High-throughput kinase assays with protein substrates using fluorescent polymer superquenching

Background  

High-throughput screening is used by the pharmaceutical industry for identifying lead compounds that interact with targets of pharmacological interest. Because of the key role that aberrant regulation of protein phosphorylation plays in diseases such as cancer, diabetes and hypertension, kinases have become one of the main drug targets. With the exception of antibody-based assays, methods to screen for specific kinase activity are generally restricted to the use of small synthetic peptides as substrates. However, the use of natural protein substrates has the advantage that potential inhibitors can be detected that affect enzyme activity by binding to a site other than the catalytic site. We have previously reported a non-radioactive and non-antibody-based fluorescence quench assay for detection of phosphorylation or dephosphorylation using synthetic peptide substrates. The aim of this work is to develop an assay for detection of phosphorylation of chemically unmodified proteins based on this polymer superquenching platform.     

Identification of CDK2 substrates in human cell lysates

Background  

Protein phosphorylation regulates a multitude of biological processes. However, the large number of protein kinases and their substrates generates an enormously complex phosphoproteome. The cyclin-dependent kinases - the CDKs - comprise a class of enzymes that regulate cell cycle progression and play important roles in tumorigenesis. However, despite intense study, only a limited number of mammalian CDK substrates are known. A comprehensive understanding of CDK function requires the identification of their substrate network.     

Phosphorylation of p130Cas initiates Rac activation and membrane ruffling

Background  

Non-receptor tyrosine kinases (NTKs) regulate physiological processes such as cell migration, differentiation, proliferation, and survival by interacting with and phosphorylating a large number of substrates simultaneously. This makes it difficult to attribute a particular biological effect to the phosphorylation of a particular substrate. We developed the Functional Interaction Trap (FIT) method to phosphorylate specifically a single substrate of choice in living cells, thereby allowing the biological effect(s) of that phosphorylation to be assessed. In this study we have used FIT to investigate the effects of specific phosphorylation of p130Cas, a protein implicated in cell migration. We have also used this approach to address a controversy regarding whether it is Src family kinases or focal adhesion kinase (FAK) that phosphorylates p130Cas in the trimolecular Src-FAK-p130Cas complex.     

Protein kinases associated with the yeast phosphoproteome

Background  

Protein phosphorylation is an extremely important mechanism of cellular regulation. A large-scale study of phosphoproteins in a whole-cell lysate of Saccharomyces cerevisiae has previously identified 383 phosphorylation sites in 216 peptide sequences. However, the protein kinases responsible for the phosphorylation of the identified proteins have not previously been assigned.     

Identification of the phosphorylation targets of symbiotic receptor‐like kinases using a high‐throughput multiplexed assay for kinase specificity

Detecting the phosphorylation substrates of multiple kinases in a single experiment is a challenge, and new techniques are being developed to overcome this challenge. Here, we used a multiplexed assay for kinase specificity (MAKS) to identify the substrates directly and to map the phosphorylation site(s) of plant symbiotic receptor‐like kinases. The symbiotic receptor‐like kinases nodulation receptor‐like kinase (NORK) and lysin motif domain‐containing receptor‐like kinase 3 (LYK3) are indispensable for the establishment of root nodule symbiosis. Although some interacting proteins have been identified for these symbiotic receptor‐like kinases, very little is known about their phosphorylation substrates. Using this high‐throughput approach, we identified several other potential phosphorylation targets for both these symbiotic receptor‐like kinases. In particular, we also discovered the phosphorylation of LYK3 by NORK itself, which was also confirmed by pairwise kinase assays. Motif analysis of potential targets for these kinases revealed that the acidic motif xxxsDxxx was common to both of them. In summary, this high‐throughput technique catalogs the potential phosphorylation substrates of multiple kinases in a single efficient experiment, the biological characterization of which should provide a better understanding of phosphorylation signaling cascade in symbiosis.     

A p130 Cas tyrosine phosphorylated substrate domain decoy disrupts v-Crk signaling

Background  

The adaptor protein p130 ^Cas (Cas) has been shown to be involved in different cellular processes including cell adhesion, migration and transformation. This protein has a substrate domain with up to 15 tyrosines that are potential kinase substrates, able to serve as docking sites for proteins with SH2 or PTB domains. Cas interacts with focal adhesion plaques and is phosphorylated by the tyrosine kinases FAK and Src. A number of effector molecules have been shown to interact with Cas and play a role in its function, including c-crk and v-crk, two adaptor proteins involved in intracellular signaling. Cas function is dependent on tyrosine phosphorylation of its substrate domain, suggesting that tyrosine phosphorylation of Cas in part regulates its control of adhesion and migration. To determine whether the substrate domain alone when tyrosine phosphorylated could signal, we have constructed a chimeric Cas molecule that is phosphorylated independently of upstream signals.     

