isabelle lucet

Structure-Guided Prediction of the Functional Impact of DCLK1 Mutations on Tumorigenesis

Publisher: MDPI AG

Date: 22-03-2023

DOI: 10.3390/BIOMEDICINES11030990

Abstract: Doublecortin-like kinase 1 (DCLK1) is a functional serine/threonine (S/T)-kinase and a member of the doublecortin family of proteins which are characterized by their ability to bind to microtubules (MTs). DCLK1 is a proposed cancer driver gene, and its upregulation is associated with poor overall survival in several solid cancer types. However, how DCLK1 associates with MTs and how its kinase function contributes to pro-tumorigenic processes is poorly understood. This review builds on structural models to propose not only the specific functions of the domains but also attempts to predict the impact of in idual somatic missense mutations on DCLK1 functions. Somatic missense mutations in DCLK1 are most frequently located within the N-terminal MT binding region and likely impact on the ability of DCLK1 to bind to αβ-tubulin and to polymerize and stabilize MTs. Moreover, the MT binding affinity of DCLK1 is negatively regulated by its auto-phosphorylation, and therefore mutations that affect kinase activity are predicted to indirectly alter MT dynamics. The emerging picture portrays DCLK1 as an MT-associated protein whose interactions with tubulin heterodimers and MTs are tightly controlled processes which, when disrupted, may confer pro-tumorigenic properties.

The intracellular domains of the EphB6 and EphA10 receptor tyrosine pseudokinases function as dynamic signalling hubs

Publisher: Portland Press Ltd.

Date: 14-09-2021

DOI: 10.1042/BCJ20210572

Abstract: EphB6 and EphA10 are two poorly characterised pseudokinase members of the Eph receptor family, which collectively serves as mediators of contact-dependent cell–cell communication to transmit extracellular cues into intracellular signals. As per their active counterparts, EphB6 and EphA10 deregulation is strongly linked to proliferative diseases. However, unlike active Eph receptors, whose catalytic activities are thought to initiate an intracellular signalling cascade, EphB6 and EphA10 are classified as catalytically dead, raising the question of how non-catalytic functions contribute to Eph receptor signalling homeostasis. In this study, we have characterised the biochemical properties and topology of the EphB6 and EphA10 intracellular regions comprising the juxtamembrane (JM) region, pseudokinase and SAM domains. Using small-angle X-ray scattering and cross-linking-mass spectrometry, we observed high flexibility within their intracellular regions in solution and a propensity for interaction between the component domains. We identified tyrosine residues in the JM region of EphB6 as EphB4 substrates, which can bind the SH2 domains of signalling effectors, including Abl, Src and Vav3, consistent with cellular roles in recruiting these proteins for downstream signalling. Furthermore, our finding that EphB6 and EphA10 can bind ATP and ATP-competitive small molecules raises the prospect that these pseudokinase domains could be pharmacologically targeted to counter oncogenic signalling.

Structural basis for small molecule targeting of Doublecortin Like Kinase 1 with DCLK1-IN-1.

Publisher: Springer Science and Business Media LLC

Date: 20-09-2021

DOI: 10.1038/S42003-021-02631-Y

Abstract: Doublecortin-like kinase 1 (DCLK1) is an understudied bi-functional kinase with a proven role in tumour growth and development. However, the presence of tissue-specific spliced DCLK1 isoforms with distinct biological functions have challenged the development of effective strategies to understand the role of DCLK1 in oncogenesis. Recently, DCLK1-IN-1 was reported as a highly selective DCLK1 inhibitor, a powerful tool to dissect DCLK1 biological functions. Here, we report the crystal structures of DCLK1 kinase domain in complex with DCLK1-IN-1 and its precursors. Combined, our data rationalises the structure-activity relationship that informed the development of DCLK1-IN-1 and provides the basis for the high selectivity of DCLK1-IN-1, with DCLK1-IN-1 inducing a drastic conformational change of the ATP binding site. We demonstrate that DCLK1-IN-1 binds DCLK1 long isoforms but does not prevent DCLK1’s Microtubule-Associated Protein (MAP) function. Together, our work provides an invaluable structural platform to further the design of isoform-specific DCLK1 modulators for therapeutic intervention.

