Innovative strategies for measuring kinase activity to accelerate the next wave of novel kinase inhibitors.

The development of protein kinase inhibitors (PKIs) has gained significance owing to their therapeutic potential for diseases like cancer. In addition, there has been a rise in refining kinase activity assays, each possessing unique biological and analytical characteristics crucial for PKI development. However, the PKI development pipeline experiences high attrition rates and approved PKIs exhibit unexploited potential because of variable patient responses. Enhancing PKI development efficiency involves addressing challenges related to understanding the PKI mechanism of action and employing biomarkers for precision medicine. Selecting appropriate kinase activity assays for these challenges can overcome these attrition rate issues. This review delves into the current obstacles in kinase inhibitor development and elucidates kinase activity assays that can provide solutions.

Phenotypic screening in Organ-on-a-Chip systems: a 1537 kinase inhibitor library screen on a 3D angiogenesis assay.

Modern drug development increasingly requires comprehensive models that can be utilized in the earliest stages of compound and target discovery. Here we report a phenotypic screening exercise in a high-throughput Organ-on-a-Chip setup. We assessed the inhibitory effect of 1537 protein kinase inhibitors in an angiogenesis assay. Over 4000 micro-vessels were grown under perfusion flow in microfluidic chips, exposed to a cocktail of pro-angiogenic factors and subsequently exposed to the respective kinase inhibitors. Efficacy of compounds was evaluated by reduced angiogenic sprouting, whereas reduced integrity of the main micro-vessel was taken as a measure for toxicity. The screen yielded 53 hits with high anti-angiogenicity and low toxicity, of which 44 were previously unassociated with angiogenic pathways. This study demonstrates that Organ-on-a-Chip models can be screened in high numbers to identify novel compounds and targets. This will ultimately reduce bias in early-stage drug development and increases probability to identify first in class compounds and targets for today's intractable diseases.

An Intestine-on-a-Chip Model of Plug-and-Play Modularity to Study Inflammatory Processes.

Development of efficient drugs and therapies for the treatment of inflammatory conditions in the intestine is often hampered by the lack of reliable, robust, and high-throughput in vitro and in vivo models. Current models generally fail to recapitulate key aspects of the intestine, resulting in low translatability to the human situation. Here, an immunocompetent 3D perfused intestine-on-a-chip platform was developed and characterized for studying intestinal inflammation. Forty independent polarized 3D perfused epithelial tubular structures were grown from cells of mixed epithelial origin, including enterocytes (Caco-2) and goblet cells (HT29-MTX-E12). Immune cells THP-1 and MUTZ-3, which can be activated, were added to the system and assessed for cytokine release. Intestinal inflammation was mimicked through exposure to tumor necrosis factor-α (TNFα) and interleukin (IL)-1ß. The effects were quantified by measuring transepithelial electrical resistance (TEER) and proinflammatory cytokine secretion on the apical and basal sides. Cytokines induced an inflammatory state in the culture, as demonstrated by the impaired barrier function and increased IL-8 secretion. Exposure to the known anti-inflammatory drug TPCA-1 prevented the inflammatory state. The model provides biological modularity for key aspects of intestinal inflammation, making use of well-established cell lines. This allows robust assays that can be tailored in complexity to serve all preclinical stages in the drug discovery and development process.

3D human microvessel-on-a-chip model for studying monocyte-to-endothelium adhesion under flow - application in systems toxicology.

