Discovery of novel TAOK2 inhibitor scaffolds from high-throughput screening.

The MAP3K (Mitogen Activated Protein Kinase Kinase Kinase) TAOK2 (Thousand-And-One Kinase 2) is an activator of p38 MAP kinase cascade that is up-regulated in response to environmental stresses. A synthetic lethal screen performed using a NSCLC (non-small cell lung cancer) cell line, and a second screen identifying potential modulators of autophagy have implicated TAOK2 as a potential cancer therapeutic target. Using a 200,000 compound high throughput screen, we identified three specific small molecule compounds that inhibit the kinase activity of TAOK2. These compounds also showed inhibition of autophagy. Based on SAR (structure-activity relationship) studies, we have predicted the modifications on the reactive groups for the three compounds.

MAP kinase modules: the excursion model and the steps that count.

MAP kinase modules propagate diverse extracellular signals to downstream effectors. The two dual phosphorylation reactions catalyzed by the modules are thought to control the switch behavior of the pathway. Here we review recent approaches to understand these pathways through signal-to-response studies in cells and in vitro. These data are reconciled with physical models as well as predictions made on mathematical and theoretical grounds. Biochemical analysis has shown recently that the dual phosphorylation reactions catalyzed by MAP kinase modules are sequential at both levels of the cascade. The observed order of phosphorylation events suggests an excursion from the Ser/Thr kinase activity of the MAP3K into Tyr kinase activity of the central dual specificity MAP2K. How the order of events might be encoded in the structures and interactions is discussed. The ordered mechanism confirms predictions that reactions should be sequential to generate the steep signal-to-response curves and delayed responses observed in cells.

Precisely ordered phosphorylation reactions in the p38 mitogen-activated protein (MAP) kinase cascade.

The MAP kinase cascades, composed of a MAP3K, a MAP2K, and a MAPK, control switch responses to extracellular stimuli and stress in eukaryotes. The most important feature of these modules is thought to be the two double phosphorylation reactions catalyzed by MAP3Ks and MAP2Ks. We addressed whether the reactions are sequential or random in the p38 MAP kinase module. Mass spectrometry was used to track the phosphorylation of the MAP2K MEK6 by two MAP3Ks, TAO2 and ASK1, and the subsequent phosphorylation of p38α by MEK6/S*T* (where S (Ser) and T (Thr) are the two phosphorylation sites and * denotes phosphorylation). Both double phosphorylation reactions are precisely ordered. MEK6 is phosphorylated first on Thr-211 and then on Ser-207 by both MAP3Ks. This is the first demonstration of a precise reaction order for a MAP2K. p38α is phosphorylated first on Tyr-182 and then on Thr-180, the same reaction order observed previously in ERK2. Thus, intermediates were MEK6/ST* and p38α/TY*. Similarly, the phosphorylation of the p38α transcription factor substrate ATF2 occurs in a precise sequence. Progress curves for the appearance of intermediates were fit to kinetic models. The models confirmed the reaction order, revealed processivity in the phosphorylation of MEK6 by ASK1, and suggested that the order of phosphorylation is dictated by both binding and catalysis rates.

Chloride sensing by WNK1 involves inhibition of autophosphorylation.

WNK1 [with no lysine (K)] is a serine-threonine kinase associated with a form of familial hypertension. WNK1 is at the top of a kinase cascade, leading to phosphorylation of several cotransporters, in particular those transporting sodium, potassium, and chloride (NKCC), sodium and chloride (NCC), and potassium and chloride (KCC). The responsiveness of NKCC, NCC, and KCC to changes in extracellular chloride parallels their phosphorylation state, provoking the proposal that these transporters are controlled by a chloride-sensitive protein kinase. We found that chloride stabilizes the inactive conformation of WNK1, preventing kinase autophosphorylation and activation. Crystallographic studies of inactive WNK1 in the presence of chloride revealed that chloride binds directly to the catalytic site, providing a basis for the unique position of the catalytic lysine. Mutagenesis of the chloride-binding site rendered the kinase less sensitive to inhibition of autophosphorylation by chloride, validating the binding site. Thus, these data suggest that WNK1 functions as a chloride sensor through direct binding of a regulatory chloride ion to the active site, which inhibits autophosphorylation.

Solution structure of the WNK1 autoinhibitory domain, a WNK-specific PF2 domain.

WNK1 [with no lysine (K)-1] is a 250-kDa serine/threonine protein kinase involved in the maintenance of cellular salt levels and is directly linked to a hereditary form of hypertension. Here, we report the solution NMR structure of the autoinhibitory domain of WNK1 (WNK1-AI), a small regulatory subunit that lies immediately C-terminal of the kinase domain. We show that this domain is a homolog of the RFXV-binding PASK/FRAY homology 2 (PF2) domain found in OSR (oxidative stress responsive) and SPAK (serine/threonine proline-alanine-rich) kinases, which are substrates of WNK1. The WNK1-AI has a circularly permuted topology relative to the OSR1-PF2 domain. Nevertheless, like PF2 domains, WNK1-AI binds peptides that contain an RFXV motif with micromolar affinities as assessed by changes in (1)H,(15)N heteronuclear single quantum coherence spectra. Mutations to the WNK1-AI and binding peptides confirm a similar binding mode.