Identification of Abscisic Acid-Dependent Phosphorylated Basic Helix-Loop-Helix Transcription Factors in Guard Cells of Vicia faba by Mass Spectrometry

An unknown 61 kDa protein is phosphorylated by abscisic acid (ABA)-activated protein kinase in response to ABA and binds to 14-3-3 protein in a phosphorylation-dependent manner in guard-cell protoplasts (GCPs) from Vicia faba. Subsequently, ABA-dependent phosphorylated proteins were identified as basic helix–loop–helix transcription factors, named ABA-responsive kinase substrates (AKSs) in GCPs from Arabidopsis thaliana. However, whether the 61 kDa protein in Vicia GCPs is an AKS is unclear. We performed immunoprecipitation of ABA-treated Vicia GCPs using anti-14-3-3 protein antibodies and identified several AKS isoforms in V. faba (VfAKSs) by mass spectrometry. The 61 kDa protein was identified as VfAKS1. Phosphoproteomic analysis revealed that VfAKSs are phosphorylated at Ser residues, which are important for 14-3-3 protein binding and monomerisation, in response to ABA in GCPs. Orthologs of AtABCG40, an ABA importer in guard cells, and CHC1, a clathrin heavy chain and a regulator of stomatal movement, also co-immunoprecipitated with 14-3-3 protein from guard cells.

LRRK2 causes centrosomal deficits via phosphorylated Rab10 and RILPL1 at centriolar subdistal appendages

The Parkinson′s disease-associated LRRK2 kinase phosphorylates multiple Rab GTPases including Rab8 and Rab10, which enhances their binding to RILPL1 and RILPL2. The nascent interaction between phospho-Rab10 and RILPL1 blocks ciliogenesis in vitro and in the intact brain, and interferes with the cohesion of duplicated centrosomes in dividing cells. We show here that various LRRK2 risk variants and all currently described regulators of the LRRK2 signaling pathway converge upon causing centrosomal cohesion deficits. The cohesion deficits do not require the presence of RILPL2 or of other LRRK2 kinase substrates including Rab12, Rab35 and Rab43. Rather, they depend on the RILPL1-mediated centrosomal accumulation of phosphorylated Rab10. RILPL1 localizes to the subdistal appendages of the mother centriole, followed by recruitment of the LRRK2-phosphorylated Rab protein to cause the centrosomal defects. These data reveal a common molecular pathway by which alterations in the LRRK2 kinase activity impact upon centrosome-related events.

KSTAR: An algorithm to predict patient-specific kinase activities from phosphoproteomic data

Kinase inhibitors are one of the largest classes of FDA-approved drugs and are major targets in oncology. Although kinase inhibitors have played an important role in improving cancer outcomes, major challenges still exist, including the development of resistance and failure to respond to treatments. Improvements for tumor profiling of kinase activity would be an important step in improving treatment outcomes and identifying effective kinase targets. Here, we present a graph- and statistics-based algorithm, called KSTAR, which harnesses the phosphoproteomic profiling of human cells and tissues by predicting kinase activity profiles from the observed phosphorylation of kinase substrates. The algorithm is based on the hypothesis that the more active a kinase is, the more of its substrates will be observed in a phosphoproteomic experiment. This method is error- and bias-aware in its approach, overcoming the challenges presented by the variability of phosphoproteomic pipelines, the limited information about kinase-substrate relationships, and limitations of global kinase-substrate predictions, such as training set bias and high overlap between predicted kinase networks. We demonstrate that the predicted kinase activities: 1) reproduce physiologically-relevant expectations and generates novel hypotheses within cell-specific experiments, 2) improve the ability to compare phosphoproteomic samples on the same tissues from different labs, and 3) identify tissue-specific kinase profiles. Finally, we apply the approach to complex human tissue biopsies in breast cancer, where we find that KSTAR activity predictions complement current clinical standards for identifying HER2-status -- KSTAR can identify clinical false positives, patients who will fail to respond to inhibitor therapy, and clinically defined HER2-negative patients that might benefit from HER2-targeted therapy. KSTAR will be useful for both basic biological understanding of signaling networks and for improving clinical outcomes through improved clinical trial design, identification of new and/or combination therapies, and for identifying the failure to respond to targeted kinase therapies.