Potent Inhibition of Necroptosis by Simultaneously Targeting Multiple Effectors of the Pathway

Publisher: American Chemical Society (ACS)

Date: 09-09-2020

DOI: 10.1021/ACSCHEMBIO.0C00482

The PEAK family of pseudokinases, their role in cell signalling and cancer

Publisher: Wiley

Date: 06-11-2019

DOI: 10.1111/FEBS.15087

Abstract: The study of pseudokinases has uncovered that catalysis-independent functions play a critical role in cell signalling regulation. However, how pseudokinases dynamically assemble and regulate oncogenic signalling pathways remains, in most cases, unclear due to a limited knowledge of the structural determinants that are critical for their functions. Here, we review the recent progress made to unravel the role of the PEAK family of pseudokinases, which comprises SgK269, SgK223 and the recently identified PEAK3, in assembling specific oncogenic signalling pathways that contribute to the progression of several aggressive cancers. We focus on recent structural advances revealing that SgK269 and SgK223 can homo- and heteroassociate via a unique dimerisation domain, comprising conserved regulatory helices directly surrounding the pseudokinase domain, which is also conserved in PEAK3. We also highlight a potential oligomerisation mechanism driven by the pseudokinase domain. While it is likely that homo- or heterodimerisation and oligomerisation mechanisms contribute to the assembly of complex signalling hubs and provide a means to spatially and temporally modulate and ersify signalling outputs, the exact role that these oncogenic scaffolds play in regulating cell migration, invasion and morphology remains unclear. Here, we attempt to link their structural characteristics to their cellular functions by providing a thorough analysis of the signalling transduction pathways they are known to modulate.

Structure of SgK223 pseudokinase reveals novel mechanisms of homotypic and heterotypic association

Publisher: Springer Science and Business Media LLC

Date: 27-10-2017

DOI: 10.1038/S41467-017-01279-9

Abstract: The mammalian pseudokinase SgK223, and its structurally related homologue SgK269, are oncogenic scaffolds that nucleate the assembly of specific signalling complexes and regulate tyrosine kinase signalling. Both SgK223 and SgK269 form homo- and hetero-oligomers, a mechanism that underpins a ersity of signalling outputs. However, mechanistic insights into SgK223 and SgK269 homo- and heterotypic association are lacking. Here we present the crystal structure of SgK223 pseudokinase domain and its adjacent N- and C-terminal helices. The structure reveals how the N- and C-regulatory helices engage in a novel fold to mediate the assembly of a high-affinity dimer. In addition, we identified regulatory interfaces on the pseudokinase domain required for the self-assembly of large open-ended oligomers. This study highlights the ersity in how the kinase fold mediates non-catalytic functions and provides mechanistic insights into how the assembly of these two oncogenic scaffolds is achieved in order to regulate signalling output.

N-Terminomic Changes in Neurons During Excitotoxicity Reveal Proteolytic Events Associated With Synaptic Dysfunctions and Potential Targets for Neuroprotection

Publisher: Elsevier BV

Date: 05-2023

DOI: 10.1016/J.MCPRO.2023.100543

Co-clustering of EphB6 and ephrinB1 in trans suppresses cancer cell invasion

Publisher: Research Square Platform LLC

Date: 26-09-2022

DOI: 10.21203/RS.3.RS-2064774/V1

Abstract: EphB6 is an understudied ephrin receptor tyrosine pseudokinase, the expression of which is downregulated in multiple types of metastatic cancers. Compared to its kinase-active counterparts, how EphB6 regulates signal transduction in the absence of intrinsic kinase activity remains unknown. Here, we unveil the molecular details of key signal transduction mechanisms driven by EphB6. We identify ephrinB1 as a cognate ligand of EphB6 and demonstrate EphB6’s ability to form extensive co-clusters at the plasma membrane upon binding ephrinB1 in trans across cell-cell contacts, which are resistant to endocytosis. Using super resolution microscopy, correlative light and electron microscopy and cryo-electron tomography, we reveal that co clustering of EphB6 and ephrinB1 promotes the formation of unprecedented double membrane tubular structure interconnecting cells. Importantly, phenotypically, co-clustering of EphB6 and ephrinB1 and the resulting tubular structures suppress cancer cell invasion by prolonging cell-cell contacts, rationalising a role for EphB6 pseudokinase as a tumor suppressor when interacting with its ligands in trans .

