ABSTRACT
We report a novel protein kinase assay designed for high-throughput detection of one or many kinases in a complex mixture. A solution-phase phosphorylation reaction is performed on 900 different peptide substrates, each covalently linked to an oligonucleotide tag. After incubation, phosphoserine, phosphothreonine, and phosphotyrosine are chemically labeled, and the substrates are hybridized to a microarray with oligonucleotides complementary to the tags to read out the phosphorylation state of each peptide. Because protein kinases act on more than one peptide sequence, each kinase can be characterized by a unique signature of phosphorylation activity on multiple substrates. Using this method, we determined signatures for 26 purified kinases and demonstrated that enzyme mixtures can be screened for activity and selectivity of inhibition.
Subject(s)
Molecular Probe Techniques , Protein Kinases/chemistry , Cell Extracts , DNA/chemistry , Enzyme Activation , HeLa Cells , Humans , Isotope Labeling , Oligonucleotides/chemistry , Peptides/chemistry , Phosphorylation , Phosphoserine/chemistry , Phosphothreonine/chemistry , Phosphotyrosine/chemistry , Protein Kinases/analysis , Sensitivity and Specificity , Solutions/chemistry , Substrate SpecificityABSTRACT
Fluorescent chemosensors of protein kinase activity provide a continuous, high-throughput sensing format for the study of the roles of these enzymes, which are crucial for regulating cellular function. Specifically, chemosensors using the nonnatural amino acid, Sox, and physiological Mg(2+) levels report phosphorylation with dramatic fluorescence changes that are amenable to real-time and high-throughput analysis. In this article, we report 15 probes for a total of six distinct serine/threonine kinases with large fluorescence increases and good reactivity toward the target kinase. The sensing mechanism is detailed, and the optimal sensing motif is determined. These versatile and powerful sensors provide tools for researchers studying the roles of the targeted kinases in signal transduction, and the design principles provide guidelines for the generation of future fluorescent chemosensors for any serine/threonine kinase.
Subject(s)
Fluorescent Dyes/chemistry , Protein Serine-Threonine Kinases/chemistry , Transcription Factors/chemistry , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , Fluorescence , Indoles/pharmacology , Kinetics , Maleimides/pharmacology , Phosphorylation , Protein Serine-Threonine Kinases/antagonists & inhibitors , Sensitivity and Specificity , Spectrometry, Fluorescence , Spectrophotometry, Ultraviolet/methods , Structure-Activity Relationship , Transcription Factors/chemical synthesisABSTRACT
The power and scope of chemical synthesis offer considerable opportunities to broaden the lexicon of chemical tools that can be implemented for the study of complex biological systems. To investigate individual signaling proteins and pathways, chemical tools provide a powerful complement to existing genetic, chemical genetic and immunologic methods. In particular, understanding phosphorylation-mediated signaling in real time yields important information about the regulation of cellular function and insights into the origin of disease. Recent advances in the development of photolabile caged analogs of bioactive species and fluorescence-based sensors of protein kinase activities are useful for investigating protein phosphorylation and the roles of phosphoproteins. Photolabile caged analogs allow spatial and temporal control over the release of a compound, while fluorescence-based sensors allow the real-time visualization of kinase activity. Here, we discuss recent advances that have increased the specificity and availability of these tools.
Subject(s)
Protein Kinases/metabolism , Signal Transduction/physiology , Animals , Fluorescence Resonance Energy Transfer , Fluorescent Dyes/chemistry , Green Fluorescent Proteins/chemistry , Humans , Models, Chemical , Molecular Probes/chemistry , Molecular Structure , Peptides/chemistry , Phosphoproteins/chemistry , Phosphorylation , Photochemistry , Protein Kinases/chemistry , Substrate SpecificityABSTRACT
New methods to quantify protein kinase activities directly from complex cellular mixtures are critical for understanding biological regulatory pathways. Herein, a fluorescence-based chemosensor strategy for the direct measurement of kinase activities in crude mammalian cell lysates is described. We first designed a new fluorescent peptide reporter substrate for each target kinase. These kinase chemosensors were readily phosphorylated by recombinant target enzyme and underwent a several-fold fluorescence increase upon phosphorylation. Then, using unfractionated cell lysates, a homogeneous kinase assay was developed that was reproducible, linear and highly preferential for monitoring changes in cellular activity of the target kinase. The general protocol was developed for the kinase Akt and then easily extended to measure protein kinase A (PKA) and mitogen-activated protein kinase-associated protein kinase 2 (MK2) activities. This assay platform is immediately useful for studying protein kinase signaling in crude cellular extracts.
Subject(s)
Biosensing Techniques/methods , Cell Extracts/analysis , Cell-Free System/metabolism , Epithelial Cells/enzymology , Protein Kinases/analysis , Protein Kinases/metabolism , Spectrometry, Fluorescence/methods , Cell Line , Enzyme Activation , HumansABSTRACT
We introduce a versatile fluorescent peptide reporter of protein kinase activity. The probe can be modified to target a desired kinase by changing the kinase recognition motif in the peptide sequence. The reporter motif contains the Sox amino acid, which generates a fluorescence signal when bound to Mg2+ present in the reaction mixture. The phosphorylated peptide exhibits a much greater affinity for Mg2+ than its unphosphorylated analogue and, thus, a greater fluorescence intensity. Product formation during phosphorylation by the kinase is easily followed by the increase in fluorescence intensity over time. These probes exhibit a 3-5-fold increase in fluorescence intensity upon phosphorylation, the magnitude of which depends on the substrate. Peptides containing the reporter functionality are phosphorylated on serine by Protein Kinase C and cAMP-dependent protein kinase and are shown to be good substrates for these enzymes. The principle of this design extends to peptides phosphorylated on threonine and tyrosine.
Subject(s)
Fluorescent Dyes/chemistry , Protein Kinases/metabolism , Fluorescent Dyes/chemical synthesis , Kinetics , Peptides/chemistry , Peptides/metabolism , Phosphorylation , Protein Kinases/chemistry , Spectrometry, FluorescenceABSTRACT
A modular peptide scaffold has been developed for fluorescent sensing of divalent zinc. The signaling component of the chemosensor is the chelation-sensitive fluorophore 8-hydroxy-5-(N,N-dimethylsulfonamido)-2-methylquinoline, which is prepared as the protected amino acid derivative Fmoc-Sox-OH and integrated into peptide sequences. Nineteen synthetic peptides incorporating the signaling element exhibit a range of affinities for Zn(2+) through variation of the type and number of Zn(2+) ligands, ligand arrangement and the beta-turn sequence that acts as a preorganization element between the ligands. The stoichiometry of the peptide-Zn(2+) complexes is evaluated by several criteria. The fluorescence response of these peptides to pH and various important metal ions is reported. Eleven of these sequences form only 1:1 complexes with Zn(2+) and their affinities range from 10 nM to nearly 1 microM. When used in concert, these sensors can provide Zn(2+) concentration information in a valuable range.
