The choreography of protein kinase PDK1 and its diverse substrate dance partners.

The protein kinase PDK1 phosphorylates at least 24 distinct substrates, all of which belong to the AGC protein kinase group. Some substrates, such as conventional PKCs, undergo phosphorylation by PDK1 during their synthesis and subsequently get activated by DAG and Calcium. On the other hand, other substrates, including members of the Akt/PKB, S6K, SGK, and RSK families, undergo phosphorylation and activation downstream of PI3-kinase signaling. This review presents two accepted molecular mechanisms that determine the precise and timely phosphorylation of different substrates by PDK1. The first mechanism involves the colocalization of PDK1 with Akt/PKB in the presence of PIP3. The second mechanism involves the regulated docking interaction between the hydrophobic motif (HM) of substrates and the PIF-pocket of PDK1. This interaction, in trans, is equivalent to the molecular mechanism that governs the activity of AGC kinases through their HMs intramolecularly. PDK1 has been instrumental in illustrating the bi-directional allosteric communication between the PIF-pocket and the ATP-binding site and the potential of the system for drug discovery. PDK1's interaction with substrates is not solely regulated by the substrates themselves. Recent research indicates that full-length PDK1 can adopt various conformations based on the positioning of the PH domain relative to the catalytic domain. These distinct conformations of full-length PDK1 can influence the interaction and phosphorylation of substrates. Finally, we critically discuss recent findings proposing that PIP3 can directly regulate the activity of PDK1, which contradicts extensive in vitro and in vivo studies conducted over the years.

Development of 2-(4-pyridyl)-benzimidazoles as PKN2 chemical tools to probe cancer.

Kinases are signalling proteins which have proven to be successful targets for the treatment of a variety of diseases, predominantly in cancers. However, only a small proportion of kinases (<20%) have been investigated for their therapeutic viability, likely due to the lack of available chemical tools across the kinome. In this work we describe initial efforts in the development of a selective chemical tool for protein kinase N2 (PKN2), a relatively unexplored kinase of interest in several types of cancer. The most successful compound, 5, has a measured IC50 of 0.064 µM against PKN2, with ca. 17-fold selectivity over close homologue, PKN1.

Allosteric Regulation of Protein Kinases Downstream of PI3-Kinase Signalling.

Allostery is a basic principle that enables proteins to process and transmit cellular information. Protein kinases evolved allosteric mechanisms to transduce cellular signals to downstream signalling components or effector molecules. Protein kinases catalyse the transfer of the terminal phosphate from ATP to protein substrates upon specific stimuli. Protein kinases are targets for the development of small molecule inhibitors for the treatment of human diseases. Drug development has focussed on ATP-binding site, while there is increase interest in the development of drugs targeting alternative sites, i.e. allosteric sites. Here, we review the mechanism of regulation of protein kinases, which often involve the allosteric modulation of the ATP-binding site, enhancing or inhibiting activity. We exemplify the molecular mechanism of allostery in protein kinases downstream of PI3-kinase signalling with a focus on phosphoinositide-dependent protein kinase 1 (PDK1), a model kinase where small compounds can allosterically modulate the conformation of the kinase bidirectionally.

A Critical View on ABC Transporters and Their Interacting Partners in Auxin Transport.

Different subclasses of ATP-binding cassette (ABC) transporters have been implicated in the transport of native variants of the phytohormone auxin. Here, the putative, individual roles of key members belonging to the ABCB, ABCD and ABCG families, respectively, are highlighted and the knowledge of their assumed expression and transport routes is reviewed and compared with their mutant phenotypes. Protein-protein interactions between ABC transporters and regulatory components during auxin transport are summarized and their importance is critically discussed. There is a focus on the functional interaction between members of the ABCB family and the FKBP42, TWISTED DWARF1, acting as a chaperone during plasma membrane trafficking of ABCBs. Further, the mode and relevance of functional ABCB-PIN interactions is diagnostically re-evaluated. A new nomenclature describing precisely the most likely ABCB-PIN interaction scenarios is suggested. Finally, available tools for the detection and prediction of ABC transporter interactomes are summarized and the potential of future ABC transporter interactome maps is highlighted.

The wheat AGC kinase TaAGC1 is a positive contributor to host resistance to the necrotrophic pathogen Rhizoctonia cerealis.

