Publications by authors named "Linda Truebestein"

9 Publications

  • Page 1 of 1

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

J Biol Chem 2021 Jun 25;297(2):100919. Epub 2021 Jun 25.

Department of Structural and Computational Biology, Max Perutz Labs, Vienna, Austria; Department of Medical Biochemistry, Medical University of Vienna, Vienna, Austria. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jbc.2021.100919DOI Listing
June 2021

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

Bioessays 2020 04 29;42(4):e1900222. Epub 2020 Jan 29.

Department of Structural and Computational Biology, Max Perutz Labs, Vienna Biocenter, 1030, Vienna, Austria.

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.
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http://dx.doi.org/10.1002/bies.201900222DOI Listing
April 2020

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

Proc Natl Acad Sci U S A 2018 04 9;115(17):E3940-E3949. Epub 2018 Apr 9.

Department of Structural and Computational Biology, Max F. Perutz Laboratories, 1030 Vienna, Austria;

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)P 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 PIP, 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 PIP 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.
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http://dx.doi.org/10.1073/pnas.1716109115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5924885PMC
April 2018

Coiled-coils: The long and short of it.

Bioessays 2016 09 5;38(9):903-16. Epub 2016 Aug 5.

Department of Structural and Computational Biology, Max F. Perutz Laboratories (MFPL), Vienna Biocenter (VBC), Vienna, Austria.

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.
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http://dx.doi.org/10.1002/bies.201600062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5082667PMC
September 2016

Made to measure - keeping Rho kinase at a distance.

Small GTPases 2016 04 12;7(2):82-92. Epub 2016 Apr 12.

a Department of Structural and Computational Biology , Max F. Perutz Laboratories (MFPL), Vienna Biocenter (VBC) , Vienna , Austria.

The Rho-associated coiled-coil containing kinases (ROCK) were first identified as effectors of the small GTPase RhoA, hence their nomenclature. Since their discovery, two decades ago, scientists have sought to unravel the structure, regulation, and function of these essential kinases. During that time, a consensus model has formed, in which ROCK activity is regulated via both Rho-dependent and independent mechanisms. However, recent findings have raised significant questions regarding this model. In their recent publication in Nature Communications, Truebestein and colleagues present the structure of a full-length Rho kinase for the first time. In contrast to previous reports, the authors could find no evidence for autoinhibition, RhoA binding, or regulation of kinase activity by phosphorylation. Instead, they propose that ROCK functions as a molecular ruler, in which the central coiled-coil bridges the membrane-binding regulatory domains to the kinase domains at a fixed distance from the plasma membrane. Here, we explore the consequences of the new findings, re-examine old data in the context of this model, and emphasize outstanding questions in the field.
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http://dx.doi.org/10.1080/21541248.2016.1173770DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4905274PMC
April 2016

A molecular ruler regulates cytoskeletal remodelling by the Rho kinases.

Nat Commun 2015 Dec 1;6:10029. Epub 2015 Dec 1.

Department of Structural and Computational Biology, Max F. Perutz Laboratories, Vienna Biocenter, 1030 Vienna, Austria.

The Rho-associated coiled-coil kinases (ROCK) are essential regulators of the actin cytoskeleton; however, the structure of a full-length ROCK is unknown and the mechanisms by which its kinase activity is controlled are not well understood. Here we determine the low-resolution structure of human ROCK2 using electron microscopy, revealing it to be a constitutive dimer, 120 nm in length, with a long coiled-coil tether linking the kinase and membrane-binding domains. We find, in contrast to previous reports, that ROCK2 activity does not appear to be directly regulated by binding to membranes, RhoA, or by phosphorylation. Instead, we show that changing the length of the tether modulates ROCK2 function in cells, suggesting that it acts as a molecular ruler. We present a model in which ROCK activity is restricted to a discrete region of the actin cytoskeleton, governed by the length of its coiled-coil. This represents a new type of spatial control, and hence a new paradigm for kinase regulation.
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http://dx.doi.org/10.1038/ncomms10029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4686654PMC
December 2015

Novel Features of DAG-Activated PKC Isozymes Reveal a Conserved 3-D Architecture.

