Evaluation of protein kinase activities of cell lysates using peptide microarrays based on surface plasmon resonance imaging

We developed a peptide microarray based on surface plasmon resonance (SPR) imaging for monitoring protein kinase activities in cell lysates. The substrate peptides of kinases were tethered to the microarray surface modified with a self-assembled monolayer of an alkanethiol with triethylene glycol terminus to create a low nonspecific binding surface. The phosphorylation of the substrate peptides immobilized on the surface was detected with the following phosphate specific binders by amplifying SPR signals: anti-phosphotyrosine antibody for tyrosine kinases and Phos-tag biotin (a phosphate-specific ligand with biotin tag) for serine/threonine kinases. Using the microarray, 9 kinds of protein kinases were evaluated as a pattern of phosphorylation of 26 kinds of substrate peptides. The pattern was unique for each protein kinase. The microarray could be used to evaluate the inhibitory activities of kinase inhibitors. The microarray was applied successfully for kinase activity monitoring of cell lysates. The chemical stimuli responsive activity changes of protein kinases in cell lysates could also be monitored by the peptide microarray. Thus, the peptide microarray based on SPR imaging would be applicable to cell-based drug discovery, diagnosis using tissue lysates, and biochemical studies to reveal signal transduction pathways.     

Detection of Rho GEF and GAP activity through a sensitive split luciferase assay system

Rho GTPases regulate the assembly of cellular actin structures and are activated by GEFs (guanine-nucleotide-exchange factors) and rendered inactive by GAPs (GTPase-activating proteins). Using the Rho GTPases Cdc42, Rac1 and RhoA, and the GTPase-binding portions of the effector proteins p21-activated kinase and Rhophilin1, we have developed split luciferase assays for detecting both GEF and GAP regulation of these GTPases. The system relies on purifying split luciferase fusion proteins of the GTPases and effectors from bacteria, and our results show that the assays replicate GEF and GAP specificities at nanomolar concentrations for several previously characterized Rho family GEFs (Dbl, Vav2, Trio and Asef) and GAPs [p190, Cdc42 GAP and PTPL1-associated RhoGAP]. The assay detected activities associated with purified recombinant GEFs and GAPs, cell lysates expressing exogenous proteins, and immunoprecipitates of endogenous Vav1 and p190. The results demonstrate that the split luciferase system provides an effective sensitive alternative to radioactivity-based assays for detecting GTPase regulatory protein activities and is adaptable to a variety of assay conditions.     

Quantitative NMR analysis of Erk activity and inhibition by U0126 in a panel of patient-derived colorectal cancer cell lines

We comparatively analyzed the basal activity of extra-cellular signal-regulated kinase (Erk1/2) in lysates of 10 human colorectal cancer cell lines by semi-quantitative Western blotting and time-resolved NMR spectroscopy. Both methods revealed heterogeneous levels of endogenous Erk1/2 activities in a highly consistent manner. Upon treatment with U0126, an inhibitor of mitogen-activated protein kinase kinase (MEK) acting upstream of Erk1/2, Western-blotting and NMR congruently reported specific modulations of cellular phospho-Erk levels that translated into reduced kinase activities. Results obtained in this study highlight the complementary nature of antibody- and NMR-based phospho-detection techniques. They further exemplify the usefulness of time-resolved NMR measurements in providing fast and quantitative readouts of kinase activities and kinase inhibitor efficacies in native cellular environments. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).     

The kinase activity of SV40 large T antigen is mediated by a cellular kinase.

