Structural Basis for Auto-Inhibition of the NDR1 Kinase Domain by an Atypically Long Activation Segment

AbstractUpon ligand binding, bone morphogenetic protein (BMP) receptors form active tetrameric complexes, comprised of two type I and two type II receptors, which then transmit signals to SMAD proteins. The link between receptor tetramerization and the mechanism of kinase activation, however, has not been elucidated. Here, using hydrogen deuterium exchange mass spectrometry (HDX-MS), small angle X-ray scattering (SAXS) and molecular dynamics (MD) simulations, combined with analysis of SMAD signaling, we show that the kinase domain of the type I receptor ALK2 and type II receptor BMPR2 form a heterodimeric complex via their C-terminal lobes. Formation of this dimer is essential for ligand-induced receptor signaling and is targeted by mutations in BMPR2 in patients with pulmonary arterial hypertension (PAH). We further show that the type I/type II kinase domain heterodimer serves as the scaffold for assembly of the active tetrameric receptor complexes to enable phosphorylation of the GS domain and activation of SMADs.

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Structural basis for VPS34 kinase activation by Rab1 and Rab5 on membranes

Nature Communications ◽

10.1038/s41467-021-21695-2 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Shirley Tremel ◽

Yohei Ohashi ◽

Dustin R. Morado ◽

Jessie Bertram ◽

Olga Perisic ◽

...

Keyword(s):

Complex I ◽

Kinase Domain ◽

Beclin 1 ◽

Structural Basis ◽

Rab Gtpases ◽

Lipid Kinase ◽

Complex Ii ◽

Cellular Processes ◽

Specific Complex ◽

Electron Cryotomography

AbstractThe lipid phosphatidylinositol-3-phosphate (PI3P) is a regulator of two fundamental but distinct cellular processes, endocytosis and autophagy, so its generation needs to be under precise temporal and spatial control. PI3P is generated by two complexes that both contain the lipid kinase VPS34: complex II on endosomes (VPS34/VPS15/Beclin 1/UVRAG), and complex I on autophagosomes (VPS34/VPS15/Beclin 1/ATG14L). The endosomal GTPase Rab5 binds complex II, but the mechanism of VPS34 activation by Rab5 has remained elusive, and no GTPase is known to bind complex I. Here we show that Rab5a–GTP recruits endocytic complex II to membranes and activates it by binding between the VPS34 C2 and VPS15 WD40 domains. Electron cryotomography of complex II on Rab5a-decorated vesicles shows that the VPS34 kinase domain is released from inhibition by VPS15 and hovers over the lipid bilayer, poised for catalysis. We also show that the GTPase Rab1a, which is known to be involved in autophagy, recruits and activates the autophagy-specific complex I, but not complex II. Both Rabs bind to the same VPS34 interface but in a manner unique for each. These findings reveal how VPS34 complexes are activated on membranes by specific Rab GTPases and how they are recruited to unique cellular locations.

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Phosphorylation of Tyrosine Residues in the Kinase Domain and Juxtamembrane Region Regulates the Biological and Catalytic Activities of Eph Receptors

Molecular and Cellular Biology ◽

10.1128/mcb.20.13.4791-4805.2000 ◽

2000 ◽

Vol 20 (13) ◽

pp. 4791-4805 ◽

Cited By ~ 123

Author(s):

Kathleen L. Binns ◽

Paul P. Taylor ◽

Frank Sicheri ◽

Tony Pawson ◽

Sacha J. Holland

Keyword(s):

