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The faithful propagation of cellular signals in most organisms relies on the coordinated functions of a large family of protein kinases that share a conserved catalytic domain. The catalytic domain is a dynamic scaffold that undergoes large conformational changes upon activation. Most of these conformational changes, such as movement of the regulatory αC-helix from an "out" to "in" conformation, hinge on a conserved, but understudied, loop termed the αC-β4 loop, which mediates conserved interactions to tether flexible structural elements to the kinase core. We previously showed that the αC-β4 loop is a unique feature of eukaryotic protein kinases. Here, we review the emerging roles of this loop in kinase structure, function, regulation, and diseases. Through a kinome-wide analysis, we define the boundaries of the loop for the first time and show that sequence and structural variation in the loop correlate with conformational and regulatory variation. Many recurrent disease mutations map to the αC-β4 loop and contribute to drug resistance and abnormal kinase activation by relieving key auto-inhibitory interactions associated with αC-helix and inter-lobe movement. The αC-β4 loop is a hotspot for post-translational modifications, protein-protein interaction, and Hsp90 mediated folding. Our kinome-wide analysis provides insights for hypothesis-driven characterization of understudied kinases and the development of allosteric protein kinase inhibitors., (© 2020 International Union of Biochemistry and Molecular Biology.)

Many bioinformatics resources with unique perspectives on the protein landscape are currently available. However, generating new knowledge from these resources requires interoperable workflows that support cross-resource queries. In this study, we employ federated queries linking information from the Protein Kinase Ontology, iPTMnet, Protein Ontology, neXtProt, and the Mouse Genome Informatics to identify key knowledge gaps in the functional coverage of the human kinome and prioritize understudied kinases, cancer variants and post-translational modifications (PTMs) for functional studies. We identify 32 functional domains enriched in cancer variants and PTMs and generate mechanistic hypotheses on overlapping variant and PTM sites by aggregating information at the residue, protein, pathway and species level from these resources. We experimentally test the hypothesis that S768 phosphorylation in the C-helix of EGFR is inhibitory by showing that oncogenic variants altering S768 phosphorylation increase basal EGFR activity. In contrast, oncogenic variants altering conserved phosphorylation sites in the 'hydrophobic motif' of PKCβII (S660F and S660C) are loss-of-function in that they reduce kinase activity and enhance membrane translocation. Our studies provide a framework for integrative, consistent, and reproducible annotation of the cancer kinomes.

Protein tyrosine kinases (PTKs) are a group of closely related enzymes that have evolutionarily diverged from serine/threonine kinases (STKs) to regulate pathways associated with multi-cellularity. Evolutionary divergence of PTKs from STKs has occurred through accumulation of mutations in the active site as well as in the commonly conserved hydrophobic core. While the functional significance of active site variations is well understood, relatively little is known about how hydrophobic core variations contribute to PTK evolutionary divergence. Here, using a combination of statistical sequence comparisons, molecular dynamics simulations, mutational analysis and in vitro thermostability and kinase assays, we investigate the structural and functional significance of key PTK-specific variations in the kinase core. We find that the nature of residues and interactions in the hydrophobic core of PTKs is strikingly different from other protein kinases, and PTK-specific variations in the core contribute to functional divergence by altering the stability and dynamics of the kinase domain. In particular, a functionally critical STK-conserved histidine that stabilizes the regulatory spine in STKs is selectively mutated to an alanine, serine or glutamate in PTKs, and this loss-of-function mutation is accommodated, in part, through compensatory PTK-specific interactions in the core. In particular, a PTK-conserved phenylalanine in the I-helix appears to structurally and functionally compensate for the loss of STK-histidine by interacting with the regulatory spine, which has far-reaching effects on enzyme activity, inhibitor sensing, and stability. We propose that hydrophobic core variations provide a selective advantage during PTK evolution by increasing the conformational flexibility, and therefore the allosteric potential of the kinase domain. Our studies also suggest that Tyrosine Kinase Like kinases such as RAF are intermediates in PTK evolutionary divergence inasmuch as they share features of both PTKs and STKs in the core. Finally, our studies provide an evolutionary framework for identifying and characterizing disease and drug resistance mutations in the kinase core. [ABSTRACT FROM AUTHOR]

Abstract: Human immunodeficiency virus (HIV) regulatory protein Tat has pro-oxidant property, which might contribute to Tat-induced long terminal repeat region (LTR) transactivation. However, the intracellular mechanisms whereby Tat triggers ROS production, and the relationship between Tat-induced ROS production and LTR transactivation, are still subject to debate. The present study was undertaken to evaluate the specific effects of Tat on nicotinamide adenine denucleotide phosphate (NADPH) oxidase in MAGI cells, and to determine the specific role of NADPH oxidase in Tat-induced LTR transactivation. Application of Tat to MAGI cells caused increases in ROS formation that were prevented by both pharmacologic NADPH oxidase inhibitors and by siRNA Nox2, but not by other inhibitors of pro-oxidant enzymes or siRNA Nox4. Furthermore, inhibition of NADPH oxidase by both pharmacologic NADPH oxidase inhibitors and by siRNA Nox2 attenuated Tat-induced p65 phosphorylation and IKK phosphorylation. Phosphatidylinositol 3-kinase/Akt signaling pathway was involved in Tat-induced NADPH oxidase stimulation. Finally, NADPH oxidase inhibitors or Nox2 siRNA, but not control siRNA, inhibited Tat-induced LTR transactivation. Tat-induced HIV-1 LTR transactivation was inhibited in wortmannin or LY294002 treated cells compared to control cells. Together, these data describe a specific and biologically significant signaling component of the MAGI cells response to Tat, and suggest the PI3K/Akt signaling pathway might originate in part with Tat-induced activation of NADPH oxidase and LTR transactivation. [ABSTRACT FROM AUTHOR]