The Candida albicans Cdk8-dependent phosphoproteome reveals repression of hyphal growth through a Flo8-dependent pathway

Ssn3, also known as Cdk8, is a member of the four protein Cdk8 submodule within the multi-subunit Mediator complex involved in the co-regulation of transcription. In Candida albicans , the loss of Ssn3 kinase activity affects multiple phenotypes including cellular morphology, metabolism, nutrient acquisition, immune cell interactions, and drug resistance. In these studies, we generated a strain in which Ssn3 was replaced with a functional variant of Ssn3 that can be rapidly and selectively inhibited by the ATP analog 3-MB-PP1 . Consistent with ssn3 null mutant and kinase dead phenotypes, inhibition of Ssn3 kinase activity promoted hypha formation. Furthermore, the increased expression of hypha-specific genes was the strongest transcriptional signal upon inhibition of Ssn3 in transcriptomics analyses. Rapid inactivation of Ssn3 was used for phosphoproteomic studies performed to identify Ssn3 kinase substrates associated with filamentation potential. Both previously validated and novel Ssn3 targets were identified. Protein phosphorylation sites that were reduced specifically upon Ssn3 inhibition included two sites in Flo8 which is a transcription factor known to positively regulate C. albicans morphology. Mutation of the two Flo8 phosphosites (threonine 589 and serine 620) was sufficient to increase Flo8-HA levels and Flo8 dependent activity, suggesting that Ssn3 kinase activity negatively regulates Flo8. Previous work has also shown that loss of Ssn3 activity leads to increased alkalinization of medium with amino acids. Here, we show that FLO8 and STP2 , a transcription factor involved in amino acid utilization, are required for ssn3 ?/? phenotype, but that loss of the Ssn3 phosphosites identified in Flo8 was not sufficient to phenocopy the ssn3 ?/? mutant. These data highlight the spectrum of processes affected by the modulation of Ssn3 activity and underscore the importance of considering Ssn3 function in the control of transcription factor activities.

Signal Transduction Profiling of Angiotensin II Type 1 Receptor With Mutations Associated to Atrial Fibrillation in Humans

The AT1 receptor (AT1R) has a major role in the Renin-Angiotensin System, being involved in several physiological events including blood pressure control and electrolyte balance. The AT1R is a member of the G protein coupled receptors (GPCR) family, classically known to couple Gαq and engage β-arrestin recruitment. Both G protein and arrestin signaling pathways are involved in modulation of different downstream kinases. A previous study reported that mutations in the AT1R (A244S and I103T-A244S) were positively correlated with higher risk of atrial fibrillation in men. Based on that report, we aimed to investigate if these mutations, including I103T only, could affect AT1R signal transduction profile, and consequently, implicate in atrial fibrillation outcome. To address that, we engineered an AT1R carrying the above-mentioned mutations, and functionally evaluated different signaling pathways. Phosphokinase profiler array to assess the mutations downstream effects on kinases and kinase substrates phosphorylation levels was used. Our results show that the I103T-A244S mutant receptor presents decreased β-arrestin 2 recruitment, which could lead to a harmful condition of sustained Gαq signaling. Moreover, the phosphokinase profiler array revealed that the same mutation led to downstream modulation of kinase pathways that are linked to physiological responses such as fibrous tissue formation, apoptosis and cell proliferation.

Advances in Understanding TKS4 and TKS5: Molecular Scaffolds Regulating Cellular Processes from Podosome and Invadopodium Formation to Differentiation and Tissue Homeostasis

Scaffold proteins are typically thought of as multi-domain “bridging molecules.” They serve as crucial regulators of key signaling events by simultaneously binding multiple participants involved in specific signaling pathways. In the case of epidermal growth factor (EGF)-epidermal growth factor receptor (EGFR) binding, the activated EGFR contacts cytosolic SRC tyrosine-kinase, which then becomes activated. This process leads to the phosphorylation of SRC-substrates, including the tyrosine kinase substrates (TKS) scaffold proteins. The TKS proteins serve as a platform for the recruitment of key players in EGFR signal transduction, promoting cell spreading and migration. The TKS4 and the TKS5 scaffold proteins are tyrosine kinase substrates with four or five SH3 domains, respectively. Their structural features allow them to recruit and bind a variety of signaling proteins and to anchor them to the cytoplasmic surface of the cell membrane. Until recently, TKS4 and TKS5 had been recognized for their involvement in cellular motility, reactive oxygen species-dependent processes, and embryonic development, among others. However, a number of novel functions have been discovered for these molecules in recent years. In this review, we attempt to cover the diverse nature of the TKS molecules by discussing their structure, regulation by SRC kinase, relevant signaling pathways, and interaction partners, as well as their involvement in cellular processes, including migration, invasion, differentiation, and adipose tissue and bone homeostasis. We also describe related pathologies and the established mouse models.

Phosphoproteomics Meets Chemical Genetics: Approaches for Global Mapping and Deciphering the Phosphoproteome

Protein kinases are important enzymes involved in the regulation of various cellular processes. To function properly, each protein kinase phosphorylates only a limited number of proteins among the thousands present in the cell. This provides a rapid and dynamic regulatory mechanism that controls biological functions of the proteins. Despite the importance of protein kinases, most of their substrates remain unknown. Recently, the advances in the fields of protein engineering, chemical genetics, and mass spectrometry have boosted studies on identification of bona fide substrates of protein kinases. Among the various methods in protein kinase specific substrate identification, genetically engineered protein kinases and quantitative phosphoproteomics have become promising tools. Herein, we review the current advances in the field of chemical genetics in analog-sensitive protein kinase mutants and highlight selected strategies for identifying protein kinase substrates and studying the dynamic nature of protein phosphorylation.