kinase; small-molecule inhibition; inhibitor selectivity; inhibitor screening; kinase domain; kinase assay
Eukaryotic protein kinases are generally classified as being either tyrosine or serine-threonine specific. Though not evident from inspection of their primary sequences, many serine-threonine kinases display a significant preference for serine or threonine as the phosphoacceptor residue. Here we show that a residue located in the kinase activation segment, which we term the “DFG+1” residue, acts as a major determinant for serine-threonine phosphorylation site specificity. Mutation of this residue was sufficient to switch the phosphorylation site preference for multiple kinases, including the serine-specific kinase PAK4 and the threonine-specific kinase MST4. Kinetic analysis of peptide substrate phosphorylation and crystal structures of PAK4-peptide complexes suggested that phosphoacceptor residue preference is not mediated by stronger binding of the favored substrate. Rather, favored kinase-phosphoacceptor combinations likely promote a conformation optimal for catalysis. Understanding the rules governing kinase phosphoacceptor preference allows kinases to be classified as serine or threonine specific based on their sequence.
•A single active site residue can determine kinase phosphoacceptor specificity•Favored and disfavored substrates promote distinct kinase-bound conformations•A simple rule predicts kinase phosphoacceptor preference from its DFG+1 residue
Group I p21-activated kinases (PAKs) are important effectors of the small GTPases Rac and Cdc42, which regulate cell motility/migration, survival, proliferation and gene transcription. Hyperactivation of these kinases have been reported in many tumor types, making PAKs attractive targets for therapeutic intervention. PAKs are activated by growth factor-mediated signaling and are negatively regulated by the tumor suppressor NF2/Merlin. Thus, tumors characterized by NF2 inactivation would be expected to show hyperactivated PAK signaling. Based on this rationale, we evaluated the status of PAK signaling in malignant mesothelioma (MM), an aggressive neoplasm that is resistant to current therapies and shows frequent inactivation of NF2. We demonstrate that group I PAKs are activated in most MMs and MM cell lines and that genetic or pharmacological inhibition of PAKs is sufficient to inhibit MM cell proliferation and survival. We also identify downstream effectors and signaling pathways that may contribute mechanistically to PAK-related tumorigenesis. Specifically, we show that inhibition of PAK results in attenuation of AKT and Raf-MAPK signaling and decreased tumor cell viability. Collectively, these data suggest that pharmacological inhibition of group I PAKs may have therapeutic efficacy in tumors characterized by PAK activation.
p21-activated kinase (PAK); malignant mesothelioma; small molecule inhibitor; targeted therapy; NF2/Merlin; AKT; mitogen-activated protein kinases (MAPK); Raf-1
Identification of small molecule targets remains an important challenge for chemical genetics. We report a new approach for target identification and protein discovery based on functional suppression of chemical inhibition in vitro. We discovered pirl1, an inhibitor of actin assembly, in a screen conducted with cytoplasmic extracts. Pirl1 was used to partially inhibit actin assembly in the same assay and concentrated biochemical fractions of cytoplasmic extracts were added to find activities that suppressed pirl1 inhibition. Two activities were detected, separately purified, and identified as Arp2/3 complex and Cdc42/RhoGDI complex, both known regulators of actin assembly. We show that pirl1 directly inhibits activation of Cdc42/RhoGDI but that Arp2/3 complex represents a downstream suppressor. This work introduces a general method for using low micromolar chemical inhibitors to identify both inhibitor targets and other components of a signaling pathway.
Cyclooxygenase (COX)-derived prostanoids have long been implicated in blood pressure (BP) regulation. Recently prostaglandin E2 (PGE2) and its receptor EP1R have emerged as key players in angiotensin II (Ang-II)-dependent hypertension (HTN) and related end-organ damage. However, the enzymatic source of PGE2, ie COX-1 or COX-2, and its site(s) of action are not known. The subfornical organ (SFO) is a key forebrain region that mediates systemic Ang-II-dependent HTN via reactive oxygen species (ROS). We tested the hypothesis that cross-talk between PGE2/EP1R and ROS signaling in the SFO is required for Ang-II HTN. Radiotelemetric assessment of BP revealed that HTN induced by infusion of systemic “slow-pressor” doses of Ang-II was abolished in mice with null mutations in EP1R or COX-1 but not COX-2. Slow-pressor Ang-II-evoked HTN and ROS formation in the SFO were prevented when the EP1R antagonist SC-51089 was infused directly into brains of wild-type mice, and Ang-II-induced ROS production was blunted in cells dissociated from SFO of EP1R−/− and COX-1−/− but not COX-2−/− mice. In addition, slow-pressor Ang-II infusion caused a ~3-fold increase in PGE2 levels in the SFO but not in other brain regions. Finally, genetic reconstitution of EP1R selectively in the SFO of EP1R-null mice was sufficient to rescue slow-pressor AngII-elicited HTN and ROS formation in the SFO of this model. Thus, COX-1-derived PGE2 signaling through EP1R in the SFO is required for the ROS-mediated HTN induced by systemic infusion of Ang-II, and suggests that EP1R in the SFO may provide a novel target for antihypertensive therapy.