Considerable progress has been made in understanding the roles of AGC kinases in mammalian systems. However, very little is known about the roles of AGC kinases in wheat (Triticum aestivum). The necrotrophic fungus Rhizoctonia cerealis is the major pathogen of the destructive disease sharp eyespot of wheat. In this study, the wheat AGC kinase gene TaAGC1, responding to R. cerealis infection, was isolated, and its properties and role in wheat defence were characterized. R. cerealis-resistant wheat lines expressed TaAGC1 at higher levels than susceptible wheat lines. Sequence and phylogenetic analyses showed that the TaAGC1 protein is a serine/threonine kinase belonging to the NDR (nuclear Dbf2-related) subgroup of AGC kinases. Kinase activity assays proved that TaAGC1 is a functional kinase and the Asp-239 residue located in the conserved serine/threonine kinase domain of TaAGC1 is required for the kinase activity. Subcellular localization assays indicated that TaAGC1 localized in the cytoplasm and nucleus. Virus-induced TaAGC1 silencing revealed that the down-regulation of TaAGC1 transcripts significantly impaired wheat resistance to R. cerealis. The molecular characterization and responses of TaAGC1 overexpressing transgenic wheat plants indicated that TaAGC1 overexpression significantly enhanced resistance to sharp eyespot and reduced the accumulation of reactive oxygen species (ROS) in wheat plants challenged with R. cerealis. Furthermore, ROS-scavenging and certain defence-associated genes were up-regulated in resistant plants overexpressing TaAGC1 but down-regulated in susceptible knock-down plants. These results suggested that the kinase TaAGC1 positively contributes to wheat immunity to R. cerealis through regulating expression of ROS-related and defence-associated genes.

Partially knocking out NtPDK1a/1b/1c/1d simultaneously in Nicotiana tabacum using CRISPR/CAS9 technology results in auxin-related developmental defects.

The eukaryotic AGC protein kinase subfamily (protein kinase A/ protein kinase G/ protein kinase C-family) is involved in regulating numerous biological processes across kingdoms, including growth and development, and apoptosis. PDK1(3-phosphoinositide-dependent protein kinase 1) is a conserved serine/threonine kinase in eukaryotes, which is both a member of AGC kinase and a major regulator of many other downstream AGC protein kinase family members. Although extensively investigated in model plant Arabidopsis, detailed reports for tobacco PDK1s have been limited. To better understand the functions of PDK1s in tobacco, CRISPR/CAS9 transgenic lines were generated in tetraploid N. tabacum, cv. Samsun (NN) with 5-7 of the 8 copies of 4 homologous PDK1 genes in tobacco genome (NtPDK1a/1b/1c/1d homologs) simultaneously knocked out. Numerous developmental defects were observed in these NtPDK1a/1b/1c/1d CRISPR/CAS9 lines, including cotyledon fusion leaf shrinkage, uneven distribution of leaf veins, convex veins, root growth retardation, and reduced fertility, all of which reminiscence of impaired polar auxin transport. The severity of these defects was correlated with the number of knocked out alleles of NtPDK1a/1b/1c/1d. Consistent with the observation in Arabidopsis, it was found that the polar auxin transport, and not auxin biosynthesis, was significantly compromised in these knockout lines compared with the wild type tobacco plants. The fact that no homozygous plant with all 8 NtPDK1a/1b/1c/1d alleles being knocked out suggested that knocking out 8 alleles of NtPDK1a/1b/1c/1d could be lethal. In conclusion, our results indicated that NtPDK1s are versatile AGC kinases that participate in regulation of tobacco growth and development via modulating polar auxin transport. Our results also indicated that CRISPR/CAS9 technology is a powerful tool in resolving gene redundancy in polyploidy plants.

Master kinase PDK1 in tumorigenesis.

