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Cell Cycle (Georgetown, Tex.) Dec 2016
Topics: Phosphorylation; Proteins
PubMed: 27589382
DOI: 10.1080/15384101.2016.1220714 -
Scientific Reports Apr 2022The role of TIRAP (toll/interleukin-1 receptor (TIR) domain-containing adapter protein) in macrophage inflammatory signalling has been significantly evolved since its...
The role of TIRAP (toll/interleukin-1 receptor (TIR) domain-containing adapter protein) in macrophage inflammatory signalling has been significantly evolved since its discovery in 2001 due to its dynamic nature and subcellular localization to regulate multiple signaling through several protein-protein interactions (PPIs). Structural analysis of these interactions can reveal a better understanding of their conformational dynamics and the nature of their binding. Tyrosine phosphorylation in the TIR domain of TIRAP is very critical for its function. In toll-like receptor (TLR) 4/2 signalling, Bruton's tyrosine kinase (BTK) and Protein kinase C delta (PKCδ) are known to phosphorylate the Y86, Y106, Y159, and Y187 of TIRAP which is crucial for the downstream function of MAPKs (mitogen-activated protein kinases) activation. The objective of this study is to understand the interaction of TIRAP with p38 MAPK through molecular docking and identify the importance of TIRAP tyrosine phosphorylation in p38 MAPK interaction. In this structural study, we performed an in-silico molecular docking using HADDOCK 2.4, pyDockWEB, ClusPro 2.0, and ZDOCK 3.0.2 tools to unravel the interaction between TIRAP and p38 MAPK. Further, manual in-silico phosphorylations of TIRAP tyrosines; Y86, Y106, Y159, and Y187 was created in the Discovery Studio tool to study the conformational changes in protein docking and their binding affinities with p38 MAPK in comparison to non-phosphorylated state. Our molecular docking and 500 ns of molecular dynamic (MD) simulation study demonstrates that the Y86 phosphorylation (pY86) in TIRAP is crucial in promoting the higher binding affinity (∆G) with p38 MAPK. The conformational changes due to the tyrosine phosphorylation mainly at the Y86 site pull the TIRAP closer to the active site in the kinase domain of p38 MAPK and plays a significant role at the interface site which is reversed in its dephosphorylated state. The heatmap of interactions between the TIRAP and p38 MAPK after the MD simulation shows that the TIRAP pY86 structure makes the highest number of stable hydrogen bonds with p38 MAPK residues. Our findings may further be validated in an in-vitro system and would be crucial for targeting the TIRAP and p38 MAPK interaction for therapeutic purposes against the chronic inflammatory response and associated diseases.
Topics: Membrane Glycoproteins; Mitogen-Activated Protein Kinases; Molecular Docking Simulation; Phosphorylation; Receptors, Interleukin-1; Signal Transduction; p38 Mitogen-Activated Protein Kinases
PubMed: 35379857
DOI: 10.1038/s41598-022-09528-8 -
Cells Feb 2023The scaffold protein IQGAP1 associates with over 150 interactors to influence multiple biological processes. The molecular mechanisms that underly spatial and temporal...
The scaffold protein IQGAP1 associates with over 150 interactors to influence multiple biological processes. The molecular mechanisms that underly spatial and temporal regulation of these interactions, which are crucial for proper cell functions, remain poorly understood. The receptor tyrosine kinase MET phosphorylates IQGAP1 on Tyr. Separately, Src homology 2 (SH2) domains mediate protein-protein interactions by binding specific phosphotyrosine residues. Here, we investigate whether MET-catalyzed phosphorylation of Tyr of IQGAP1 regulates the docking of SH2-containing proteins. Using a peptide array, we identified SH2 domains from several proteins, including the non-receptor tyrosine kinases Abl1 and Abl2, that bind to the Tyr of IQGAP1 in a phosphorylation-dependent manner. Using pure proteins, we validated that full-length Abl1 and Abl2 bind directly to phosphorylated Tyr of IQGAP1. In cells, MET inhibition decreases endogenous IQGAP1 phosphorylation and interaction with endogenous Abl1 and Abl2, indicating that binding is regulated by MET-catalyzed phosphorylation of IQGAP1. Functionally, IQGAP1 modulates basal and HGF-stimulated Abl signaling. Moreover, IQGAP1 binds directly to MET, inhibiting its activation and signaling. Collectively, our study demonstrates that IQGAP1 is a phosphotyrosine-regulated scaffold for SH2-containing proteins, thereby uncovering a previously unidentified mechanism by which IQGAP1 coordinates intracellular signaling.