Lifestyle and genetic factors can lead to the development of atherosclerosis and, ultimately, cardiovascular adverse events. Rodent models are commonly used to investigate mechanism(s) of atherogenesis. However, the 3Rs principles, aiming to limit animal testing, encourage the scientific community to develop new physiologically relevant in vitro alternatives. Leveraging the 96-chip OrganoPlate®, a microfluidic platform, we have established a three-dimensional (3D) model of endothelial microvessels-on-a-chip under flow using primary human coronary arterial endothelial cells. As functional readout, we have set up an assay to measure the adhesion of monocytes to the lumen of perfused microvessels. For monitoring molecular changes in microvessels, we have established the staining and quantification of specific protein markers of inflammation and oxidative stress using high content imaging, as well as analyzed transcriptome changes using microarrays. To demonstrate its usefulness in systems toxicology, we leveraged our 3D vasculature-on-a-chip model to assess the impact of the Tobacco Heating System (THS) 2.2, a candidate modified risk tobacco product, and the 3R4F reference cigarette on the adhesion of monocytic cells to endothelial microvessels. Our results show that THS 2.2 aerosol-conditioned medium had a reduced effect on monocyte-endothelium adhesion compared with 3R4F smoke-conditioned medium. In conclusion, we have established a relevant 3D vasculature-on-a-chip model for investigating leukocyte-endothelial microvessel adhesion. A case study illustrates how the model can be used for product testing in the context of systems toxicology-based risk assessment. The current model and its potential further development options also open perspectives of applications in vascular disease research and drug discovery.

Development of a Gut-On-A-Chip Model for High Throughput Disease Modeling and Drug Discovery.

A common bottleneck in any drug development process is finding sufficiently accurate models that capture key aspects of disease development and progression. Conventional drug screening models often rely on simple 2D culture systems that fail to recapitulate the complexity of the organ situation. In this study, we show the application of a robust high throughput 3D gut-on-a-chip model for investigating hallmarks of inflammatory bowel disease (IBD). Using the OrganoPlate platform, we subjected enterocyte-like cells to an immune-relevant inflammatory trigger in order to recapitulate key events of IBD and to further investigate the suitability of this model for compound discovery and target validation activities. The induction of inflammatory conditions caused a loss of barrier function of the intestinal epithelium and its activation by increased cytokine production, two events observed in IBD physiopathology. More importantly, anti-inflammatory compound exposure prevented the loss of barrier function and the increased cytokine release. Furthermore, knockdown of key inflammatory regulators RELA and MYD88 through on-chip adenoviral shRNA transduction alleviated IBD phenotype by decreasing cytokine production. In summary, we demonstrate the routine use of a gut-on-a-chip platform for disease-specific aspects modeling. The approach can be used for larger scale disease modeling, target validation and drug discovery purposes.

Constitutively active GSK3ß as a means to bolster dendritic cell functionality in the face of tumour-mediated immune suppression.

In patients with cancer, the functionality of Dendritic Cells (DC) is hampered by high levels of tumor-derived suppressive cytokines, which interfere with DC development and maturation. Poor DC development can limit the efficacy of immune checkpoint blockade and in vivo vaccination approaches. Interference in intracellular signaling cascades downstream from the receptors of major tumor-associated suppressive cytokines like IL-10 and IL-6, might improve DC development and activation, and thus enhance immunotherapy efficacy. We performed exploratory functional screens on arrays consisting of >1000 human kinase peptide substrates to identify pathways involved in DC development and its inhibition by IL-10 or IL-6. The resulting alterations in phosphorylation of the kinome substrate profile pointed to glycogen-synthase kinase-3ß (GSK3ß) as a pivotal kinase in both DC development and suppression. GSK3ß inhibition blocked human DC differentiation in vitro, which was accompanied by decreased levels of IL-12p70 secretion, and a reduced capacity for T cell priming. More importantly, adenoviral transduction of monocytes with a constitutively active form of GSK3ß induced resistance to the suppressive effects of IL-10 and melanoma-derived supernatants alike, resulting in improved DC development, accompanied by up-regulation of co-stimulatory markers, an increase in CD83 expression levels in mature DC, and diminished release of IL-10. Moreover, adenovirus-mediated intratumoral manipulation of this pathway in an in vivo melanoma model resulted in DC activation and recruitment, and in improved immune surveillance and tumor control. We propose the induction of constitutive GSK3ß activity as a novel therapeutic means to bolster DC functionality in the tumor microenvironment.

Interstitial Flow Recapitulates Gemcitabine Chemoresistance in A 3D Microfluidic Pancreatic Ductal Adenocarcinoma Model by Induction of Multidrug Resistance Proteins.

