases has been implicated in a spectrum of human neurodevelopmental disorders. However, little is known about their biological function or regulation. Seeking to fill these gaps in our knowledge, we have identified upstream and downstream partners of MAST1. 14-3-3, a neuronal 14-3-3 paralog, specifically interacts with MAST1 at two regulatory serines, S90 and S161. PAK, a neuronal regulator of the actin cytoskeleton, phosphorylates MAST1 to regulate its interaction with 14-3-3. Exploiting mouse models of human Mega Corpus Callosum Syndrome (MCC) and whole brain phosphoproteomics, we identify the microtubule-associated protein Tau as a substrate of MAST1. We show that pathogenic MAST1 mutations perturb protein function either through misfolding or attenuation of kinase activity. Our data is consistent with a model in which the MAST kinases couple PAK, a neuronal regulator of the actin cytoskeleton, to microtubule remodeling during the differentiation and specification of cortical neurons.

molecular biology↗

bioRxiv2025

Mechanism of activation of an ancestral Tec kinase by PIP3

The TEC kinases are a family of five paralogous mammalian genes that play crucial roles in cell growth, proliferation and differentiation, particularly in immune cells. The recruitment and activation of the TEC kinases depend on the generation of the lipid second messenger, PIP3, in the plasma membrane. However, the mechanisms by which PIP3 activates the TEC kinases are not well understood. We have elucidated the autoinhibited conformation of an ancestral TEC kinase from the choanoflagellate Monosiga brevicollis. We demonstrate that PIP3 relieves autoinhibition of MbTEC by displacing its PH domain from an evolutionarily conserved inhibitory interaction with its kinase domain. We also show that a conserved polyproline motif within MbTEC promotes its activation in a kinase-intrinsic mechanism. Finally, we show that the PH domain is sufficient to restore autoinhibition in a constitutively active mutant of MbTEC. Our findings reveal that PIP3 is necessary and sufficient for both MbTEC activation and inactivation. Significance StatementThe Tec family of protein kinases plays an essential role in cell signaling, particularly in the proliferation and differentiation of immune cells. Consequently, their dysregulation is causative of inherited immunodeficiency, while the Tec kinases are also therapeutic targets in the control of hematological malignancies. We have elucidated a conserved mechanism by which the Tec kinases are activated by the lipid second messenger PIP3. PIP3 is necessary and sufficient for Tec activation, while its turnover is sufficient for Tec inactivation. Our work identifies PIP3 as the ultimate gatekeeper of Tec activity in cells, with implications for the rationalization and treatment of human disease.

biochemistry↗

bioRxiv2024

PHLPP2 is a pseudophosphatase that lost activity in the metazoan ancestor

The phosphoinositide 3-kinase (PI3K) pathway is a major regulator of cell and organismal growth. Consequently, hyperactivation of PI3K and its downstream effector kinase, Akt, is observed in many human cancers. PH domain leucine-rich repeat-containing protein phosphatases (PHLPP), two paralogous members of the metal-dependent protein phosphatase family, have been reported as negative regulators of Akt signaling and, therefore, tumor suppressors. However, the stoichiometry and identity of the bound metal ion(s), mechanism of action, and enzymatic specificity of these proteins are not known. Seeking to fill these gaps in our understanding of PHLPP biology, we unexpectedly discovered that PHLPP2 has no catalytic activity against the regulatory phosphorylation sites of Akt, nor the generic substrate para -nitrophenylphosphate. Instead, we found that PHLPP2 is a pseudophosphatase with a single zinc ion bound in its catalytic center. Furthermore, we found that current cancer genomics data do not support the proposed role of PHLPP1 or PHLPP2 as tumor suppressors. Phylogenetic analyses revealed an ancestral phosphatase that arose more than 1 Mya, but that lost activity at the base of the metazoan lineage. In summary, our results provide a molecular explanation for the inconclusive results that have hampered research on PHLPP and argue for a new focus on non-catalytic roles of PHLPP1 and PHLPP2. Significance StatementPHLPP1 and PHLPP2 have previously been reported as protein phosphatases that specifically inactivate Akt, a pro-growth and survival kinase hyperactivated in many human cancers. Unexpectedly, we found that purified PHLPP2 has no detectable enzymatic activity in vitro, an observation which can be rationalized by its unusual active site, which has diverged significantly from that of canonical metal-dependent phosphatases. Furthermore, we show that cancer genomics do not support a role for either PHLPP1 or PHLPP2 in cancer. Our findings argue for the exploration of alternative hypotheses regarding the role of PHLPP in Akt signaling and cancer, with a focus on its non-catalytic functions.