Distinct pseudokinase domain conformations underlie divergent activation mechanisms among vertebrate MLKL orthologues

Publisher: Springer Science and Business Media LLC

Date: 19-06-2020

DOI: 10.1038/S41467-020-16823-3

Abstract: The MLKL pseudokinase is the terminal effector in the necroptosis cell death pathway. Phosphorylation by its upstream regulator, RIPK3, triggers MLKL’s conversion from a dormant cytoplasmic protein into oligomers that translocate to, and permeabilize, the plasma membrane to kill cells. The precise mechanisms underlying these processes are incompletely understood, and were proposed to differ between mouse and human cells. Here, we examine the ergence of activation mechanisms among nine vertebrate MLKL orthologues, revealing remarkable specificity of mouse and human RIPK3 for MLKL orthologues. Pig MLKL can restore necroptotic signaling in human cells while horse and pig, but not rat, MLKL can reconstitute the mouse pathway. This selectivity can be rationalized from the distinct conformations observed in the crystal structures of horse and rat MLKL pseudokinase domains. These studies identify important differences in necroptotic signaling between species, and suggest that, more broadly, ergent regulatory mechanisms may exist among orthologous pseudoenzymes.

Mapping and functional analysis of heterochromatin protein 1 phosphorylation in the malaria parasite Plasmodium falciparum

Publisher: Springer Science and Business Media LLC

Date: 13-11-2019

DOI: 10.1038/S41598-019-53325-9

Abstract: Previous studies in model eukaryotes have demonstrated that phosphorylation of heterochromatin protein 1 (HP1) is important for dynamically regulating its various functions. However, in the malaria parasite Plasmodium falciparum both the function of HP1 phosphorylation and the identity of the protein kinases targeting HP1 are still elusive. In order to functionally analyze phosphorylation of P. falciparum HP1 (PfHP1), we first mapped PfHP1 phosphorylation sites by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of native PfHP1, which identified motifs from which potential kinases could be predicted in particular, several phosphorylated residues were embedded in motifs rich in acidic residues, reminiscent of targets for P. falciparum casein kinase 2 (PfCK2). Secondly, we tested recombinant PfCK2 and a number of additional protein kinases for their ability to phosphorylate PfHP1 in in vitro kinase assays. These experiments validated our prediction that PfHP1 acts as a substrate for PfCK2. Furthermore, LC-MS/MS analysis showed that PfCK2 phosphorylates three clustered serine residues in an acidic motif within the central hinge region of PfHP1. To study the role of PfHP1 phosphorylation in live parasites we used CRISPR/Cas9-mediated genome editing to generate a number of conditional PfHP1 phosphomutants based on the DiCre/LoxP system. Our studies revealed that neither PfCK2-dependent phosphorylation of PfHP1, nor phosphorylation of the hinge domain in general, affect PfHP1′s ability to localize to heterochromatin, and that PfHP1 phosphorylation in this region is dispensable for the proliferation of P. falciparum blood stage parasites.

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

Publisher: Research Square Platform LLC

Date: 15-01-2021

DOI: 10.21203/RS.3.RS-133153/V1

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.

Granulovirus PK-1 kinase activity relies on a side-to-side dimerization mode centered on the regulatory αC helix

Publisher: Springer Science and Business Media LLC

Date: 12-02-2021

DOI: 10.1038/S41467-021-21191-7

Abstract: The life cycle of Baculoviridae family insect viruses depends on the viral protein kinase, PK-1, to phosphorylate the regulatory protein, p6.9, to induce baculoviral genome release. Here, we report the crystal structure of Cydia pomenella granulovirus PK-1, which, owing to its likely ancestral origin among host cell AGC kinases, exhibits a eukaryotic protein kinase fold. PK-1 occurs as a rigid dimer, where an antiparallel arrangement of the αC helices at the dimer core stabilizes PK-1 in a closed, active conformation. Dimerization is facilitated by C-lobe:C-lobe and N-lobe:N-lobe interactions between protomers, including the domain-swapping of an N-terminal helix that crowns a contiguous β-sheet formed by the two N-lobes. PK-1 retains a dimeric conformation in solution, which is crucial for catalytic activity. Our studies raise the prospect that parallel, side-to-side dimeric arrangements that lock kinase domains in a catalytically-active conformation could function more broadly as a regulatory mechanism among eukaryotic protein kinases.