Pancreatic Ductal Adenocarcinoma (PDAC) is one of the most lethal cancers due to a high chemoresistance and poor vascularization, which results in an ineffective systemic therapy. PDAC is characterized by a high intratumoral pressure, which is not captured by current 2D and 3D in vitro models. Here, we demonstrated a 3D microfluidic interstitial flow model to mimic the intratumoral pressure in PDAC. We found that subjecting the S2-028 PDAC cell line to interstitial flow inhibits the proliferation, while maintaining a high viability. We observed increased gemcitabine chemoresistance, with an almost nine-fold higher EC50 as compared to a monolayer culture (31 nM versus 277 nM), and an alleviated expression and function of the multidrug resistance protein (MRP) family. In conclusion, we developed a 3D cell culture modality for studying intratissue pressure and flow that exhibits more predictive capabilities than conventional 2D cell culture and is less time-consuming, and more scalable and accessible than animal models. This increase in microphysiological relevance might support improved efficiency in the drug development pipeline.

Nephrotoxicity and Kidney Transport Assessment on 3D Perfused Proximal Tubules.

Proximal tubules in the kidney play a crucial role in reabsorbing and eliminating substrates from the body into the urine, leading to high local concentrations of xenobiotics. This makes the proximal tubule a major target for drug toxicity that needs to be evaluated during the drug development process. Here, we describe an advanced in vitro model consisting of fully polarized renal proximal tubular epithelial cells cultured in a microfluidic system. Up to 40 leak-tight tubules were cultured on this platform that provides access to the basolateral as well as the apical side of the epithelial cells. Exposure to the nephrotoxicant cisplatin caused a dose-dependent disruption of the epithelial barrier, a decrease in viability, an increase in effluent LDH activity, and changes in expression of tight-junction marker zona-occludence 1, actin, and DNA-damage marker H2A.X, as detected by immunostaining. Activity and inhibition of the efflux pumps P-glycoprotein (P-gp) and multidrug resistance protein (MRP) were demonstrated using fluorescence-based transporter assays. In addition, the transepithelial transport function from the basolateral to the apical side of the proximal tubule was studied. The apparent permeability of the fluorescent P-gp substrate rhodamine 123 was decreased by 35% by co-incubation with cyclosporin A. Furthermore, the activity of the glucose transporter SGLT2 was demonstrated using the fluorescent glucose analog 6-NBDG which was sensitive to inhibition by phlorizin. Our results demonstrate that we developed a functional 3D perfused proximal tubule model with advanced renal epithelial characteristics that can be used for drug screening studies.

Determination of Site-Specific Phosphorylation Ratios in Proteins with Targeted Mass Spectrometry.

We show that parallel reaction monitoring (PRM) can be used for exact quantification of phosphorylation ratios of proteins using stable-isotope-labeled peptides. We have compared two different PRM approaches on a digest of a U87 cell culture, namely, direct-PRM (tryptic digest measured by PRM without any further sample preparation) and TiO2-PRM (tryptic digest enriched with TiO2 cartridges, followed by PRM measurement); these approaches are compared for the following phosphorylation sites: neuroblast differentiation-associated protein (AHNAK S5480-p), calcium/calmodulin-dependent protein kinase type II subunit delta (CAMK2D T337-p), and epidermal growth factor receptor (EGFR S1166-p). A reproducible percentage of phosphorylation could be determined (CV 6-13%) using direct-PRM or TiO2-PRM. In addition, we tested the approaches in a cell culture experiment in which U87 cells were deprived of serum. As a "gold standard" we included immune precipitation of EGFR followed by PRM (IP-PRM). For EGFR (S1166) and AHNAK (S5480) a statistical significant change in the percentage of phosphorylation could be observed as a result of serum deprivation; for EGFR (S1166) this change was observed for both TiO2-PRM and IP-PRM. The presented approach has the potential to multiplex and to quantify the ratio of phosphorylation in a single analysis.

Therapy response testing of breast cancer in a 3D high-throughput perfused microfluidic platform.