3-phosphoinositide-dependent protein kinase 1 (PDK1) is considered as master kinase regulating AGC kinase family members such as AKT, SGK, PLK, S6K and RSK. Although autophosphorylation regulates PDK1 activity, accumulating evidence suggests that PDK1 is manipulated by many other mechanisms, including S6K-mediated phosphorylation, and the E3 ligase SPOP-mediated ubiquitination and degradation. Dysregulation of these upstream regulators or downstream signals involves in cancer development, as PDK1 regulating cell growth, metastasis, invasion, apoptosis and survival time. Meanwhile, overexpression of PDK1 is also exposed in a plethora of cancers, whereas inhibition of PDK1 reduces cell size and inhibits tumor growth and progression. More importantly, PDK1 also modulates the tumor microenvironments and markedly influences tumor immunotherapies. In summary, we comprehensively summarize the downstream signals, upstream regulators, mouse models, inhibitors, tumor microenvironment and clinical treatments for PDK1, and highlight PDK1 as a potential cancer therapeutic target.

Activation of σ1-Receptors by R-Ketamine May Enhance the Antidepressant Effect of S-Ketamine.

Ketamine is a racemic mixture composed of two enantiomers, S-ketamine and R-ketamine. In preclinical studies, both enantiomers have exhibited antidepressant effects, but these effects are attributed to distinct pharmacological activities. The S-enantiomer acts as an NMDA-channel blocker and as an opioid µ-receptor agonist, whereas the R-enantiomer binds to σ1-receptors and is believed to act as an agonist. As racemate, ketamine potentially triggers four biochemical pathways involving the AGC-kinases, PKA, Akt (PKB), PKC and RSK that ultimately lead to inhibitory phosphorylation of GSK3ß in microglia. In patients with major depressive disorder, S-ketamine administered as a nasal spray has shown clear antidepressant activity. However, when compared to intravenously infused racemic ketamine, the response rate, duration of action and anti-suicidal activity of S-ketamine appear to be less pronounced. The σ1-protein interacts with µ-opioid and TrkB-receptors, whereas in preclinical experiments σ1-agonists reduce µ-receptor desensitization and improve TrkB signal transduction. TrkB activation occurs as a response to NMDA blockade. So, the σ1-activity of R-ketamine may not only enhance two pathways via which S-ketamine produces an antidepressant response, but it furthermore provides an antidepressant activity in its own right. These two factors could explain the apparently superior antidepressant effect observed with racemic ketamine compared to S-ketamine alone.

An essential Trypanosoma brucei protein kinase: a functional analysis of regulation and the identification of inhibitors.

Introduction: The protein serine/threonine kinase AEK1 is essential in the pathogenic stage of Trypanosoma brucei, the causative agent of African trypanosomiasis. AEK1 is a member of the AGC protein kinase family, although it is not closely related to a specific human AGC kinase. Our previous chemical genetic studies showed that targeted inhibition of AEK1 in parasites expressing analog-sensitive AEK1 blocked parasite growth and enhanced survival of infected mice. Methods: To further validate AEK1 as a drug target, we used the chemical genetic system to determine the effect of a 24 hour loss of AEK1 activity on cell viability at the clonal level. A panel of 429 protein kinase inhibitors were screened against the wild-type protein for binding, using time-resolved fluorescence energy transfer (TR-FRET). The role of phosphorylation sites and motifs was probed by determining whether expression of proteins harboring mutations in these sequences could rescue AEK1 conditional knockout parasites. To determine the effect that mutations in the phosphosites have on the kinase activity of cellular AEK1 we compared the in vitro kinase activity of mutant and wild-type proteins immunoprecipitated from parasite lysates using the exogenous substrate MBP. Finally, the tagged AEK1 protein was localized by deconvolution microscopy. Results: After a 24 hour exposure to an AEK1 inhibitory analog in the chemical genetic system, less than five percent of the remaining live cells can clonally expand, further validating AEK1 as a drug target. In the AEK1 inhibitor screening assay, we identified 17 hit compounds. Complementation studies showed that of the two known phosphorylation sites in the activation loop; mutation of one abolished function while mutation of the other had no discernable effect. Mutation of the other two AEK1 phosphosites gave intermediate phenotypes. Mutations in either the hydrophobic motif at the C-terminus of the protein or in the region of AEK1 predicted to bind the hydrophobic motif were also required for function. All parasites with defective AEK1 showed reduced proliferation and defects in cytokinesis, although the tested mutations differed in terms of the extent of cell death. Kinase activity of immunoprecipitated AEK1 phosphosite mutants largely paralleled the effects seen in complementation studies, although the mutation of the phosphosite adjacent to the hydrophobic motif had a greater impact on activity than predicted by the complementation studies. AEK1 was localized to cytoplasmic puncta distinct from glycosomes and acidocalcisomes. Discussion: The rapid loss of viability of cells inhibited for AEK1 supports the idea that a short course of treatment that target AEK1 may be sufficient for treatment of people or animals infected with T. brucei. Key regulatory elements between AEK1 and its closest mammalian homolog appear to be largely conserved despite the vast evolutionary distance between mammals and T. brucei. The presence of AEK1 in cytoplasmic puncta raises the possibility that its localization may also play a role in functional activity.