Eph receptor signalling: from catalytic to non-catalytic functions

Publisher: Springer Science and Business Media LLC

Date: 12-08-2019

DOI: 10.1038/S41388-019-0931-2

Abstract: Eph receptors, the largest subfamily of receptor tyrosine kinases, are linked with proliferative disease, such as cancer, as a result of their deregulated expression or mutation. Unlike other tyrosine kinases that have been clinically targeted, the development of therapeutics against Eph receptors remains at a relatively early stage. The major reason is the limited understanding on the Eph receptor regulatory mechanisms at a molecular level. The complexity in understanding Eph signalling in cells arises due to following reasons: (1) Eph receptors comprise 14 members, two of which are pseudokinases, EphA10 and EphB6, with relatively uncharacterised function (2) activation of Eph receptors results in dimerisation, oligomerisation and formation of clustered signalling centres at the plasma membrane, which can comprise different combinations of Eph receptors, leading to erse downstream signalling outputs (3) the non-catalytic functions of Eph receptors have been overlooked. This review provides a structural perspective of the intricate molecular mechanisms that drive Eph receptor signalling, and investigates the contribution of intra- and inter-molecular interactions between Eph receptors intracellular domains and their major binding partners. We focus on the non-catalytic functions of Eph receptors with relevance to cancer, which are further substantiated by exploring the role of the two pseudokinase Eph receptors, EphA10 and EphB6. Throughout this review, we carefully analyse and reconcile the existing/conflicting data in the field, to allow researchers to further the current understanding of Eph receptor signalling.

Statistical characterisation and analysis of differential correlation‐based frame detector

Publisher: Institution of Engineering and Technology (IET)

Date: 10-2019

DOI: 10.1049/IET-COM.2018.5882

Ubiquitylation of MLKL at lysine 219 positively regulates necroptosis-induced tissue injury and pathogen clearance

Publisher: Springer Science and Business Media LLC

Date: 07-06-2021

DOI: 10.1038/S41467-021-23474-5

Abstract: Necroptosis is a lytic, inflammatory form of cell death that not only contributes to pathogen clearance but can also lead to disease pathogenesis. Necroptosis is triggered by RIPK3-mediated phosphorylation of MLKL, which is thought to initiate MLKL oligomerisation, membrane translocation and membrane rupture, although the precise mechanism is incompletely understood. Here, we show that K63-linked ubiquitin chains are attached to MLKL during necroptosis and that ubiquitylation of MLKL at K219 significantly contributes to the cytotoxic potential of phosphorylated MLKL. The K219R MLKL mutation protects animals from necroptosis-induced skin damage and renders cells resistant to pathogen-induced necroptosis. Mechanistically, we show that ubiquitylation of MLKL at K219 is required for higher-order assembly of MLKL at membranes, facilitating its rupture and necroptosis. We demonstrate that K219 ubiquitylation licenses MLKL activity to induce lytic cell death, suggesting that necroptotic clearance of pathogens as well as MLKL-dependent pathologies are influenced by the ubiquitin-signalling system.

PEAK3 pseudokinase represents a pro-migratory and -invasive signalling scaffold

Publisher: Cold Spring Harbor Laboratory

Date: 19-02-2021

DOI: 10.1101/2021.02.17.431740

Abstract: The PEAK family of pseudokinases comprises PEAK1 and PEAK2 as well as the recently-identified PEAK3. PEAK1/2 play fundamental roles in regulating tyrosine kinase signal output and oncogenesis, while PEAK3 remains poorly-characterized. Here, we demonstrate that PEAK3 undergoes homotypic association as well as heterotypic interaction with PEAK1/2. PEAK3 also recruits ASAP1/2, Cbl and PYK2 and the adaptors Grb2 and CrkII, with binding dependent on PEAK3 dimerization. PEAK3 tyrosine phosphorylation on Y24 is also dependent on dimerization as well as Src family kinase activity, and interestingly, is decreased via PTPN12 in response to EGF treatment. Y24 phosphorylation is required for binding of Grb2 and ASAP1. Overexpression of PEAK3 in MDA-MB-231 breast cancer cells enhanced cell elongation and cell motility, while knockdown of endogenous PEAK3 decreased cell migration. In addition, overexpression of PEAK3 in PEAK1/2 compound knock-out MCF-10A breast epithelial cells enhanced acinar growth and invasion in 3D culture, with the latter phenotype dependent on PEAK3 tyrosine phosphorylation and binding of Grb2 and ASAP1. These findings characterize PEAK3 as an integral member of the PEAK family with scaffolding roles that promote cell proliferation, migration and invasion.