BACKGROUND: Breast cancer is the most common invasive cancer among women. Currently, there are only a few models used for therapy selection, and they are often poor predictors of therapeutic response or take months to set up and assay. In this report, we introduce a microfluidic OrganoPlate® platform for extracellular matrix (ECM) embedded tumor culture under perfusion as an initial study designed to investigate the feasibility of adapting this technology for therapy selection. METHODS: The triple negative breast cancer cell lines MDA-MB-453, MDA-MB-231 and HCC1937 were selected based on their different BRCA1 and P53 status, and were seeded in the platform. We evaluate seeding densities, ECM composition (Matrigel®, BME2rgf, collagen I) and biomechanical (perfusion vs static) conditions. We then exposed the cells to a series of anti-cancer drugs (paclitaxel, olaparib, cisplatin) and compared their responses to those in 2D cultures. Finally, we generated cisplatin dose responses in 3D cultures of breast cancer cells derived from 2 PDX models. RESULTS: The microfluidic platform allows the simultaneous culture of 96 perfused micro tissues, using limited amounts of material, enabling drug screening of patient-derived material. 3D cell culture viability is improved by constant perfusion of the medium. Furthermore, the drug response of these triple negative breast cancer cells was attenuated by culture in 3D and differed from that observed in 2D substrates. CONCLUSIONS: We have investigated the use of a high-throughput organ-on-a-chip platform to select therapies. Our results have raised the possibility to use this technology in personalized medicine to support selection of appropriate drugs and to predict response to therapy in a real time fashion.

TP53 mutated glioblastoma stem-like cell cultures are sensitive to dual mTORC1/2 inhibition while resistance in TP53 wild type cultures can be overcome by combined inhibition of mTORC1/2 and Bcl-2.

BACKGROUND: Glioblastoma is the most malignant tumor of the central nervous system and still lacks effective treatment. This study explores mutational biomarkers of 11 drugs targeting either the RTK/Ras/PI3K, the p53 or the Rb pathway using 25 patient-derived glioblastoma stem-like cell cultures (GSCs). RESULTS: We found that TP53 mutated GSCs were approximately 3.5 fold more sensitive to dual inhibition of mammalian target of rapamycin complex 1 and 2 (mTORC1/2) compared to wild type GSCs. We identified that Bcl-2(Thr56/Ser70) phosphorylation contributed to the resistance of TP53 wild type GSCs against dual mTORC1/2 inhibition. The Bcl-2 inhibitor ABT-263 (navitoclax) increased sensitivity to the mTORC1/2 inhibitor AZD8055 in TP53 wild type GSCs, while sensitivity to AZD8055 in TP53 mutated GSCs remained unchanged. CONCLUSION: Our data suggest that Bcl-2 confers resistance to mTORC1/2 inhibitors in TP53 wild type GSCs and that combined inhibition of both mTORC1/2 and Bcl-2 is worthwhile to explore further in TP53 wild type glioblastomas, whereas in TP53 mutated glioblastomas dual mTORC1/2 inhibitors should be explored.

Kinome Profiling of Regulatory T Cells: A Closer Look into a Complex Intracellular Network.

Regulatory T cells (Treg) are essential for T cell homeostasis and maintenance of peripheral tolerance. They prevent activation of auto-reactive T effector cells (Teff) in the context of autoimmunity and allergy. Otherwise, Treg also inhibit effective immune responses against tumors. Besides a number of Treg-associated molecules such as Foxp3, CTLA-4 or GARP, known to play critical roles in Treg differentiation, activation and function, the involvement of additional regulatory elements is suggested. Herein, kinase activities seem to play an important role in Treg fine tuning. Nevertheless, our knowledge regarding the complex intracellular signaling pathways controlling phenotype and function of Treg is still limited and based on single kinase cascades so far. To gain a more comprehensive insight into the pathways determining Treg function we performed kinome profiling using a phosphorylation-based kinome array in human Treg at different activation stages compared to Teff. Here we have determined intriguing quantitative differences in both populations. Resting and activated Treg showed an altered pattern of CD28-dependent kinases as well as of those involved in cell cycle progression. Additionally, significant up-regulation of distinct kinases such as EGFR or CK2 in activated Treg but not in Teff not only resemble data we obtained in previous studies in the murine system but also suggest that those specific molecular activation patterns can be used for definition of the activation and functional state of human Treg. Taken together, detailed investigation of kinome profiles opens the possibility to identify novel molecular mechanisms for a better understanding of Treg biology but also for development of effective immunotherapies against unwanted T cell responses in allergy, autoimmunity and cancer.