Large T antigen (large T) extracted from SV40-infected or transformed cells exhibits an in vitro protein kinase activity, whose origin and biological significance up to now had been obscure. We have addressed the questions of whether this activity is intrinsic to large T or arises by association with a cellular kinase, and, furthermore, whether this activity might play a biological role in vivo. Instead of analyzing large T from whole-cell lysates, where non-specific association of a cellular kinase(s) with large T might easily occur, we analyzed individual cellular subclasses of large T, isolated from their in vivo locations. In contrast to large T isolated from whole-cell lysates which was always kinase positive, none of the cellular subclasses of large T prepared by in situ fractionation of SV40-transformed mKSA cells exhibited detectable in vitro kinase activity. We could demonstrate that our fractionation conditions neither inactivated the large T-associated kinase activity nor dissociated it from large T when they were applied to kinase-positive large T isolated from whole-cell lysates. We conclude that large T does not contain an intrinsic kinase activity. This conclusion was further supported by our finding that it was possible to remove the large T-associated kinase activity from kinase-positive large T preparations and to reconstitute it by incubating the kinase-negative large T with cell lysates from various cell lines. Therefore, the simplest way of interpreting our results is that the in vitro kinase activity measured with large T preparations from whole-cell lysates is the result of an in vitro association of a cellular kinase(s) with large T during certain conditions of cell lysis.     

Reconstitution of killer cell inhibitory receptor-mediated signal transduction machinery in a cell-free model system.

Recognition of class I MHC molecules on target cells by killer cell inhibitory receptors (KIRs) blocks natural cytotoxicity and antibody-dependent cell cytotoxicity of NK cells and CD3/TCR dependent cytotoxicity of T cells. The inhibitory effect of KIR ligation requires phosphorylation of the cytoplasmic tail of KIR and subsequent recruitment of an SH2-containing protein tyrosine phosphatase, SHP-1. To better understand the molecular mechanism of the KIR-mediated inhibitory signal transduction, we developed an in vitro assay system using a purified His-tag fusion protein of KIR cytoplasmic tail (His-CytKIR) and Jurkat T cell lysates. We identified a target molecule of SHP-1 by comparing the phosphorylation of major cellular substrates following in vitro phosphorylation of Jurkat cell lysates in the presence and absence of the His-CytKIR in this cell-free model system. The His-CytKIR was tyrosine phosphorylated by Lck in vitro, and the phosphorylated His-CytKIR recruited SHP-1. Interestingly, we observed that among major substrates phosphorylated in vitro, PLC-gamma exhibited a dramatic decrease in phosphorylation when the His-CytKIR was mixed with Jurkat T cell lysates. However, PLC-gamma exhibited no decrease in phosphorylation when SHP-1 or Lck was depleted or deficient in this reaction mixture, suggesting that the SHP-1 recruited by the phosphorylated His-CytKIR directly mediate the dephosphorylation of PLC-gamma. The cell-free model system could be used to reveal the detailed molecular interactions in the KIR-mediated signal transduction.     

Characterization of synthetic peptide substrates for p34cdc2 protein kinase

Synthetic peptide substrates for the cell division cycle regulated protein kinase, p34cdc2, have been developed and characterized. These peptides are based on the sequences of two known substrates of the enzyme, Simian Virus 40 Large T antigen and the human cellular recessive oncogene product, p53. The peptide sequences are H-A-D-A-Q-H-A-T-P-P-K-K-K-R-K-V-E-D-P-K-D-F-OH (T antigen) and H-K-R-A-L-P-N-N-T-S-S-S-P-Q-P-K-K-K-P-L-D-G-E-Y-NH2 (p53), and they have been employed in a rapid assay of phosphorylation in vitro. Both peptides show linear kinetics and an apparent Km of 74 and 120 microM, respectively, for the purified human enzyme. The T antigen peptide is specifically phosphorylated by p34cdc2 and not by seven other protein serine/threonine kinases, chosen because they represent major classes of such enzymes. The peptides have been used in whole cell lysates to detect protein kinase activity, and the cell cycle variation of this activity is comparable to that measured with specific immune and affinity complexes of p34cdc2. In addition, the peptide phosphorylation detected in mitotic cells is depleted by affinity adsorption of p34cdc2 using either antibodies to p34cdc2 or by immobilized p13, a p34cdc2-binding protein. Purification of peptide kinase activity from mitotic HeLa cells yields an enzyme indistinguishable from p34cdc2. These peptides should be useful in the investigation of p34cdc2 protein kinase and their regulation throughout the cell division cycle.     