Prostanoids; PGE2; COX; reactive oxygen species; blood pressure; central nervous system
Small-molecule protein kinase inhibitors are central tools for elucidating cellular signaling pathways and are promising therapeutic agents. Due to evolutionary conservation of the ATP-binding site, most kinase inhibitors that target this site promiscuously inhibit multiple kinases. Interpretation of experiments utilizing these compounds is confounded by a lack of data on the comprehensive kinase selectivity of most inhibitors. Here we profiled the activity of 178 commercially available kinase inhibitors against a panel of 300 recombinant protein kinases using a functional assay. Quantitative analysis revealed complex and often unexpected kinase-inhibitor interactions, with a wide spectrum of promiscuity. Many off-target interactions occur with seemingly unrelated kinases, revealing how large-scale profiling can be used to identify multi-targeted inhibitors of specific, diverse kinases. The results have significant implications for drug development and provide a resource for selecting compounds to elucidate kinase function and for interpreting the results of experiments that use them.
Methylthioadenosine phosphorylase (MTAP), a key enzyme in the methionine salvage pathway, is inactivated in a variety of human cancers. Since all human tissues express MTAP, it would be of potential interest to identify compounds that selectively inhibit the growth of MTAP deficient cells. To determine if MTAP inactivation could be targeted, we have performed a differential chemical genetic screen in isogenic MTAP+ and MTAP− S. cerevisiae. A low molecular weight compound library containing 30,080 unique compounds was screened for those that selectively inhibit growth of MTAP− yeast using a differential growth assay. One compound, containing a 1,3,4-thiadiazine ring, repeatedly showed a differential dose response, with MTAP− cells exhibiting a four-fold shift in IC50 compared to MTAP+ cells. Several structurally related derivatives of this compound also showed enhanced growth inhibition in MTAP− yeast. These compounds were also examined for growth inhibition of isogenic MTAP+ and MTAP− HT1080 fibrosarcoma cells, and four of the five compounds exhibited evidence of modest, but significant, increased potency in MTAP− cells. In summary, these studies show the feasibility of differential growth screening technology and have identified a novel class of compounds that can preferentially inhibit growth of MTAP− cells.
Methionine Salvage Pathway; Drug screening; Yeast; Genetic-chemical interaction
p21-activated kinases are essential for spatial and temporal coordination of cytoskeletal dynamics with cellular adhesion during cell migration.
Cell motility requires the spatial and temporal coordination of forces in the actomyosin cytoskeleton with extracellular adhesion. The biochemical mechanism that coordinates filamentous actin (F-actin) assembly, myosin contractility, adhesion dynamics, and motility to maintain the balance between adhesion and contraction remains unknown. In this paper, we show that p21-activated kinases (Paks), downstream effectors of the small guanosine triphosphatases Rac and Cdc42, biochemically couple leading-edge actin dynamics to focal adhesion (FA) dynamics. Quantitative live cell microscopy assays revealed that the inhibition of Paks abolished F-actin flow in the lamella, displaced myosin IIA from the cell edge, and decreased FA turnover. We show that, by controlling the dynamics of these three systems, Paks regulate the protrusive activity and migration of epithelial cells. Furthermore, we found that expressing Pak1 was sufficient to overcome the inhibitory effects of excess adhesion strength on cell motility. These findings establish Paks as critical molecules coordinating cytoskeletal systems for efficient cell migration.