Microfluidic 3D cell culture: from tools to tissue models.

The transition from 2D to 3D cell culture techniques is an important step in a trend towards better biomimetic tissue models. Microfluidics allows spatial control over fluids in micrometer-sized channels has become a valuable tool to further increase the physiological relevance of 3D cell culture by enabling spatially controlled co-cultures, perfusion flow and spatial control over of signaling gradients. This paper reviews most important developments in microfluidic 3D culture since 2012. Most efforts were exerted in the field of vasculature, both as a tissue on its own and as part of cancer models. We observe that the focus is shifting from tool building to implementation of specific tissue models. The next big challenge for the field is the full validation of these models and subsequently the implementation of these models in drug development pipelines of the pharmaceutical industry and ultimately in personalized medicine applications.

Microfluidic titer plate for stratified 3D cell culture.

Human tissues and organs are inherently heterogeneous. Their functionality is determined by the interplay between different cell types, their secondary architecture, vascular system and gradients of signaling molecules and metabolites. Here we propose a stratified 3D cell culture platform, in which adjacent lanes of gels and liquids are patterned by phaseguides to capture this tissue heterogeneity. We demonstrate 3D cell culture of HepG2 hepatocytes under continuous perfusion, a rifampicin toxicity assay and co-culture with fibroblasts. 4T1 breast cancer cells are used to demonstrate invasion and aggregation models. The platform is incorporated in a microtiter plate format that renders it fully compatible with automation and high-content screening equipment. The extended functionality, ease of handling and full compatibility to standard equipment is an important step towards adoption of Organ-on-a-Chip technology for screening in an industrial setting.

Kinome profiling of non-canonical TRAIL signaling reveals RIP1-Src-STAT3-dependent invasion in resistant non-small cell lung cancer cells.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) triggers apoptosis selectively in tumor cells through interaction with TRAIL-R1/DR4 or TRAIL-R2/DR5 and this process is considered a promising avenue for cancer treatment. TRAIL resistance, however, is frequently encountered and hampers anti-cancer activity. Here we show that whereas H460 non-small cell lung cancer (NSCLC) cells display canonical TRAIL-dependent apoptosis, A549 and SW1573 NSCLC cells are TRAIL resistant and display pro-tumorigenic activity, in particular invasion, following TRAIL treatment. We exploit this situation to contrast TRAIL effects on the kinome of apoptosis-sensitive cells to that of NSCLC cells in which non-canonical effects predominate, employing peptide arrays displaying 1024 different kinase pseudosubstrates more or less comprehensively covering the human kinome. We observed that failure of a therapeutic response to TRAIL coincides with the activation of a non-canonical TRAIL-induced signaling pathway involving, amongst others, Src, STAT3, FAK, ERK and Akt. The use of selective TRAIL variants against TRAIL-R1 or TRAIL-R2 subsequently showed that this non-canonical migration and invasion is mediated through TRAIL-R2. Short-hairpin-mediated silencing of RIP1 kinase prevented TRAIL-induced Src and STAT3 phosphorylation and reduced TRAIL-induced migration and invasion of A549 cells. Inhibition of Src or STAT3 by shRNA or chemical inhibitors including dasatinib and 5,15-diphenylporphyrin blocked TRAIL-induced invasion. FAK, AKT and ERK were activated in a RIP1-independent way and inhibition of AKT sensitized A549 cells to TRAIL-induced apoptosis. We thus identified RIP1-dependent and -independent non-canonical TRAIL kinase cascades in which Src and AKT are instrumental and could be exploited as co-targets in TRAIL therapy for NSCLC.