Tyrosine Kinases ◽

Biological Activities ◽

Phosphorylation Site ◽

Kinase Domain ◽

Kinase Assay ◽

Eph Receptors ◽

Amino Acid Homology ◽

Tyrosine Residues ◽

Juxtamembrane Region ◽

Activation Segment

ABSTRACT Members of the Eph family of receptor tyrosine kinases exhibit a striking degree of amino acid homology, particularly notable in the kinase and membrane-proximal regions. A mutagenesis approach was taken to address the functions of specific conserved tyrosine residues within these catalytic and juxtamembrane domains. Ligand stimulation of wild-type EphB2 in neuronal NG108-15 cells resulted in an upregulation of catalytic activity and an increase in cellular tyrosine phosphorylation, accompanied by a retraction of neuritic processes. Tyrosine-to-phenylalanine substitutions within the conserved juxtamembrane motif abolished these responses. The mechanistic basis for these observations was examined using the highly related EphA4 receptor in a continuous coupled kinase assay. Tandem mass spectrometry experiments confirmed autophosphorylation of the two juxtamembrane tyrosine residues and also identified a tyrosine within the kinase domain activation segment as a phosphorylation site. Kinetic analysis revealed a decreased affinity for peptide substrate upon substitution of activation segment or juxtamembrane tyrosines. Together, our data suggest that the catalytic and therefore biological activities of Eph receptors are controlled by a two-component inhibitory mechanism, which is released by phosphorylation of the juxtamembrane and activation segment tyrosine residues.

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Tyr198 is the Essential Autophosphorylation Site for STK16 Localization and Kinase Activity

International Journal of Molecular Sciences ◽

10.3390/ijms20194852 ◽

2019 ◽

Vol 20 (19) ◽

pp. 4852 ◽

Cited By ~ 1

Author(s):

Junjun Wang ◽

Juanjuan Liu ◽

Xinmiao Ji ◽

Xin Zhang

Keyword(s):

Cell Cycle ◽

Cell Cycle Progression ◽

Kinase Activity ◽

Kinase Domain ◽

Serine Threonine Kinase ◽

Cycle Progression ◽

Threonine Kinase ◽

Cellular Processes ◽

Activation Segment ◽

And Function

STK16, reported as a Golgi localized serine/threonine kinase, has been shown to participate in multiple cellular processes, including the TGF-β signaling pathway, TGN protein secretion and sorting, as well as cell cycle and Golgi assembly regulation. However, the mechanisms of the regulation of its kinase activity remain underexplored. It was known that STK16 is autophosphorylated at Thr185, Ser197, and Tyr198 of the activation segment in its kinase domain. We found that STK16 localizes to the cell membrane and the Golgi throughout the cell cycle, but mutations in the auto-phosphorylation sites not only alter its subcellular localization but also affect its kinase activity. In particular, the Tyr198 mutation alone significantly reduced the kinase activity of STK16, abolished its Golgi and membrane localization, and affected the cell cycle progression. This study demonstrates that a single site autophosphorylation of STK16 could affect its localization and function, which provides insights into the molecular regulatory mechanism of STK16’s kinase activity.

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Structural basis for the autoinhibition of the C-terminal kinase domain of human RSK1

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s0907444912007457 ◽

2012 ◽

Vol 68 (6) ◽

pp. 680-685 ◽

Cited By ~ 5

Author(s):

Dan Li ◽

Tian-Min Fu ◽

Jie Nan ◽

Cong Liu ◽

Lan-Fen Li ◽

...

Keyword(s):

Kinase Domain ◽

Structural Basis

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Structural basis for small molecule targeting of Doublecortin Like Kinase 1 DCLK1

10.21203/rs.3.rs-133153/v1 ◽

2021 ◽

Author(s):

Onisha Patel ◽

Michael Roy ◽

Ashleigh Kropp ◽

Weiwen Dai ◽

Isabelle Lucet

Keyword(s):