A positive feedback circuit for ROP-mediated polar growth.

Tip growth is a special type of polarized growth in which a single and unique polarization site is established and maintained. Rho of Plants (ROP) proteins, which represent the only class of Rho GTPases in plants, regulate tip growth. The dynamic and asymmetric distribution of ROPs is critical for the establishment and maintenance of tip growth, and requires at least one positive feedback loop, which is still elusive. Here, we report a positive feedback circuit essential for tip growth of root hairs, in which ROPs, ROP activators and effectors, and AGC1.5 subfamily kinases are interconnected by sequential oligomerization and phosphorylation. AGC1.5 subfamily kinases interact with and phosphorylate two guanine nucleotide exchange factors (GEFs) of ROPs, RopGEF4 and RopGEF10. They also interact with two ROP effectors, ICR2/RIP3 and MIDD1/RIP4, which bridge active ROPs with AGC1.5. Functional loss of the AGC1.5 subfamily kinases or ICR2 and MIDD1 compromised root hair growth due to reduced ROP signaling. We found that asymmetric targeting of RopGEF4 and RopGEF10 is controlled by AGC1.5-dependent phosphorylation. Interestingly, we discovered that the ROP effectors recruit AGC1.5 to active ROP domains at the plasma membrane during root hair growth and are critical for AGC1.5-dependent phosphorylation of RopGEFs. Given the large number of AGC kinases in plants, this positive feedback circuit may be a universal theme for plant cell polar growth.

Drug Target Validation of the Protein Kinase AEK1, Essential for Proliferation, Host Cell Invasion, and Intracellular Replication of the Human Pathogen Trypanosoma cruzi.

Protein phosphorylation is involved in several key biological roles in the complex life cycle of Trypanosoma cruzi, the etiological agent of Chagas disease, and protein kinases are potential drug targets. Here, we report that the AGC essential kinase 1 (TcAEK1) exhibits a cytosolic localization and a higher level of expression in the replicative stages of the parasite. A CRISPR/Cas9 editing technique was used to generate ATP analog-sensitive TcAEK1 gatekeeper residue mutants that were selectively and acutely inhibited by bumped kinase inhibitors (BKIs). Analysis of a single allele deletion cell line (TcAEK1-SKO), and gatekeeper mutants upon treatment with inhibitor, showed that epimastigote forms exhibited a severe defect in cytokinesis. Moreover, we also demonstrated that TcAEK1 is essential for epimastigote proliferation, trypomastigote host cell invasion, and amastigote replication. We suggest that TcAEK1 is a pleiotropic player involved in cytokinesis regulation in T. cruzi and thus validate TcAEK1 as a drug target for further exploration. The gene editing strategy we applied to construct the ATP analog-sensitive enzyme could be appropriate for the study of other proteins of the T. cruzi kinome. IMPORTANCE Chagas disease affects 6 to 7 million people in the Americas, and its treatment has been limited to drugs with relatively high toxicity and low efficacy in the chronic phase of the infection. New validated targets are needed to combat this disease. In this work, we report the chemical and genetic validation of the protein kinase AEK1, which is essential for cytokinesis and infectivity, using a novel gene editing strategy.

Akt Isoforms: A Family Affair in Breast Cancer.

