Looking lively: emerging principles of pseudokinase signaling.

Progress towards understanding catalytically 'dead' protein kinases - pseudokinases - in biology and disease has hastened over the past decade. An especially lively area for structural biology, pseudokinases appear to be strikingly similar to their kinase relatives, despite lacking key catalytic residues. Distinct active- and inactive-like conformation states, which are crucial for regulating bona fide protein kinases, are conserved in pseudokinases and appear to be essential for function. We discuss recent structural data on conformational transitions and nucleotide binding by pseudokinases, from which some common principles emerge. In both pseudokinases and bona fide kinases, a conformational toggle appears to control the ability to interact with signaling effectors. We also discuss how biasing this conformational toggle may provide opportunities to target pseudokinases pharmacologically in disease.

Dynamics of protein kinases and pseudokinases by HDX-MS.

Dynamics of the protein kinase fold are deeply intertwined with its structure. The past three decades of kinase biophysical studies revealed key dynamic features of the kinase domain and, more recently, how these features may endow catalytically impaired kinases-or pseudokinases-with signaling properties. Hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) is proving to be a valuable approach for studies of kinase and pseudokinase domain dynamics. Here, we briefly discuss the methods that have provided insights into protein kinase dynamics, describe how HDX-MS is being used to answer questions in the kinase/pseudokinase field, and provide a detailed protocol for collecting an HDX-MS dataset to study the impacts of small molecule binding to a pseudokinase domain. As more small molecules are discovered that can disrupt pseudokinase conformations, HDX-MS is likely to be a powerful approach for exploring drug-induced changes in pseudokinase dynamics and structure.

ROR and RYK extracellular region structures suggest that receptor tyrosine kinases have distinct WNT-recognition modes.

WNTs play key roles in development and disease, signaling through Frizzled (FZD) seven-pass transmembrane receptors and numerous co-receptors including ROR and RYK family receptor tyrosine kinases (RTKs). We describe crystal structures and WNT-binding characteristics of extracellular regions from the Drosophila ROR and RYK orthologs Nrk (neurospecific receptor tyrosine kinase) and Derailed-2 (Drl-2), which bind WNTs though a FZD-related cysteine-rich domain (CRD) and WNT-inhibitory factor (WIF) domain respectively. Our crystal structures suggest that neither Nrk nor Drl-2 can accommodate the acyl chain typically attached to WNTs. The Nrk CRD contains a deeply buried bound fatty acid, unlikely to be exchangeable. The Drl-2 WIF domain lacks the lipid-binding site seen in WIF-1. We also find that recombinant DWnt-5 can bind Drosophila ROR and RYK orthologs despite lacking an acyl chain. Alongside analyses of WNT/receptor interaction sites, our structures provide further insight into how WNTs may recruit RTK co-receptors into signaling complexes.

Structural Insights into Pseudokinase Domains of Receptor Tyrosine Kinases.

Despite their apparent lack of catalytic activity, pseudokinases are essential signaling molecules. Here, we describe the structural and dynamic properties of pseudokinase domains from the Wnt-binding receptor tyrosine kinases (PTK7, ROR1, ROR2, and RYK), which play important roles in development. We determined structures of all pseudokinase domains in this family and found that they share a conserved inactive conformation in their activation loop that resembles the autoinhibited insulin receptor kinase (IRK). They also have inaccessible ATP-binding pockets, occluded by aromatic residues that mimic a cofactor-bound state. Structural comparisons revealed significant domain plasticity and alternative interactions that substitute for absent conserved motifs. The pseudokinases also showed dynamic properties that were strikingly similar to those of IRK. Despite the inaccessible ATP site, screening identified ATP-competitive type-II inhibitors for ROR1. Our results set the stage for an emerging therapeutic modality of "conformational disruptors" to inhibit or modulate non-catalytic functions of pseudokinases deregulated in disease.

Distinct Hepatic PKA and CDK Signaling Pathways Control Activity-Independent Pyruvate Kinase Phosphorylation and Hepatic Glucose Production.

Pyruvate kinase is an important enzyme in glycolysis and a key metabolic control point. We recently observed a pyruvate kinase liver isoform (PKL) phosphorylation site at S113 that correlates with insulin resistance in rats on a 3 day high-fat diet (HFD) and suggests additional control points for PKL activity. However, in contrast to the classical model of PKL regulation, neither authentically phosphorylated PKL at S12 nor S113 alone is sufficient to alter enzyme kinetics or structure. Instead, we show that cyclin-dependent kinases (CDKs) are activated by the HFD and responsible for PKL phosphorylation at position S113 in addition to other targets. These CDKs control PKL nuclear retention, alter cytosolic PKL activity, and ultimately influence glucose production. These results change our view of PKL regulation and highlight a previously unrecognized pathway of hepatic CDK activity and metabolic control points that may be important in insulin resistance and type 2 diabetes.