Polybrominated diphenyl ethers (PBDEs) are persistent organic pollutants (POPs). They are constantly detected in foods. PBDEs can disrupt the intestinal flora, but enterotoxicity is unknown. Luteolin, one kind of flavonoid, has drawn increasing interest as an agent that strengthens the intestinal barrier. This study aimed to evaluate the mitigating effect of luteolin on damage to the intestinal barrier induced by decabromodiphenyl ether (BDE-209) in a Caco-2 cell monolayer model. Results showed that luteolin mitigated BDE-209-induced damage to intestinal epithelial barrier by reducing the levels of reactive oxygen species (ROS), increasing the activity of superoxide dismutase (SOD) and glutathione (GSH), suppressed the secretion of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β), and increased the expression of tight junction (TJ) proteins (ZO-1, occludin, and claudin-1). Furthermore, the protective effects were related to the inhibition of extracellular regulated protein kinases (ERK) and nuclear factor kappa-B (NF-κB)/myosin light chain kinase (MLCK) signaling pathways, and the activation of nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathways. This study is the first to provide strong evidence that BDE-209 can damage the intestinal barrier, and we here investigated the important protective effect of luteolin, which may lay the foundation for the development of luteolin as a dietary supplement to strengthen the intestinal barrier. [Display omitted] • BDE-209 induced intestinal epithelial dysfunction. • Luteolin regulated oxidative damage by activating the Nrf2/ARE signaling pathways. • Luteolin relieved TJ damage by inhibiting the ERK and NF-κB/MLCK signaling pathways. [ABSTRACT FROM AUTHOR]

Convergent evidence links traditional brominated flame retardants (BFRs) exposure to weight gain, while the obesogenic potency of new BFRs (NBFRs) remain largely unknown. Aiding by luciferase-reporter gene assay, the present study revealed only pentabromoethylbenzene (PBEB), an alternative for penta-BDEs, binds with retinoid X receptor α (RXRα) but not peroxisome proliferator receptor γ (PPARγ) among the seven testing NBFRs. An apparent induction of adipogenesis in 3T3-L1 cells was observed at nanomolar of PBEB, much lower than penta-BFRs. Mechanistic research uncovered PBEB initiated the adipogenesis by demethylated CpG sites in the PPARγ promoter region. Specifically, activation RXRα by PBEB strengthened the activity of RXRα/PPARγ heterodimer, tightened the interaction between the heterodimer and PPAR response elements, and further enhanced adipogenesis. RNA sequencing combined with k -means clustering analysis exposed adenosine 5′-monophosphate (AMP)-activated protein kinase and phosphoinositide-3-kinase (PI3K)/protein kinase B (AKT) signaling as two predominant pathways that enriched in PBEB-induced lipogenesis. The obesogenic outcome was further corroborated in offspring mice when the maternal mice exposed to environmental relevant doses of PBEB. We found the male offspring exhibited adipocyte hypertrophy and increased weight gain in the epididymal white adipose tissue (eWAT). Consistent with in vitro findings, the reduction in protein phosphorylation of both AMPK and PI3K/AKT were observed within eWAT. Thus, we posited PBEB disrupts the pathways controlling adipogenesis and adipose tissue maintenance, supporting its potential as an environmental obesogen. [ABSTRACT FROM AUTHOR]

Summary The UbiB protein kinase-like (PKL) family is widespread, comprising one-quarter of microbial PKLs and five human homologs, yet its biochemical activities remain obscure. COQ8A (ADCK3) is a mammalian UbiB protein associated with ubiquinone (CoQ) biosynthesis and an ataxia (ARCA2) through unclear means. We show that mice lacking COQ8A develop a slowly progressive cerebellar ataxia linked to Purkinje cell dysfunction and mild exercise intolerance, recapitulating ARCA2. Interspecies biochemical analyses show that COQ8A and yeast Coq8p specifically stabilize a CoQ biosynthesis complex through unorthodox PKL functions. Although COQ8 was predicted to be a protein kinase, we demonstrate that it lacks canonical protein kinase activity in trans . Instead, COQ8 has ATPase activity and interacts with lipid CoQ intermediates, functions that are likely conserved across all domains of life. Collectively, our results lend insight into the molecular activities of the ancient UbiB family and elucidate the biochemical underpinnings of a human disease. [ABSTRACT FROM AUTHOR]