biochemistry↗

bioRxiv2022

ATP-competitive and allosteric inhibitors induce differential conformational changes at the auto-inhibitory interface of Akt

The protein kinase Akt is a master regulator of pro-growth signalling in the cell. Akt is activated through its targeted recruitment to phosphoinositides, leading to disruption of the autoinhibitory interface between the kinase and pleckstrin homology (PH) domains. Hyper activation of Akt is common in oncogenic transformation, with multiple oncogenic activating mutants identified in Akt. This has led to the development of potent and selective ATP-competitive and allosteric inhibitors for Akt. Paradoxically, some ATP-competitive Akt inhibitors cause hyperphosphorylation of Akt. Here, using hydrogen deuterium exchange mass spectrometry (HDX-MS), we interrogated the conformational changes upon binding to the Akt active site inhibitor A-443654, and the Akt allosteric inhibitor MK-2206. We compared the conformational changes that occurred for each inhibitor under three different states of Akt: i-inactive monophosphorylated, ii-partially active tris-phosphorylated [T308, T450, S473], and iii-fully activated, tris-phosphorylated bound to PIP3 membranes. The allosteric MK-2206 inhibitor results in large scale allosteric conformational changes in all states, and restricts membrane binding through sequestration of the PH domain. Binding of the A-443654 inhibitor led to large scale allosteric conformational changes in both the monophosphorylated and phosphorylated states, leading to an alteration in the autoinhibitory PH-kinase interface. We also observed increased protection in the PH domain upon membrane binding in the presence of A-443654, 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 dynamic conformational changes induced by Akt inhibitors, and has important implications for the design of Akt targeted therapeutics.

biochemistry↗

bioRxiv2022

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 phospho-transfer reaction are, themselves, often regulated by phosphorylation. Paradoxically, however, a substantial fraction of the 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 auto- 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 have profound implications for the regulation of many other eukaryotic kinases.

biochemistry↗

bioRxiv2022

Structure and regulation of the Myotonic dystrophy kinase-Related Cdc42-binding Kinase

Remodeling of the cytoskeleton underlies myriad processes essential for life. Protein kinases of the DMPK family are critical regulators of actomyosin contractility in cells. In the nematode worm, Caenorhabditis elegans, 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 C. elegans MRCK1. MRCK1 forms an obligate homodimer with N-terminal kinase domains, a parallel coiled-coil of 55 nm, and a C-terminal tripartite module of C1, PH and CNH domains. High-throughput liposome binding assays indicate binding to specific phosphoinositides, while the C-terminal Cdc42-binding (CRIB) motif binds specifically to activated Cdc42. The length of the coiled-coil domain of MRCK, as well as those of the related DMPK kinases ROCK, CRIK and DMPK, is remarkably conserved over millions of years of evolution, suggesting that they may function as molecular rulers to precisely position kinase activity at a fixed distance from the membrane.

biochemistry↗

bioRxiv2021

Activation of the essential kinase PDK1 by phosphoinositide-driven 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. Our work implies 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.

biochemistry↗

bioRxiv2021

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

Serum- and glucocorticoid-regulated kinase 3 (Sgk3) is activated by the phospholipid phosphatidylinositol-3-phosphate (PI3P) downstream of growth factor signaling and by Vps34-mediated PI3P production on endosomes. Upregulation of Sgk3 activity has recently been linked to a number of human cancers. Here, we show that Sgk3 is regulated by a combination of phosphorylation and allosteric activation by PI3P. We demonstrate that PI3P binding 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 reconstituted Sgk3 activation via Vps34-mediated PI3P synthesis on phosphatidylinositol liposomes in vitro. In addition to defining the mechanism of Sgk3 activation by PI3P, our findings open up potential therapeutic avenues in allosteric inhibitor development to target Sgk3 in cancer.

biochemistry↗