Phospholipid-enriched membranes such as the plasma membrane can serve as direct regulators of kinase signaling. Pak1 is involved in growth factor signaling at the plasma membrane and its dysregulation is implicated in cancer. Pak1 adopts an autoinhibited conformation that is relieved upon binding to membrane-bound Rho GTPases Rac1 or Cdc42, but whether lipids also regulate Pak1 in vivo is unknown. We show here that phosphoinositides, particularly PIP2, potentiate Rho-GTPase-mediated Pak1 activity. A positively charged region of Pak1 binds to phosphoinositide-containing membranes, and this interaction is essential for membrane recruitment and activation of Pak1 in response to extracellular signals. Our results highlight an active role for lipids as allosteric regulators of Pak1 and suggest that Pak1 is a “coincidence detector” whose activation depends on GTPases present in phosphoinositide-rich membranes. These findings expand the role of phosphoinositides in kinase signaling and suggest how altered phosphoinositide metabolism may upregulate Pak1 activity in cancer cells.
The renin angiotensin system (RAS) exerts a tremendous influence over fluid balance and arterial pressure. Angiotensin II (Ang-II), the effector peptide of the RAS, acts in the CNS to regulate neurohumoral outflow and thirst. Dysregulation of Ang-II signaling in the CNS is implicated in cardiovascular diseases, however the mechanisms remain poorly understood. Recently we established that NADPH oxidase (Nox)-derived superoxide acting in the forebrain subfornical organ (SFO) is critical in the physiologic responses to central Ang-II. In addition, we have found that Nox2 and Nox4 are the most abundantly expressed Nox homologues within Ang-II-sensitive sites in the forebrain. To dissect out the functional importance and unique roles of these Nox enzymes in the pressor and dipsogenic effects of central Ang-II, we developed adenoviral vectors expressing siRNA to selectively silence Nox2 or Nox4 expression in the SFO. Our results demonstrate that both Nox2 and Nox4 are required for the full vasopressor effects of brain Ang-II, but that only Nox2 is coupled to the Ang-II-induced water intake response. These studies establish the importance of both Nox2- and Nox4-containing NADPH oxidases in the actions of Ang-II in the CNS, and are the first to reveal differential involvement of these Nox enzymes in the various physiologic effects of central Ang-II.
hypertension; blood pressure; water intake; subfornical organ; adenovirus; siRNA
Formins are potent actin assembly factors. Diaphanous formins, including mDia1, mDia2, and mDia3 in mammals, are implicated in mitosis and cytokinesis but no chemical interactors have been reported. We developed an in vitro screen for inhibitors of actin assembly by mDia1, and identified an inhibitor of mDia1 and mDia2 that does not inhibit mDia3 at the concentrations tested. These results establish the druggability of mDia formins and introduce a first generation inhibitor.
Kinases are important therapeutic targets in oncology due to their frequent deregulation in cancer. Typical ATP-competitive kinase inhibitors, however, also inhibit off-target kinases that could lead to drug toxicity. Allosteric inhibitors represent an alternative approach to achieve greater kinase selectivity although examples of such compounds are few. Here we elucidate the mechanism of action of IPA-3, an allosteric inhibitor of Pak kinase activation. We demonstrate that IPA-3 binds covalently to the Pak1 regulatory domain and prevents binding to the upstream activator Cdc42. Pre-activated Pak1, however, is neither inhibited nor bound significantly by IPA-3, demonstrating exquisite conformational specificity of the interaction. Using radiolabeled IPA-3 we show that inhibitor binding is specific and reversible in reducing environments. Finally, cell experiments using IPA-3 implicate Pak1 in phorbol-ester stimulated membrane ruffling. This study reveals a novel allosteric mechanism for kinase inhibition through covalent targeting of a regulatory domain.
small molecule; mechanism of action; Pak kinase; kinase inhibitor
p21-activated kinases have been classified into two groups based on their domain architecture. Group II PAKs (PAK4–6) regulate a wide variety of cellular functions, and PAK deregulation has been linked to tumor development. Structural comparison of five high-resolution structures comprising all active, monophosphorylated group II catalytic domains revealed a surprising degree of domain plasticity, including a number of catalytically productive and nonproductive conformers. Rearrangements of helix αC, a key regulatory element of kinase function, resulted in an additional helical turn at the αC N terminus and a distortion of its C terminus, a movement hitherto unseen in protein kinases. The observed structural changes led to the formation of interactions between conserved residues that structurally link the glycine-rich loop, αC, and the activation segment and firmly anchor αC in an active conformation. Inhibitor screening identified six potent PAK inhibitors from which a tri-substituted purine inhibitor was cocrystallized with PAK4 and PAK5.