Systematic analysis of F-box proteins reveals a new branch of the yeast mating pathway.

The mating pathway in yeast Saccharomyces cerevisiae has long been used to reveal new mechanisms of signal transduction. The pathway comprises a pheromone receptor, a heterotrimeric G protein, and intracellular effectors of morphogenesis and transcription. Polarized cell growth, in the direction of a potential mating partner, is accomplished by the G-protein ßγ subunits and the small G-protein Cdc42. Transcription induction, needed for cell-cell fusion, is mediated by Gßγ and the mitogen-activated protein kinase (MAPK) scaffold protein Ste5. A potential third pathway is initiated by the G-protein α subunit Gpa1. Gpa1 signaling was shown previously to involve the F-box adaptor protein Dia2 and an endosomal effector protein, the phosphatidylinositol 3-kinase Vps34. Vps34 is also required for proper vacuolar sorting and autophagy. Here, using a panel of reporter assays, we demonstrate that mating pheromone stimulates vacuolar targeting of a cytoplasmic reporter protein and that this process depends on Vps34. Through a systematic analysis of F-box deletion mutants, we show that Dia2 is required to sustain pheromone-induced vacuolar targeting. We also found that other F-box proteins selectively regulate morphogenesis (Ydr306, renamed Pfu1) and transcription (Ucc1). These findings point to the existence of a new and distinct branch of the pheromone-signaling pathway, one that likely leads to vacuolar engulfment of cytoplasmic proteins and recycling of cellular contents in preparation for mating.

Flipping ATP to AMPlify Kinase Functions.

Understanding protein kinase family members that lack key catalytic residues-or pseudokinases-is a major challenge in cell signaling. In this issue of Cell, Sreelatha et al. (2018) describe how one pseudokinase transfers adenosine monophosphate (AMP) rather than phosphate to protein substrates, revealing unexpected catalytic diversity for the kinase fold.

PKCε contributes to lipid-induced insulin resistance through cross talk with p70S6K and through previously unknown regulators of insulin signaling.

Insulin resistance drives the development of type 2 diabetes (T2D). In liver, diacylglycerol (DAG) is a key mediator of lipid-induced insulin resistance. DAG activates protein kinase C ε (PKCε), which phosphorylates and inhibits the insulin receptor. In rats, a 3-day high-fat diet produces hepatic insulin resistance through this mechanism, and knockdown of hepatic PKCε protects against high-fat diet-induced hepatic insulin resistance. Here, we employed a systems-level approach to uncover additional signaling pathways involved in high-fat diet-induced hepatic insulin resistance. We used quantitative phosphoproteomics to map global in vivo changes in hepatic protein phosphorylation in chow-fed, high-fat-fed, and high-fat-fed with PKCε knockdown rats to distinguish the impact of lipid- and PKCε-induced protein phosphorylation. This was followed by a functional siRNA-based screen to determine which dynamically regulated phosphoproteins may be involved in canonical insulin signaling. Direct PKCε substrates were identified by motif analysis of phosphoproteomics data and validated using a large-scale in vitro kinase assay. These substrates included the p70S6K substrates RPS6 and IRS1, which suggested cross talk between PKCε and p70S6K in high-fat diet-induced hepatic insulin resistance. These results identify an expanded set of proteins through which PKCε may drive high-fat diet-induced hepatic insulin resistance that may direct new therapeutic approaches for T2D.

RGS proteins and septins cooperate to promote chemotropism by regulating polar cap mobility.

BACKGROUND: Septins are well known to form a boundary between mother and daughter cells in mitosis, but their role in other morphogenic states is poorly understood. RESULTS: Using microfluidics and live-cell microscopy, coupled with new computational methods for image analysis, we investigated septin function during pheromone-dependent chemotropic growth in yeast. We show that septins colocalize with the regulator of G protein signaling (RGS) Sst2, a GTPase-activating protein that dampens pheromone receptor signaling. We show further that the septin structure surrounds the polar cap, ensuring that cell growth is directed toward the source of pheromone. When RGS activity is abrogated, septins are partially disorganized. Under these circumstances, the polar cap travels toward septin structures and away from sites of exocytosis, resulting in a loss of gradient tracking. CONCLUSIONS: Septin organization is dependent on RGS protein activity. When assembled correctly, septins promote turning of the polar cap and proper tracking of a pheromone gradient.