Rational design of homogenous protein kinase assay platforms that allow both fluorometric and colorimetric signal readouts

Protein kinases play important roles in signaling pathways that regulate many cellular biological processes, including apoptosis, cell growth, and differentiation in response to extracellular stimuli. Design of homogenous protein kinase assay platforms including design of potent protein kinase substrates is essential for exploration of the phosphoproteome. Here, we describe a unique chromism-based assay (CHROBA) technique for the direct measurement of protein kinase activities. The CHROBA is a novel chemosensor system that produces signals based on the photochromic and thermodynamic properties of a spiropyran derivative incorporated into peptide substrates. The CHROBA technique for detecting protein kinase activities involves the following five steps: (i) phosphorylation, (ii) photobleaching of the reaction mixture, (iii) addition of ionic polymer(s), (iv) incubation in the dark, and (v) signal readout. This simple 'end-point' assay method allows quantitative measurements of protein kinase A, Src protein tyrosine kinase, c-Abl protein tyrosine kinase, and protein kinase Calpha activities even with excess ATP. Our results showed that spiropyran-containing peptide substrates with net charges between +2 and 0 are suitable for the present CHROBA method. This information should aid in the rational design of diverse protein kinase assay platforms. The present CHROBA technique can be adapted to a microplate format with both fluorometric and colorimetric readouts and would be useful for high-throughput drug discovery and analysis of the phosphoproteome.     

Discovery, identification, and characterization of candidate pharmacodynamic markers of methionine aminopeptidase-2 inhibition

The catalytic activity of methionine aminopeptidase-2 (MetAP2) has been pharmacologically linked to cell growth, angiogenesis, and tumor progression, making this an attractive target for cancer therapy. An assay for monitoring specific protein changes in response to MetAP2 inhibition, allowing pharmacokinetic (PK)/pharmacodynamic (PD) models to be established, could dramatically improve clinical decision-making. Candidate MetAP2-specific protein substrates were discovered from undigested cell culture-derived proteomes by MALDI-/SELDI-MS profiling and a biochemical method using (35)S-Met labeled protein lysates. Substrates were identified either as intact proteins by FT-ICR-MS or applying in-gel protease digestions followed by LC-MS/MS. The combination of these approaches led to the discovery of novel MetAP2-specific substrates including thioredoxin-1 (Trx-1), SH3 binding glutamic acid rich-like protein (SH3BGRL), and eukaryotic elongation factor-2 (eEF2). These studies also confirmed glyceraldehye 3-phosphate dehydrogenase (GAPDH) and cyclophillin A (CypA) as MetAP2 substrates. Additional data in support of these proteins as MetAP2-specific substrates were provided by in vitro MetAP1/MetAP2 enzyme assays with the corresponding N-terminal derived peptides and 1D/2D Western analyses of cellular and tissue lysates. FT-ICR-MS characterization of all intact species of the 18 kDa substrate, CypA, enabled a SELDI-MS cell-based assay to be developed for correlating N-terminal processing and inhibition of proliferation. The MetAP2-specific protein substrates discovered in this study have diverse properties that should facilitate the development of reagents for testing in preclinical and clinical environments.     

Synthesis of 3′-O-Alkyl Homologues and a Biotin Probe of Isorhamnetin and Evaluation of Cytotoxic Efficacy on Cancer Cells

Isorhamnetin is a natural flavonoid which shows a variety of biological activities such as antioxidant, anti-inflammatory and antitumor. In order to identify the cellular binding protein of isorhamnetin as potential anti-cancer target, we first synthesized 3′-O-substituted quercetin as isorhamnetin homologues and evaluated the growth inhibitory activity of these derivatives on breast, colon and prostate cancer cell lines. The preliminary results showed that the 3′-O modification did not affect the cytotoxic activity of the scaffold. Analysis of the co-crystal structure and the docking pose of isorhamnetin with reported binding protein of isorhamnetin or quercetin indicated the 3′-O-substitution groups located outside of the binding pocket, which is in accordance with activity of 3′-O derivatives. Then a biotin conjugate of isorhamnetin with a tetraethylene glycol (PEG)₄ linker at the 3′ position was synthesized and the resulting probe retained the anti-proliferative activity on cancer cell lines, while the cellular fluorescence analysis showed the distribution of probe inside the cells which indicated the probe had limited cell permeability. Finally, pull down assay both in situ inside cells and in the cell lysates indicated the isorhamnetin biotin probe was capable of protein labeling in cell lysates. These findings provide the isorhamnetin 3′-O-biotin probe as a tool to reveal the target proteins of isorhamnetin.     