Topics: Phosphotyrosine; ras GTPase-Activating Proteins; Phosphorylation; Receptor Protein-Tyrosine Kinases
PubMed: 36766826
DOI: 10.3390/cells12030483 -
ELife Feb 2017The kinase Mps1, long known to be the 'boss' in mitotic checkpoint signaling, phosphorylates multiple proteins in the checkpoint signaling cascade.
The kinase Mps1, long known to be the 'boss' in mitotic checkpoint signaling, phosphorylates multiple proteins in the checkpoint signaling cascade.
Topics: Cell Cycle Proteins; Kinetochores; Phosphorylation; Protein Serine-Threonine Kinases; Signal Transduction
PubMed: 28206949
DOI: 10.7554/eLife.25001 -
MSystems May 2024For over 40 years, the two-component systems (TCSs) have taken front and center in our thinking about the signaling mechanisms by which bacteria sense and respond to... (Review)
Review
For over 40 years, the two-component systems (TCSs) have taken front and center in our thinking about the signaling mechanisms by which bacteria sense and respond to their environment. In contrast, phosphorylation on Ser/Thr and Tyr (-phosphorylation) was long thought to be mostly restricted to eukaryotes and a somewhat accessory signaling mechanism in bacteria. Several recent studies exploring systems aspects of bacterial -phosphorylation, however, now show that it is in fact pervasive, with some bacterial proteomes as highly phosphorylated as those of eukaryotes. Labile, non-canonical protein phosphorylation sites on Asp, Arg, and His are now also being identified in large numbers in bacteria and first cellular functions are discovered. Other phosphomodifications on Cys, Glu, and Lys remain largely unexplored. The surprising breadth and complexity of bacterial phosphosignaling reveals a vast signaling capacity, the full scope of which we may only now be beginning to understand but whose functions are likely to affect all aspects of bacterial physiology and pathogenesis.
Topics: Phosphorylation; Bacterial Proteins; Bacteria; Signal Transduction; Proteome
PubMed: 38591891
DOI: 10.1128/msystems.00289-24 -
International Journal of Molecular... Sep 2023Polo-Like Kinase 1 (PLK1), a key mediator of cell-cycle progression, is associated with poor prognosis and is a therapeutic target in a number of malignancies. Putative...
Polo-Like Kinase 1 (PLK1), a key mediator of cell-cycle progression, is associated with poor prognosis and is a therapeutic target in a number of malignancies. Putative phosphorylation sites for PLK1 have been identified on Drosha, the main catalytic component of the microprocessor responsible for miR biogenesis. Several kinases, including GSK3β, p70 S6 kinase, ABL, PAK5, p38 MAPK, CSNK1A1 and ANKRD52-PPP6C, have been shown to phosphorylate components of the miR biogenesis machinery, altering their activity and/or localisation, and therefore the biogenesis of distinct miR subsets. We hypothesised that PLK1 regulates miR biogenesis through Drosha phosphorylation. In vitro kinase assays confirmed PLK1 phosphorylation of Drosha at S and/or S. PLK1 inhibition reduced serine-phosphorylated levels of Drosha and its RNA-dependent association with DGCR8. In contrast, a "phospho-mimic" Drosha mutant showed increased association with DGCR8. PLK1 phosphorylation of Drosha alters Drosha Microprocessor complex subcellular localisation, since PLK1 inhibition increased cytosolic protein levels of both DGCR8 and Drosha, whilst nuclear levels were decreased. Importantly, the above effects are independent of PLK1's cell cycle-regulatory role, since altered Drosha:DGCR8 localisation upon PLK1 inhibition occurred prior to significant accumulation of cells in M-phase, and PLK1-regulated miRs were not increased in M-phase-arrested cells. Small RNA sequencing and qPCR validation were used to assess downstream consequences of PLK1 activity on miR biogenesis, identifying a set of ten miRs (miR-1248, miR-1306-5p, miR-2277-5p, miR-29c-5p, miR-93-3p, miR-152-3p, miR-509-3-5p, miR-511-5p, miR-891a-5p and miR-892a) whose expression levels were statistically significantly downregulated by two pharmacological PLK1 kinase domain inhibitors, RO-5203280 and GSK461364. Opposingly, increased levels of these miRs were observed upon transfection