Kinase Inhibitor ◽

Critical Role ◽

Structural Data ◽

Kinase Domain ◽

Microtubule Assembly ◽

Systematic Evaluation ◽

Structural Basis ◽

Allosteric Site ◽

Functional Protein ◽

Wide Range

Abstract Doublecortin-like kinase 1 (DCLK1) is a bi-functional protein classified as a Microtubule-Associated Protein (MAP) and as a serine/threonine kinase that plays a critical role in regulating microtubule assembly. This understudied kinase is upregulated or mutated in a wide range of cancers. Knockdown studies have shown that DCLK1 is functionally important for tumour growth. However, the presence of tissue and development specific spliced DCLK1 isoforms and the lack of systematic evaluation of their biological function have challenged the development of effective strategies to understand the role of DCLK1 in oncogenesis. Recently, DCLK1-IN-1 was reported as a potent and selective DCLK1 kinase inhibitor, a powerful new tool to dissect DCLK1 biological functions. Here, we report the crystal structures of DCLK1 kinase domain in complex with two DCLK1-IN-1 precursors and DCLK-IN-1. Combined, our structural data analysis illuminates and rationalises the structure-activity relationship that informed development of DCLK1-IN-1 and provides the basis for DCLK1-IN-1 increased selectivity. We show that DCLK1-IN-1 induces a drastic conformational change of the N-lobe, which uncovered a new allosteric site. In addition, we demonstrate that DCLK1-IN-1 binds DCLK1 long isoforms with high affinity but does not prevent DCLK1 MAP function. Together, our work outlines the need for in-depth studies to rationally design of isoform-specific modulators and provides an invaluable structural platform to further the design of selective DCLK1 therapeutic agents.

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Crystal structure of death-associated protein kinase 1 in complex with the dietary compound resveratrol

IUCrJ ◽

10.1107/s2052252520015614 ◽

2021 ◽

Vol 8 (1) ◽

pp. 131-138

Author(s):

Takeshi Yokoyama ◽

Ryoya Suzuki ◽

Mineyuki Mizuguchi

Keyword(s):

Catalytic Activity ◽

Protein Kinase ◽

Binding Mode ◽

Competitive Inhibitor ◽

Kinase Domain ◽

Crystallographic Analysis ◽

Structural Basis ◽

Atp Binding Site ◽

Death Associated Protein Kinase

Death-associated protein kinase 1 (DAPK1) is a large multidomain protein with an N-terminal serine/threonine protein kinase domain. DAPK1 is considered to be a promising molecular target for the treatment of Alzheimer's disease (AD). In the present study, the inhibitory potency of resveratrol (RSV), a dietary polyphenol found in red wine, against the catalytic activity of DAPK1 was investigated. Kinetic and fluorescent probe competitive binding analyses revealed that RSV directly inhibited the catalytic activity of DAPK1 by binding to the ATP-binding site. Crystallographic analysis of DAPK1 in complex with RSV revealed that the A-ring of RSV occupied the nucleobase-binding position. Determination of the binding mode provided a structural basis for the design of more potent DAPK1 inhibitors. In conclusion, the data here clearly show that RSV is an ATP-competitive inhibitor of DAPK1, encouraging speculation that RSV may be useful for the development of AD inhibitors.

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Structural basis of oncogenic activation caused by point mutations in the kinase domain of the MET proto-oncogene: Modeling studies

Proteins Structure Function and Bioinformatics ◽

10.1002/prot.1069 ◽

2001 ◽

Vol 44 (1) ◽

pp. 32-43 ◽

Cited By ~ 48

Author(s):

Maria Miller ◽

Krzysztof Ginalski ◽

Bogdan Lesyng ◽

Noboru Nakaigawa ◽

Laura Schmidt ◽

...

Keyword(s):

Point Mutations ◽

Kinase Domain ◽

Structural Basis ◽

Modeling Studies

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Structural basis for UCN-01 (7-hydroxystaurosporine) specificity and PDK1 (3-phosphoinositide-dependent protein kinase-1) inhibition

Biochemical Journal ◽

10.1042/bj20031119 ◽

2003 ◽

Vol 375 (2) ◽

pp. 255-262 ◽

Cited By ~ 73

Author(s):

David KOMANDER ◽

Gursant S. KULAR ◽

Jennifer BAIN ◽

Matthew ELLIOTT ◽

Dario R. ALESSI ◽

...