Akt, also known as protein kinase B (PKB), belongs to the AGC family of protein kinases. It acts downstream of the phosphatidylinositol 3-kinase (PI3K) and regulates diverse cellular processes, including cell proliferation, cell survival, metabolism, tumor growth and metastasis. The PI3K/Akt signaling pathway is frequently deregulated in breast cancer and plays an important role in the development and progression of breast cancer. There are three closely related members in the Akt family, namely Akt1(PKBα), Akt2(PKBß) and Akt3(PKBγ). Although Akt isoforms share similar structures, they exhibit redundant, distinct as well as opposite functions. While the Akt signaling pathway is an important target for cancer therapy, an understanding of the isoform-specific function of Akt is critical to effectively target this pathway. However, our perception regarding how Akt isoforms contribute to the genesis and progression of breast cancer changes as we gain new knowledge. The purpose of this review article is to analyze current literatures on distinct functions of Akt isoforms in breast cancer.

The Arabidopsis Rho of Plants GTPase ROP1 Is a Potential Calcium-Dependent Protein Kinase (CDPK) Substrate.

Plant Rho-type GTPases (ROPs) are versatile molecular switches involved in a number of signal transduction pathways. Although it is well known that they are indirectly linked to protein kinases, our knowledge about their direct functional interaction with upstream or downstream protein kinases is scarce. It is reasonable to suppose that similarly to their animal counterparts, ROPs might also be regulated by phosphorylation. There is only, however, very limited experimental evidence to support this view. Here, we present the analysis of two potential phosphorylation sites of AtROP1 and two types of potential ROP-kinases. The S74 site of AtROP1 has been previously shown to potentially regulate AtROP1 activation dependent on its phosphorylation state. However, the kinase phosphorylating this evolutionarily conserved site could not be identified: we show here that despite of the appropriate phosphorylation site consensus sequences around S74 neither the selected AGC nor CPK kinases phosphorylate S74 of AtROP1 in vitro. However, we identified several phosphorylation sites other than S74 for the CPK17 and 34 kinases in AtROP1. One of these sites, S97, was tested for biological relevance. Although the mutation of S97 to alanine (which cannot be phosphorylated) or glutamic acid (which mimics phosphorylation) somewhat altered the protein interaction strength of AtROP1 in yeast cells, the mutant proteins did not modify pollen tube growth in an in vivo test.

The AGC Kinase SsAgc1 Regulates Sporisorium scitamineum Mating/Filamentation and Pathogenicity.

Sporisorium scitamineum is the fungal pathogen causing severe sugarcane smut disease that leads to massive economic losses globally. S. scitamineum invades host cane by dikaryotic hyphae, formed after sexual mating of two haploid sporidia of opposite mating type. Therefore, mating/filamentation is critical for S. scitamineum pathogenicity, while its molecular mechanisms remain largely unknown. The AGC (cyclic AMP [cAMP]-dependent protein kinase 1 [protein kinase A {PKA}], cGMP-dependent protein kinase [PKG], and protein kinase C [PKC]) kinase family is a group of serine/threonine (Ser/Thr) protein kinases conserved among eukaryotic genomes, serving a variety of physiological functions, including cell growth, metabolism, differentiation, and cell death. In this study, we identified an AGC kinase, named SsAgc1 (for S. scitamineum Agc1), and characterized its function by reverse genetics. Our results showed that SsAgc1 is critical for S. scitamineum mating/filamentation and pathogenicity, and oxidative stress tolerance under some circumstances. Transcriptional profiling revealed that the SsAgc1 signaling pathway may control expression of the genes governing fungal mating/filamentation and tryptophan metabolism, especially for tryptophol production. We showed that tryptophan and tryptophol could at least partially restore ssagc1Δ mating/filamentation. Overall, our work revealed a signaling pathway mediated by AGC protein kinases to regulate fungal mating/filamentation, possibly through sensing and responding to tryptophol as signal molecules.IMPORTANCE The AGC signaling pathway represents a conserved distinct signaling pathway in regulation of fungal differentiation and virulence, while it has not been identified or characterized in the sugarcane smut fungus Sporisorium scitamineum In this study, we identified a PAS domain-containing AGC kinase, SsAgc1, in S. scitamineum Functional analysis revealed that SsAgc1 plays a regulatory role on the fungal dimorphic switch.

AGC1.5 Kinase Phosphorylates RopGEFs to Control Pollen Tube Growth.