Monitoring selectivity in kinase-promoted phosphorylation of densely packed peptide monolayers using label-free electrochemical detection.

This paper describes remarkably high sensitivities in the label-free detection of kinase-promoted phosphorylation for 14 different peptide substrates on electrode-immobilized monolayers (gold or nitride) using serine/threonine kinases PKA, PKC, and CaMK2. Peptide substrates were preselected using (33)P-labeling in a microarray of 1024 substrates. The three most active peptides (A1-A3, C1-C3, and M1-M3) were investigated using electrochemical impedance spectroscopy (EIS) and ion-sensitive field effect transistors (ISFETs). Some of the peptide substrates, for example, the PKC-specific substrate PPRRSSIRNAH (C1), showed a remarkably high sensitivity in the EIS-based sensor measurements. Our studies revealed that this high sensitivity is primarily due to the monolayer's packing density. Nanoscopic studies demonstrated a distinct disordering of the C1-monolayer upon phosphorylation, while phosphatase-promoted dephosphorylation regenerated the highly ordered peptide monolayer. As a matter of fact, the initial surface packing of the peptide monolayer mainly determined the level of sensitivity, whereas electrostatic repulsion of the redox-active species was found to be much less important.

Roscovitine blocks leukocyte extravasation by inhibition of cyclin-dependent kinases 5 and 9.

BACKGROUND AND PURPOSE: Roscovitine, a cyclin-dependent kinase (CDK) inhibitor that induces tumour cell death, is under evaluation as an anti-cancer drug. By triggering leukocyte apoptosis, roscovitine can also enhance the resolution of inflammation. Beyond death-inducing properties, we tested whether roscovitine affects leukocyte-endothelial cell interaction, a vital step in the onset of inflammation. EXPERIMENTAL APPROACH: Leukocyte-endothelial cell interactions were evaluated in venules of mouse cremaster muscle, using intravital microscopy. In primary human endothelial cells, we studied the influence of roscovitine on adhesion molecules and on the nuclear factor-κB (NF-κB) pathway. A cellular kinome array, in vitro CDK profiling and RNAi methods were used to identify targets of roscovitine. KEY RESULTS: In vivo, roscovitine attenuated the tumour necrosis factor-α (TNF-α)-induced leukocyte adherence to and transmigration through, the endothelium. In vitro, roscovitine strongly inhibited TNF-α-evoked expression of endothelial adhesion molecules (E-selectin, intercellular cell adhesion molecule, vascular cell adhesion molecule). Roscovitine blocked NF-κB-dependent gene transcription, but not the NF-κB activation cascade [inhibitor of κB (IκB) kinase activity, IκB-α degradation, p65 translocation]. Using a cellular kinome array and an in vitro CDK panel, we found that roscovitine inhibited protein kinase A, ribosomal S6 kinase and CDKs 2, 5, 7 and 9. Experiments using kinase inhibitors and siRNA showed that the decreased endothelial activation was due solely to blockade of CDK5 and CDK9 by roscovitine. CONCLUSIONS AND IMPLICATIONS: Our study highlights a novel mode of action for roscovitine, preventing endothelial activation and leukocyte-endothelial cell interaction by inhibition of CDK5 and 9. This might expand its usage as a promising anti-inflammatory compound.

Flavopiridol protects against inflammation by attenuating leukocyte-endothelial interaction via inhibition of cyclin-dependent kinase 9.