Keyword(s):

Protein Kinase ◽

Hydroxy Group ◽

Active Site ◽

Binding Pocket ◽

Kinase Domain ◽

Structural Basis ◽

Active Site Residues ◽

Dependent Protein Kinase ◽

Growth Factor Signalling ◽

Dependent Protein

PDK1 (3-phosphoinositide-dependent protein kinase-1) is a member of the AGC (cAMP-dependent, cGMP-dependent, protein kinase C) family of protein kinases, and has a key role in insulin and growth-factor signalling through phosphorylation and subsequent activation of a number of other AGC kinase family members, such as protein kinase B. The staurosporine derivative UCN-01 (7-hydroxystaurosporine) has been reported to be a potent inhibitor for PDK1, and is currently undergoing clinical trials for the treatment of cancer. Here, we report the crystal structures of staurosporine and UCN-01 in complex with the kinase domain of PDK1. We show that, although staurosporine and UCN-01 interact with the PDK1 active site in an overall similar manner, the UCN-01 7-hydroxy group, which is not present in staurosporine, generates direct and water-mediated hydrogen bonds with active-site residues. Inhibition data from UCN-01 tested against a panel of 29 different kinases show a different pattern of inhibition compared with staurosporine. We discuss how these differences in inhibition could be attributed to specific interactions with the additional 7-hydroxy group, as well as the size of the 7-hydroxy-group-binding pocket. This information could lead to opportunities for structure-based optimization of PDK1 inhibitors.

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AtCERK1 Phosphorylation Site S493 Contributes to the Transphosphorylation of Downstream Components for Chitin-Induced Immune Signaling

Plant and Cell Physiology ◽

10.1093/pcp/pcz096 ◽

2019 ◽

Vol 60 (8) ◽

pp. 1804-1810 ◽

Cited By ~ 6

Author(s):

Maruya Suzuki ◽

Issei Yoshida ◽

Kenkichi Suto ◽

Yoshitake Desaki ◽

Naoto Shibuya ◽

...

Keyword(s):

Catalytic Activity ◽

Induced Defense ◽

Phosphorylation Site ◽

Defense Responses ◽

Kinase Domain ◽

Kinase Assay ◽

Structural Basis ◽

Downstream Signaling ◽

Signaling Components

Abstract While ligand-induced autophosphorylation of receptor-like kinases (RLKs) is known to be critical for triggering the downstream responses, biochemical mechanism by which each phosphorylation site contributes to the initiation of corresponding signaling cascades is only poorly understood, except the involvement of some phosphorylation sites in the regulation of catalytic activity of these RLKs. In this article, we first confirmed that the phosphorylation of S493 of AtCERK1 is involved in the regulation of chitin-induced defense responses by the complementation of an atcerk1 mutant with AtCERK1(S493A) cDNA. In vitro kinase assay with the heterologously expressed kinase domain of AtCERK1, GST-AtCERK1cyt, showed that the S493A mutation did not affect the autophosphorylation of AtCERK1 itself but diminished the transphosphorylation of downstream signaling components, PBL27 and PUB4. On the other hand, a phosphomimetic mutant, GST-AtCERK1(S493D)cyt, transphosphorylated these substrates as similar to the wild type AtCERK1. These results suggested that the phosphorylation of S493 does not contribute to the regulation of catalytic activity but plays an important role for the transphosphorylation of the downstream signaling components, thus contributing to the initiation of chitin signaling. To our knowledge, it is a novel finding that a specific phosphorylation site contributes to the regulation of transphosphorylation activity of RLKs. Further studies on the structural basis by which S493 phosphorylation contributes to the regulation of transphosphorylation would contribute to the understanding how the ligand-induced autophosphorylation of RLKs properly regulates the downstream signaling.

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