J Mol Biol 2016 Jan 12;428(1):121-141. Epub 2015 Nov 12.

Department of Structural and Computational Biology, Max F. Perutz Laboratories, Medical University of Vienna, 1030 Vienna, Austria. Electronic address:

Diacylglycerol (DAG) activates the eight conventional and novel isozymes of protein kinase C (PKC) by binding to their C1 domains. The crystal structure of PKCβII in a partially activated conformation showed how the C1B domain regulates activity by clamping a helix in the C-terminal AGC extension of the kinase domain. Here we show that the global three-dimensional shape of the conventional and novel PKCs is conserved despite differences in the order of the domains in their primary sequences. The membrane translocation phenotypes of mutants in the C1B clamp are consistent across all DAG-activated PKCs, demonstrating conservation of this regulatory interface. We now identify a novel interface that sequesters the C1A domain in PKCβII in a membrane-inaccessible state and we generalize this to all DAG-activated PKCs. In the conventional PKCs, we identify a novel element of their C2 domains that additionally contributes to the stability of the inactive conformation. We demonstrate that the interdomain linkers play important roles in permitting and stabilizing this state. We propose a multi-step activation mechanism in which the sequential and cooperative binding of the regulatory domains to the membrane is coupled to allosteric activation of the kinase domain by DAG and that acquisition of full catalytic activity requires DAG binding to the C1B domain. In light of the conservation of shape and intramolecular architecture, we propose that this mechanism is common to all DAG-activated PKCs.
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http://dx.doi.org/10.1016/j.jmb.2015.11.001DOI Listing
January 2016

Human high temperature requirement serine protease A1 (HTRA1) degrades tau protein aggregates.

J Biol Chem 2012 Jun 25;287(25):20931-41. Epub 2012 Apr 25.

Centre for Medical Biotechnology, Faculty of Biology, University Duisburg-Essen, Universitaetsstrasse, 45141 Essen, Germany.

Protective proteases are key elements of protein quality control pathways that are up-regulated, for example, under various protein folding stresses. These proteases are employed to prevent the accumulation and aggregation of misfolded proteins that can impose severe damage to cells. The high temperature requirement A (HtrA) family of serine proteases has evolved to perform important aspects of ATP-independent protein quality control. So far, however, no HtrA protease is known that degrades protein aggregates. We show here that human HTRA1 degrades aggregated and fibrillar tau, a protein that is critically involved in various neurological disorders. Neuronal cells and patient brains accumulate less tau, neurofibrillary tangles, and neuritic plaques, respectively, when HTRA1 is expressed at elevated levels. Furthermore, HTRA1 mRNA and HTRA1 activity are up-regulated in response to elevated tau concentrations. These data suggest that HTRA1 is performing regulated proteolysis during protein quality control, the implications of which are discussed.
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http://dx.doi.org/10.1074/jbc.M111.316232DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3375517PMC
June 2012

Substrate-induced remodeling of the active site regulates human HTRA1 activity.

Nat Struct Mol Biol 2011 Mar 6;18(3):386-8. Epub 2011 Feb 6.

Centre for Medical Biotechnology, Faculty of Biology, University Duisburg-Essen, Essen, Germany.

Crystal structures of active and inactive conformations of the human serine protease HTRA1 reveal that substrate binding to the active site is sufficient to stimulate proteolytic activity. HTRA1 attaches to liposomes, digests misfolded proteins into defined fragments and undergoes substrate-mediated oligomer conversion. In contrast to those of other serine proteases, the PDZ domain of HTRA1 is dispensable for activation or lipid attachment, indicative of different underlying mechanistic features.
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http://dx.doi.org/10.1038/nsmb.2013DOI Listing
March 2011
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