OBJECTIVE: The cyclin-dependent kinase (CDK) inhibitor flavopiridol is currently being tested in clinical trials as anticancer drug. Beyond its cell death-inducing action, we hypothesized that flavopiridol affects inflammatory processes. Therefore, we elucidated the action of flavopiridol on leukocyte-endothelial cell interaction and endothelial activation in vivo and in vitro and studied the underlying molecular mechanisms. METHODS AND RESULTS: Flavopiridol suppressed concanavalin A-induced hepatitis and neutrophil infiltration into liver tissue. Flavopiridol also inhibited tumor necrosis factor-α-induced leukocyte-endothelial cell interaction in the mouse cremaster muscle. Endothelial cells were found to be the major target of flavopiridol, which blocked the expression of endothelial cell adhesion molecules (intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin), as well as NF-κB-dependent transcription. Flavopiridol did not affect inhibitor of κB (IκB) kinase, the degradation and phosphorylation of IκBα, nuclear translocation of p65, or nuclear factor-κB (NF-κB) DNA-binding activity. By performing a cellular kinome array and a kinase activity panel, we found LIM domain kinase-1 (LIMK1), casein kinase 2, c-Jun N-terminal kinase (JNK), protein kinase C (PKC), CDK4, CDK6, CDK8, and CDK9 to be influenced by flavopiridol. Using specific inhibitors, as well as RNA interference (RNAi), we revealed that only CDK9 is responsible for the action of flavopiridol. CONCLUSIONS: Our study highlights flavopiridol as a promising antiinflammatory compound and inhibition of CDK9 as a novel approach for the treatment of inflammation-associated diseases.

Comparison of peptide array substrate phosphorylation of c-Raf and mitogen activated protein kinase kinase kinase 8.

Kinases are pivotal regulators of cellular physiology. The human genome contains more than 500 putative kinases, which exert their action via the phosphorylation of specific substrates. The determinants of this specificity are still only partly understood and as a consequence it is difficult to predict kinase substrate preferences from the primary structure, hampering the understanding of kinase function in physiology and prompting the development of technologies that allow easy assessment of kinase substrate consensus sequences. Hence, we decided to explore the usefulness of phosphorylation of peptide arrays comprising of 1176 different peptide substrates with recombinant kinases for determining kinase substrate preferences, based on the contribution of individual amino acids to total array phosphorylation. Employing this technology, we were able to determine the consensus peptide sequences for substrates of both c-Raf and Mitogen Activated Protein Kinase Kinase Kinase 8, two highly homologous kinases with distinct signalling roles in cellular physiology. The results show that although consensus sequences for these two kinases identified through our analysis share important chemical similarities, there is still some sequence specificity that could explain the different biological action of the two enzymes. Thus peptide arrays are a useful instrument for deducing substrate consensus sequences and highly homologous kinases can differ in their requirement for phosphorylation events.

Granzyme K displays highly restricted substrate specificity that only partially overlaps with granzyme A.

Granzymes are serine proteases stored in cytolytic granules of cytotoxic lymphocytes that eliminate virus-infected and tumor cells. Little is known about the molecular mechanism and function of granzyme (Gr)K. GrK is similar to GrA in that they are the only granzymes that display tryptase-like activity. Both granzymes induce cell death by single-stranded nicking of the chromosomal DNA by cleaving the same components of the endoplasmic reticulum-associated SET complex. Therefore, GrK may provide a backup and failsafe mechanism for GrA with redundant specificity. In the present study, we addressed the question of whether GrK displays identical substrate specificity as GrA. In peptide- and protease-proteomic screens, GrK and GrA displayed highly restricted substrate specificities that overlapped only partially. Whereas GrK and GrA cleave SET with similar efficiencies likely at the same sites, both granzymes cleaved the pre-mRNA-binding protein heterogeneous ribonuclear protein K with different kinetics at distinct sites. GrK was markedly more efficient in cleaving heterogeneous ribonuclear protein K than GrA. GrK, but not GrA, cleaved the microtubule network protein beta-tubulin after two distinct Arg residues. Neither GrK cleavage sites in beta-tubulin nor a peptide-based proteomic screen revealed a clear GrK consensus sequence around the P1 residue, suggesting that GrK specificity depends on electrostatic interactions between exosites of the substrate and the enzyme. We hypothesize that GrK not only constitutes a redundant functional backup mechanism that assists GrA-induced cell death but that it also displays a unique function by cleaving its own specific substrates.