Distinct structural mechanisms determine substrate affinity and kinase activity of protein kinase Cα

Lee, Sangbae; Devamani, Titu; Song, Hyun Deok; Sandhu, Manbir; Larsen, Adrien; Sommese, Ruth F.; Jain, Abhinandan; Vaidehi, Nagarajan; Sivaramakrishnan, Sivaraj

doi:10.1074/jbc.m117.804781

Cited by 17 publications

(18 citation statements)

References 40 publications

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“…Previously, we have shown that the residues in the activation loop interact with the peptide substrate and form the floor of the substrate binding site in PKC α for 14 different peptides. 8 A basic residue (K/R), three amino acids C-terminal to the phosphorylated Ser/Thr in the EGFR substrate, forms a strong electrostatic contact with D544 and a cation− π interaction with F498 in the activation loop (Figure 1B). However, if these residues in the activation loop interact with residues in the G-loop forming the closed state, they are no longer available for substrate binding.…”

Section: An Allosteric Switch Regulates the Kinase Conformation Compamentioning

confidence: 99%

“…We have recently demonstrated the combined strength of a novel FRET sensor technology and torsional molecular dynamics (MD) simulations 7 to iteratively dissect the substrate binding interface of PKC α . 8 In this study, we have used the combined approach to identify an allosteric switch region near the ATP binding site and shown that the allosteric switch region can be engaged to regulate inhibition of both ATP and substrate binding. This allosteric switch is conserved across many kinases, thus opening up the possibility of utilizing it to design bitopic inhibitors for kinases.…”

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confidence: 99%

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Bitopic Inhibition of ATP and Substrate Binding in Ser/Thr Kinases through a Conserved Allosteric Mechanism

Lippert

Devamani

et al. 2018

Biochemistry

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Protein kinases achieve substrate selective phosphorylation through their conformational flexibility and dynamic interaction with the substrate. Designing substrate selective or kinase selective small molecule inhibitors remains a challenge because of a lack of understanding of the dynamic mechanism by which substrates are selected by the kinase. Using a combination of all-atom molecular dynamics simulations and FRET sensors, we have delineated an allosteric mechanism that results in interaction among the DFG motif, G-loop, and activation loop and structurally links the nucleotide and substrate binding interfaces in protein kinase Cα and three other Ser/Thr kinases. ATP-competitive staurosporine analogues engage this allosteric switch region located just outside the ATP binding site to displace substrate binding to varying degrees. These inhibitors function as bitopic ligands by occupying the ATP binding site and interacting with the allosteric switch region. The conserved mechanism identified in this study can be exploited to select and design bitopic inhibitors for kinases.

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Section: An Allosteric Switch Regulates the Kinase Conformation Compamentioning

confidence: 99%

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confidence: 99%

Bitopic Inhibition of ATP and Substrate Binding in Ser/Thr Kinases through a Conserved Allosteric Mechanism

Lippert

Devamani

et al. 2018

Biochemistry

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“…Among these was a CHG site within the gene body region of the Protein Kinase C Alpha (PRKCA) gene. The PRKCA gene encodes for Protein Kinase C Alpha, a member of the serine-and threonine-specific protein kinases, which phosphorylates peptide substrates and is involved in various cell-signaling processes (Lee et al, 2017). The PRKCA gene has been associated with PTSD and memory in humans (De Quervain et al, 2012).…”

Section: Gene Body Regionsmentioning

confidence: 99%

Prenatal transportation stress alters genome-wide DNA methylation in suckling Brahman bull calves1,2

Littlejohn

Price

Neuendorff³

et al. 2018

Journal of Animal Science

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The objective of this experiment was to identify genome-wide differential methylation of DNA in young prenatally stressed (PNS) bull calves. Mature Brahman cows (n = 48) were transported for 2-h periods at 60 ± 5, 80 ± 5, 100 ± 5, 120 ± 5, and 140 ± 5 d of gestation or maintained as nontransported Controls (n = 48). Methylation of DNA from white blood cells from a subset of 28-d-old intact male offspring (n = 7 PNS; n = 7 Control) was assessed via reduced representation bisulfite sequencing. Samples from PNS bulls contained 16,128 CG, 226 CHG, and 391 CHH (C = cytosine; G = guanine; H = either adenine, thymine, or cytosine) sites that were differentially methylated compared to samples from Controls. Of the CG sites, 7,407 were hypermethylated (at least 10% more methylated than Controls; P ≤ 0.05) and 8,721 were hypomethylated (at least 10% less methylated than Controls; P ≤ 0.05). Increased DNA methylation in gene promoter regions typically results in decreased transcriptional activity of the region. Therefore, differentially methylated CG sites located within promoter regions (n = 1,205) were used to predict (using Ingenuity Pathway Analysis software) alterations to canonical pathways in PNS compared with Control bull calves. In PNS bull calves, 113 pathways were altered (P ≤ 0.05) compared to Controls. Among these were pathways related to behavior, stress response, metabolism, immune function, and cell signaling. Genome-wide differential DNA methylation and predicted alterations to pathways in PNS compared with Control bull calves suggest epigenetic programming of biological systems in utero.

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“…The thermostabilizing mutations in crystal structure was converted back to the wild type residues using Maestro9.2, and disulfide bonds were built accordingly. The minimizationheating-equilibration-production was carried out using the protocol used in the previous work (39)(40)(41)(42). Each of the 15 extracted all-atom simulation boxes were minimized and equilibrated using 50 ns long NPT equilibration simulations performed in steps to gradually reduce constraints on protein heavy atoms and lipid heavy atoms from 5 to 0 kcal/mol by 1 kcal/mol interval per 10 ns simulation windows.…”

Section: Coarse Grain (Cg) Simulation Followed By All Atom MD Simulatmentioning

confidence: 99%

Activation microswitches in GPCRs function as rheostats in cell membrane

Lee

Vaidehi

2020

Preprint

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Although multiple components of the cell membrane modulate the stability and activation of G protein coupled receptors (GPCRs), the activation mechanism comes from detergent studies, since it is challenging to study activation in multi-component lipid bilayer. Using the multi-scale molecular dynamics simulations(50μs), our comparative study between cell membrane and detergents shows that: the changes in inter-residue distances, known as activation microswitches, show an ensemble of states in the extent of activation in cell membrane. We forward a rheostat model of GPCR activation rather than a binary switch model. Phosphatidylinositol bisphosphate (PIP2) and calcium ions, through a tug of war, maintain a balance between the GPCR stability and activity in cell membrane. Due to the lack of receptor stiffening effects by PIP2, detergents promote more transitions among conformational states than cell membrane. These findings connect the chemistry of cell membrane lipids to receptor activity useful to design detergents mimicking cell membrane.

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Distinct structural mechanisms determine substrate affinity and kinase activity of protein kinase Cα

Cited by 17 publications

References 40 publications

Bitopic Inhibition of ATP and Substrate Binding in Ser/Thr Kinases through a Conserved Allosteric Mechanism

Bitopic Inhibition of ATP and Substrate Binding in Ser/Thr Kinases through a Conserved Allosteric Mechanism

Prenatal transportation stress alters genome-wide DNA methylation in suckling Brahman bull calves1,2

Activation microswitches in GPCRs function as rheostats in cell membrane

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