Detection and characterization of 3D-signature phosphorylation site motifs and their contribution towards improved phosphorylation site prediction in proteins

Background  

Phosphorylation of proteins plays a crucial role in the regulation and activation of metabolic and signaling pathways and constitutes an important target for pharmaceutical intervention. Central to the phosphorylation process is the recognition of specific target sites by protein kinases followed by the covalent attachment of phosphate groups to the amino acids serine, threonine, or tyrosine. The experimental identification as well as computational prediction of phosphorylation sites (P-sites) has proved to be a challenging problem. Computational methods have focused primarily on extracting predictive features from the local, one-dimensional sequence information surrounding phosphorylation sites.     

Integrin-Linked Kinase Is a Functional Mn2+-Dependent Protein Kinase that Regulates Glycogen Synthase Kinase-3β (GSK-3β) Phosphorylation

Background

Integrin-linked kinase (ILK) is a highly evolutionarily conserved, multi-domain signaling protein that localizes to focal adhesions, myofilaments and centrosomes where it forms distinct multi-protein complexes to regulate cell adhesion, cell contraction, actin cytoskeletal organization and mitotic spindle assembly. Numerous studies have demonstrated that ILK can regulate the phosphorylation of various protein and peptide substrates in vitro, as well as the phosphorylation of potential substrates and various signaling pathways in cultured cell systems. Nevertheless, the ability of ILK to function as a protein kinase has been questioned because of its atypical kinase domain.

Methodology/Principal Findings

Here, we have expressed full-length recombinant ILK, purified it to >94% homogeneity, and characterized its kinase activity. Recombinant ILK readily phosphorylates glycogen synthase kinase-3 (GSK-3) peptide and the 20-kDa regulatory light chains of myosin (LC₂₀). Phosphorylation kinetics are similar to those of other active kinases, and mutation of the ATP-binding lysine (K220 within subdomain 2) causes marked reduction in enzymatic activity. We show that ILK is a Mn-dependent kinase (the K_m for MnATP is ∼150-fold less than that for MgATP).

Conclusions/Significance

Taken together, our data demonstrate that ILK is a bona fide protein kinase with enzyme kinetic properties similar to other active protein kinases.     

Cell-free protein synthesis energized by slowly-metabolized maltodextrin

Background  

Cell-free protein synthesis (CFPS) is a rapid and high throughput technology for obtaining proteins from their genes. The primary energy source ATP is regenerated from the secondary energy source through substrate phosphorylation in CFPS.     

Cbl-associated protein is tyrosine phosphorylated by c-Abl and c-Src kinases

Background  

The c-Cbl-associated protein (CAP), also known as ponsin, localizes to focal adhesions and stress fibers and is involved in signaling events. Phosphorylation has been described for the other two members of the sorbin homology family, vinexin and ArgBP2, but no data exist about the putative phosphorylation of CAP. According to previous findings, CAP binds to tyrosine kinase c-Abl. However, it is not known if CAP is a substrate of c-Abl or other tyrosine kinases or if phosphorylation regulates its localization.     

Prediction of 492 human protein kinase substrate specificities

Background

Complex intracellular signaling networks monitor diverse environmental inputs to evoke appropriate and coordinated effector responses. Defective signal transduction underlies many pathologies, including cancer, diabetes, autoimmunity and about 400 other human diseases. Therefore, there is high impetus to define the composition and architecture of cellular communications networks in humans. The major components of intracellular signaling networks are protein kinases and protein phosphatases, which catalyze the reversible phosphorylation of proteins. Here, we have focused on identification of kinase-substrate interactions through prediction of the phosphorylation site specificity from knowledge of the primary amino acid sequence of the catalytic domain of each kinase.

Results

The presented method predicts 488 different kinase catalytic domain substrate specificity matrices in 478 typical and 4 atypical human kinases that rely on both positive and negative determinants for scoring individual phosphosites for their suitability as kinase substrates. This represents a marked advancement over existing methods such as those used in NetPhorest (179 kinases in 76 groups) and NetworKIN (123 kinases), which consider only positive determinants for kinase substrate prediction. Comparison of our predicted matrices with experimentally-derived matrices from about 9,000 known kinase-phosphosite substrate pairs revealed a high degree of concordance with the established preferences of about 150 well studied protein kinases. Furthermore for many of the better known kinases, the predicted optimal phosphosite sequences were more accurate than the consensus phosphosite sequences inferred by simple alignment of the phosphosites of known kinase substrates.