of wild-type or constitutively active PLK1. Importantly, pre-miR levels were reduced upon PLK1 inhibition, and pri-miR levels decreased upon PLK1 activation, and hence, PLK1 Drosha phosphorylation regulates MiR biogenesis at the level of pri-miR-to-pre-miR processing. In combination with prior studies, this work identifies Drosha S and S as major integration points for signalling by several kinases, whose relative activities will determine the relative biogenesis efficiency of different miR subsets. Identified kinase-regulated miRs have potential for use as kinase inhibitor response-predictive biomarkers, in cancer and other diseases.
Topics: MicroRNAs; Phosphorylation; Protein Serine-Threonine Kinases; RNA-Binding Proteins; Polo-Like Kinase 1
PubMed: 37762595
DOI: 10.3390/ijms241814290 -
The Biochemical Journal Mar 2022The RBR E3 ligase parkin is recruited to the outer mitochondrial membrane (OMM) during oxidative stress where it becomes activated and ubiquitinates numerous proteins....
The RBR E3 ligase parkin is recruited to the outer mitochondrial membrane (OMM) during oxidative stress where it becomes activated and ubiquitinates numerous proteins. Parkin activation involves binding of a phosphorylated ubiquitin (pUb), followed by phosphorylation of the Ubl domain in parkin, both mediated by the OMM kinase, PINK1. How an OMM protein is selected for ubiquitination is unclear. Parkin targeted OMM proteins have little structural or sequence similarity, with the commonality between substrates being proximity to the OMM. Here, we used chimeric proteins, tagged with ubiquitin (Ub), to evaluate parkin ubiquitination of mitochondrial acceptor proteins pre-ligated to Ub. We find that pUb tethered to the mitochondrial target proteins, Miro1 or CISD1, is necessary for parkin recruitment and essential for target protein ubiquitination. Surprisingly, phosphorylation of parkin is not necessary for the ubiquitination of either Miro1 or CISD1. Thus, parkin lacking its Ubl domain efficiently ubiquitinates a substrate tethered to pUb. Instead, phosphorylated parkin appears to stimulate free Ub chain formation. We also demonstrate that parkin ubiquitination of pUb-tethered substrates occurs on the substrate, rather than the pUb modification. We propose divergent parkin mechanisms whereby parkin-mediated ubiquitination of acceptor proteins is driven by binding to pre-existing pUb on the OMM protein and subsequent parkin phosphorylation triggers free Ub chain formation. This finding accounts for the broad spectrum of OMM proteins ubiquitinated by parkin and has implications on target design for therapeutics.
Topics: Mitochondrial Proteins; Phosphorylation; Ubiquitin; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 35262643
DOI: 10.1042/BCJ20210741 -
DNA Repair Dec 2021The cellular response to DNA damage (DDR) that causes replication collapse and/or DNA double strand breaks, is characterised by a massive change in the... (Review)
Review
The cellular response to DNA damage (DDR) that causes replication collapse and/or DNA double strand breaks, is characterised by a massive change in the post-translational modifications (PTM) of hundreds of proteins involved in the detection and repair of DNA damage, and the communication of the state of damage to the cellular systems that regulate replication and cell division. A substantial proportion of these PTMs involve targeted phosphorylation, which among other effects, promotes the formation of multiprotein complexes through the specific binding of phosphorylated motifs on one protein, by specialised domains on other proteins. Understanding the nature of these phosphorylation mediated interactions allows definition of the pathways and networks that coordinate the DDR, and helps identify new targets for therapeutic intervention that may be of benefit in the treatment of cancer, where DDR plays a key role. In this review we summarise the present understanding of how phosphorylated motifs are recognised by BRCT domains, which occur in many DDR proteins. We particularly focus on TOPBP1 - a multi-BRCT domain scaffold protein with essential roles in replication and the repair and signalling of DNA damage.