Author Reply to Peer Reviews of An Atlas of Phosphorylation and Proteolytic Processing Events During Excitotoxic Neuronal Death Reveals New Therapeutic Opportunities

Publisher: EMBO

Date: 15-09-2020

DOI: 10.15252/RC.2022884848

An Atlas of Phosphorylation and Proteolytic Processing Events During Excitotoxic Neuronal Death Reveals New Therapeutic Opportunities

Publisher: Cold Spring Harbor Laboratory

Date: 15-06-2020

DOI: 10.1101/2020.06.15.151456

Abstract: Excitotoxicity, a neuronal death process in neurological disorders, is initiated by over-stimulation of neuronal ionotropic glutamate receptors. The over-stimulated receptors dysregulate proteases, protein kinases and phosphatases, which in turn modify target neuronal proteins to induce cell death. To decipher this cell death mechanism, we used quantitative proteomics, phosphoproteomics and N-terminomics to identify modified proteins in excitotoxic neurons. Data, available in ProteomeXchange (identifiers: PXD019527 and PXD019211), enabled us to identify over one thousand such proteins with calpains, cathepsins and over twenty protein kinases as their major modifiers. These protein modification events can potentially perturb signalling pathways governing cell survival, synaptogenesis, axonal guidance and mRNA processing. Importantly, blocking the modification of Src protein kinase, a signalling hub in excitotoxic neurons, protected against neuronal loss in vivo in a rat model of neurotoxicity. Besides offering new insights into excitotoxic neuronal death mechanism, our findings suggest potential neuroprotective therapeutic targets for treating neurological disorders. Multi-dimensional proteomic analysis identified proteins modified by proteolysis and altered phosphorylation in neurons undergoing excitotoxic cell death. Calpains, cathepsins and over twenty protein kinases are major modifiers of these proteins. These protein modification events are predicted to impact cell survival, axonal guidance, synaptogenesis and mRNA processing. Blocking modification of an identified protein Src, which acts as a major signalling hub in neurons, was protective against excitotoxic injury in vivo . Using multidimensional proteomic approaches, Ameen, et al . mapped the changes of proteome, phosphoproteome and N-terminome of cultured primary neurons during excitotoxicity, a crucial neuronal death process in neurological disorders. These proteomic changes document new excitotoxicity-associated molecular events, and offer insights into how these events are organized to induce neuronal death. Potential therapeutic relevance of these molecular events is illustrated by the demonstration that in vivo blockade of one of these events could protect against excitotoxic neuronal loss.

When two’s a crowd - Structural mapping of PEAK pseudokinase interactions identifies 14 3 3 as a molecular switch for PEAK3/Crk signaling.

Publisher: Research Square Platform LLC

Date: 31-08-2022

DOI: 10.21203/RS.3.RS-1906871/V1

Abstract: PEAK pseudokinases regulate cell migration, invasion and proliferation by recruiting key signaling proteins to the cytoskeleton. Despite lacking catalytic activity, alteration in their expression level is associated with several aggressive cancers. Here, we elucidate new molecular details of key PEAK signaling interactions with the adapter proteins CrkII and Grb2 and the scaffold protein 14-3-3. Our findings rationalize why the dimerization of PEAK proteins has a crucial function in signal transduction and provide biophysical and structural data to unravel binding specificity within the PEAK interactome. We identify a conserved high affinity 14-3-3 motif on PEAK3 and demonstrate its role as a molecular switch to regulate CrkII binding. Together, our studies provide a detailed structural snapshot of PEAK interaction networks and further elucidate how PEAK proteins, especially PEAK3, act as dynamic scaffolds that exploit adapter proteins to control signal transduction in cell growth/motility and cancer.

When two’s a crowd - Structural mapping of PEAK pseudokinase interactions identifies 14-3-3 as a molecular switch for PEAK3/Crk signaling

Publisher: Cold Spring Harbor Laboratory

Date: 02-09-2022

DOI: 10.1101/2022.09.01.506260

Abstract: PEAK pseudokinases regulate cell migration, invasion and proliferation by recruiting key signaling proteins to the cytoskeleton. Despite lacking catalytic activity, alteration in their expression level is associated with several aggressive cancers. Here, we elucidate new molecular details of key PEAK signaling interactions with the adapter proteins CrkII and Grb2 and the scaffold protein 14-3-3. Our findings rationalize why the dimerization of PEAK proteins has a crucial function in signal transduction and provide biophysical and structural data to unravel binding specificity within the PEAK interactome. We identify a conserved high affinity 14-3-3 motif on PEAK3 and demonstrate its role as a molecular switch to regulate CrkII binding. Together, our studies provide a detailed structural snapshot of PEAK interaction networks and further elucidate how PEAK proteins, especially PEAK3, act as dynamic scaffolds that exploit adapter proteins to control signal transduction in cell growth/motility and cancer.