Conclusions

Application of this improved kinase substrate prediction algorithm to the primary structures of over 23, 000 proteins encoded by the human genome has permitted the identification of about 650, 000 putative phosphosites, which are posted on the open source PhosphoNET website (http://www.phosphonet.ca).

     

Myomegalin is a novel A-kinase anchoring protein involved in the phosphorylation of cardiac myosin binding protein C

Background  

Cardiac contractility is regulated by dynamic phosphorylation of sarcomeric proteins by kinases such as cAMP-activated protein kinase A (PKA). Efficient phosphorylation requires that PKA be anchored close to its targets by A-kinase anchoring proteins (AKAPs). Cardiac Myosin Binding Protein-C (cMyBPC) and cardiac troponin I (cTNI) are hypertrophic cardiomyopathy (HCM)-causing sarcomeric proteins which regulate contractility in response to PKA phosphorylation.     

Signal transduction protein array analysis links LRRK2 to Ste20 kinases and PKC zeta that modulate neuronal plasticity

Background

Dominant mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson''s disease, however, the underlying pathogenic mechanisms are poorly understood. Several in vitro studies have shown that the most frequent mutation, LRRK2(G2019S), increases kinase activity and impairs neuronal survival. LRRK2 has been linked to the mitogen-activated protein kinase kinase kinase family and the receptor-interacting protein kinases based on sequence similarity within the kinase domain and in vitro substrate phosphorylation.

Methodology/Principal Findings

We used an unbiased proteomic approach to identify the kinase signaling pathways wherein LRRK2 may be active. By incubation of protein microarrays containing 260 signal transduction proteins we detected four arrayed Ste20 serine/threonine kinase family members (TAOK3, STK3, STK24, STK25) as novel LRRK2 substrates and LRRK2 interacting proteins, respectively. Moreover, we found that protein kinase C (PKC) zeta binds and phosphorylates LRRK2 both in vitro and in vivo.

Conclusions/Significance

Ste20 kinases and PKC zeta contribute to neuronal Tau phosphorylation, neurite outgrowth and synaptic plasticity under physiological conditions. Our data suggest that these kinases may also be involved in synaptic dysfunction and neurite fragmentation in transgenic mice and in human PD patients carrying toxic gain-of-function LRRK2 mutations.     

A general approach to simultaneous model fitting and variable elimination in response models for biological data with many more variables than observations

Background  

With the advent of high throughput biotechnology data acquisition platforms such as micro arrays, SNP chips and mass spectrometers, data sets with many more variables than observations are now routinely being collected. Finding relationships between response variables of interest and variables in such data sets is an important problem akin to finding needles in a haystack. Whilst methods for a number of response types have been developed a general approach has been lacking.     

Functional classification of proteins based on projection of amino acid sequences: application for prediction of protein kinase substrates

Background  

The knowledge about proteins with specific interaction capacity to the protein partners is very important for the modeling of cell signaling networks. However, the experimentally-derived data are sufficiently not complete for the reconstruction of signaling pathways. This problem can be solved by the network enrichment with predicted protein interactions. The previously published in silico method PAAS was applied for prediction of interactions between protein kinases and their substrates.     

A novel tetratricopeptide repeat (TPR) containing PP5 serine/threonine protein phosphatase in the malaria parasite, Plasmodium falciparum

Background  

The malarial parasite, Plasmodium falciparum (Pf), is responsible for nearly 2 million deaths worldwide. However, the mechanisms of cellular signaling in the parasite remain largely unknown. Recent discovery of a few protein kinases and phosphatases point to a thriving reversible phosphorylation system in the parasite, although their function and regulation need to be determined.     

The Fer tyrosine kinase regulates an axon retraction response to Semaphorin 3A in dorsal root ganglion neurons

Background  

Fps/Fes and Fer are the only two members of a distinct subclass of cytoplasmic protein tyrosine kinases. Fps/Fes was previously implicated in Semaphorin 3A (Sema3A)-induced growth cone collapse signaling in neurons from the dorsal root ganglion (DRG) through interaction with and phosphorylation of the Sema3A receptor component PlexinA1, and members of the collapsin response mediator protein (CRMP) family of microtubule regulators. However, the potential role of the closely related Fer kinase has not been examined.