Topics: Carrier Proteins; Cell Cycle Proteins; DNA Damage; DNA Repair; DNA-Binding Proteins; Nuclear Proteins; Phosphorylation
PubMed: 34678589
DOI: 10.1016/j.dnarep.2021.103232 -
Biochemistry Aug 2022Canonically, MST1/2 functions as a core kinase of the Hippo pathway and noncanonically during both apoptotic signaling and with RASSFs in T-cells. Faithful signal...
Canonically, MST1/2 functions as a core kinase of the Hippo pathway and noncanonically during both apoptotic signaling and with RASSFs in T-cells. Faithful signal transduction by MST1/2 relies on both appropriate activation and regulated substrate phosphorylation by the activated kinase. Considerable progress has been made in understanding the molecular mechanisms regulating the activation of MST1/2 and identifying downstream signaling events. Here, we investigated the ability of MST2 to phosphorylate a peptide substrate and how that activity is regulated. Using a steady-state kinetic system, we parse the contribution of different factors to substrate phosphorylation, including the domains of MST2, phosphorylation, caspase cleavage, and complex formation. We found that in the unphosphorylated state, the SARAH domain stabilizes interactions with a peptide substrate and promotes turnover. Phosphorylation drives the activity of MST2, and once activated, MST2 is not further regulated by complex formation with other Hippo pathway components (SAV1, MOB1A, and RASSF5). We also show that the phosphorylated, caspase-cleaved MST2 is as active as the full-length one, suggesting that caspase-stimulated activity arises through noncatalytic mechanisms. The kinetic analysis presented here establishes a framework for interpreting how signaling events and post-translational modifications contribute to the signaling of MST2 in vivo.
Topics: Animals; Caspases; Kinetics; Mammals; Phosphorylation; Protein Serine-Threonine Kinases; Signal Transduction
PubMed: 35895874
DOI: 10.1021/acs.biochem.2c00022 -
Cells Feb 2021Septins are GTP-binding proteins that form heteromeric filaments for proper cell growth and migration. Among the septins, septin7 (SEPT7) is an important component of...
Septins are GTP-binding proteins that form heteromeric filaments for proper cell growth and migration. Among the septins, septin7 (SEPT7) is an important component of all septin filaments. Here we show that protein kinase A (PKA) phosphorylates SEPT7 at Thr197, thus disrupting septin filament dynamics and ciliogenesis. The Thr197 residue of SEPT7, a PKA phosphorylating site, was conserved among different species. Treatment with cAMP or overexpression of PKA catalytic subunit (PKACA2) induced SEPT7 phosphorylation, followed by disruption of septin filament formation. Constitutive phosphorylation of SEPT7 at Thr197 reduced SEPT7‒SEPT7 interaction, but did not affect SEPT7‒SEPT6‒SEPT2 or SEPT4 interaction. Moreover, we noted that SEPT7 interacted with PKACA2 via its GTP-binding domain. Furthermore, PKA-mediated SEPT7 phosphorylation disrupted primary cilia formation. Thus, our data uncover the novel biological function of SEPT7 phosphorylation in septin filament polymerization and primary cilia formation.
Topics: Amino Acid Sequence; Cell Cycle Proteins; Cilia; Conserved Sequence; Cyclic AMP-Dependent Protein Kinases; Humans; Organogenesis; Phosphorylation; Phosphothreonine; Protein Binding; Protein Domains; Septins; Species Specificity
PubMed: 33572403
DOI: 10.3390/cells10020361