Structural mapping of PEAK pseudokinase interactions identifies 14-3-3 as a molecular switch for PEAK3 signaling

Publisher: Springer Science and Business Media LLC

Date: 19-06-2023

DOI: 10.1038/S41467-023-38869-9

Abstract: PEAK pseudokinases regulate cell migration, invasion and proliferation by recruiting key signaling proteins to the cytoskeleton. Despite lacking catalytic activity, alteration in their expression level is associated with several aggressive cancers. Here, we elucidate the molecular details of key PEAK signaling interactions with the adapter proteins CrkII and Grb2 and the scaffold protein 14-3-3. Our findings rationalize why the dimerization of PEAK proteins has a crucial function in signal transduction and provide biophysical and structural data to unravel binding specificity within the PEAK interactome. We identify a conserved high affinity 14-3-3 motif on PEAK3 and demonstrate its role as a molecular switch to regulate CrkII binding and signaling via Grb2. Together, our studies provide a detailed structural snapshot of PEAK interaction networks and further elucidate how PEAK proteins, especially PEAK3, act as dynamic scaffolds that exploit adapter proteins to control signal transduction in cell growth/motility and cancer.

Distinct PEAK3 interactors and outputs expand the signaling potential of the PEAK pseudokinase family

Publisher: American Association for the Advancement of Science (AAAS)

Date: 22-02-2022

DOI: 10.1126/SCISIGNAL.ABJ3554

Abstract: The pseudokinase scaffolds PEAK1 and PEAK2 are implicated in cancer cell migration and metastasis. We characterized the regulation and role of the third family member PEAK3 in cell signaling. Similar to PEAK1 and PEAK2, PEAK3 formed both homotypic and heterotypic complexes. In addition, like PEAK1, it bound to the adaptors Grb2 and CrkII. However, unlike PEAK1 and PEAK2, homodimerized PEAK3 also interacted with the ARF GTPase-activating protein ASAP1, the E3 ubiquitin ligase Cbl, and the kinase PYK2. Dimerization and subsequent phosphorylation on Tyr 24 , likely by a Src family kinase, were required for the binding of PEAK3 to Grb2 and ASAP1. Interactions with Grb2, CrkII, ASAP1, Cbl, and PYK2 exhibited contrasting dynamics upon cell stimulation with epidermal growth factor (EGF), in part due to PEAK3 dephosphorylation mediated by the phosphatase PTPN12. Overexpressing PEAK3 in mesenchymal-like MDA-MB-231 breast cancer cells enhanced cell elongation in a manner dependent on PEAK3 dimerization, and manipulation of PEAK3 expression demonstrated a positive role for this scaffold in regulating cell migration. Overexpressing PEAK3 in PEAK1/2 double-knockout MCF-10A breast epithelial cells enhanced acinar growth, impaired basement membrane integrity, and promoted invasion in three-dimensional cultures, with the latter two effects dependent on the binding of PEAK3 to Grb2 and ASAP1. PEAK1 and PEAK2 quantitatively and temporally influenced PEAK3 function. These findings characterize PEAK3 as an integral, signal- ersifying member of the PEAK family with scaffolding roles that promote cell proliferation, migration, and invasion.

King Fahd University Of Petroleum And Minerals (KFUPM)

Location: Saudi Arabia

View Organisation

King Abdullah University Of Science And Technology (KAUST)

Location: Saudi Arabia

View Organisation

Carleton University

Location: Canada

View Organisation

Saleh Abdulaziz And Abahsain Co. Ltd.

Location: Saudi Arabia

View Organisation

Michigan State University (MSU)

Location: United States of America

View Organisation

Walter And Eliza Hall Institute Of Medical Research

Location: Australia

View Organisation

University Of Angers

Location: France

View Organisation

Discovery Projects - Grant ID: DP180102463

Start Date: 2018

End Date: 12-2020

Amount: $707,328.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE200100045

Start Date: 06-2020

End Date: 06-2021

Amount: $623,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE230100099

Start Date: 2023

End Date: 12-2023

Amount: $685,000.00

Funder: Australian Research Council

Industrial Transformation Training Centres - Grant ID: IC200100052

Start Date: 03-2021

End Date: 03-2026

Amount: $4,789,838.00

Funder: Australian Research Council