Double fertilization in angiosperms requires the targeted delivery of immotile sperm to the eggs through pollen tubes. The polarity of tip-growing pollen tubes is maintained through dynamic association of active Rho GTPases of plants (ROP-GTP) with the apical plasma membrane. Guanine nucleotide exchange factors for ROPs (RopGEFs) catalyze the activation of ROPs and thereby affect spatiotemporal ROP signaling. Whereas RopGEFs have been found to be phosphorylated proteins, the kinases responsible for their phosphorylation in vivo and biological consequences of RopGEF phosphorylation in pollen tube growth remain unclear. We report here that the Arabidopsis AGC1.5 subfamily of cytoplasmic kinases is critical for the restricted localization of ROP-GTP during pollen tube growth. Loss of AGC1.5 and AGC1.7 functions resulted in the mistargeting of active ROPs and defective events downstream of ROP signaling in pollen tubes. AGC1.5 interacts with RopGEFs via their catalytic PRONE domain and phosphorylates RopGEFs at a conserved Ser residue of PRONE domain. Loss of AGC1.5 and AGC1.7 functions resulted in the mistargeting of RopGEFs in pollen tubes, similar to the phenotype caused by the mutation that renders RopGEFs non-phosphorylatable by AGC1.5. Collectively, our results provide mechanistic insights into the spatiotemporal activation of ROPs during the polar growth of pollen tubes.

Activation and Polarity Control of PIN-FORMED Auxin Transporters by Phosphorylation.

Auxin controls almost every aspect of plant development. Auxin is distributed within the plant by passive diffusion and active cell-to-cell transport. PIN-FORMED (PIN) auxin efflux transporters are polarly distributed in the plasma membranes of many cells, and knowledge about their distribution can predict auxin transport and explain auxin distribution patterns, even in complex tissues. Recent studies have revealed that phosphorylation is essential for PIN activation, suggesting that PIN phosphorylation needs to be taken into account in understanding auxin transport. These findings also ask for a re-examination of previously proposed mechanisms for phosphorylation-dependent PIN polarity control. We provide a comprehensive summary of the current knowledge on PIN regulation by phosphorylation, and discuss possible mechanisms of PIN polarity control in the context of recent findings.

A novel protein kinase is essential in bloodstream Trypanosoma brucei.

Human African trypanosomiasis a fatal disease for which no vaccines exist and treatment regimens are difficult. Here, we evaluate a Trypanosoma brucei protein kinase, AEK1, as a potential drug target. Conditional knockouts confirmed AEK1 essentiality in bloodstream forms. For chemical validation, we overcame the lack of AEK1 inhibitors by creating parasites expressing a single, functional analog-sensitive AEK1 allele. Analog treatment of mice infected with this strain delayed parasitemia and death, with one-third of animals showing no parasitemia. These studies validate AEK1 as a drug target and highlight the need for further understanding of its function.

Bidirectional Allosteric Communication between the ATP-Binding Site and the Regulatory PIF Pocket in PDK1 Protein Kinase.

Allostery is a phenomenon observed in many proteins where binding of a macromolecular partner or a small-molecule ligand at one location leads to specific perturbations at a site not in direct contact with the region where the binding occurs. The list of proteins under allosteric regulation includes AGC protein kinases. AGC kinases have a conserved allosteric site, the phosphoinositide-dependent protein kinase 1 (PDK1)-interacting fragment (PIF) pocket, which regulates protein ATP-binding, activity, and interaction with substrates. In this study, we identify small molecules that bind to the ATP-binding site and affect the PIF pocket of AGC kinase family members, PDK1 and Aurora kinase. We describe the mechanistic details and show that although PDK1 and Aurora kinase inhibitors bind to the conserved ATP-binding site, they differentially modulate physiological interactions at the PIF-pocket site. Our work outlines a strategy for developing bidirectional small-molecule allosteric modulators of protein kinases and other signaling proteins.

Discovery of N-[4-(1H-Pyrazolo[3,4-b]pyrazin-6-yl)-phenyl]-sulfonamides as Highly Active and Selective SGK1 Inhibitors.

From a virtual screening starting point, inhibitors of the serum and glucocorticoid regulated kinase 1 were developed through a combination of classical medicinal chemistry and library approaches. This resulted in highly active small molecules with nanomolar activity and a good overall in vitro and ADME profile. Furthermore, the compounds exhibited unusually high kinase and off-target selectivity due to their rigid structure.