Use of pseudosubstrate affinity to measure active protein kinase A

Traditional cAMP-dependent protein kinase (also known as protein kinase A [PKA]) assays, which are based on substrate phosphorylation, often have high background activity from other kinases, thereby limiting sensitivity and making it difficult to detect low levels of active PKA in cell lysates. Therefore, a better technique that measures active PKA in crude cell lysates undoubtedly is necessary. We developed an efficient and sensitive assay to compare active PKA levels based on binding of the active PKA catalytic subunit to its pseudosubstrate domain inhibitor (PKI) fused with glutathione S-transferase (GST-PKI). This pseudosubstrate affinity assay can detect variations in the active PKA levels in the presence of common inducers of PKA activity such as forskolin and prostaglandins. It has resolution to detect a concentration-dependent curve of active PKA in a linear range, and it also has sensitivity to detect up to 2.5 ng of active enzyme. An observed change in the binding affinity between PKA and PKI in the presence of the PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89) shows that this assay can be successfully used to measure how active PKA is affected by specific inhibitors. We conclude that this method is a simple, inexpensive, and nonhazardous method to compare active PKA levels with high sensitivity and specificity with negligible background.     

Mass-tag technology for monitoring of protein kinase activity using mass spectrometry

Monitoring of intracellular protein kinase activity is very important for fields involving diagnosis and drug screening. However, current methods, such as radiometry using (32)P, or ELISA, are laborious and time-consuming. We have developed high-throughput assay system of protein kinase activity using mass-tagged substrate peptide probes and mass spectrometry. This assay system can easily evaluate target kinase activity and will potentially be able to simultaneously profile many protein kinase activities.     

Mevalonate kinase in lysates of cultured human fibroblasts and lymphoblasts: kinetic properties, assay conditions, carrier detection and measurement of residual activity in a patient with mevalonic aciduria

An assay has been developed for the measurement of mevalonate kinase activity in extracts of cultured human fibroblasts and lymphoblasts. Individual elements of the assay were investigated in order to achieve optimum conditions. Apparent Michaelis constants (KMapp) for the substrates mevalonic acid and adenosine-5'-triphosphate were 22 +/- 10 mumol/l and 0.42-0.53 mmol/l, respectively, in lysates of control fibroblast lines. The same values in lysates of a control lymphoblast line were 17 mumol/l and 0.23 mmol/l, respectively. Mevalonate kinase activity in extracts of cultured fibroblasts derived from 6 control individuals was 3.24 +/- (SD) 0.91 nmol/min/mg protein. The activity in extracts of fibroblasts derived from a patient with mevalonic aciduria was 0.15 +/- 0.10 nmol/min/mg protein, approximately 5% of the control mean. The parents and brother of the patient displayed mevalonate kinase activities in fibroblast extracts approximating 38-42% of the control mean. Substantially higher mevalonate kinase activity was documented in extracts of cultured lymphoblasts. When assayed on various occasions, the mean activity of mevalonate kinase in extracts of lymphoblasts derived from the parents, brother and maternal grandmother of the patient ranged from 27 to 32% of the mean activity of 9.8 +/- (SD) 3.4 nmol/min/mg protein measured in a parallel control lymphoblast line, while the mean activity in a maternal and paternal uncle approximated 65-89% of the same control mean. The mean activity in extracts of lymphoblasts derived from the patient approximated 2% of the control mean. The data suggest that the parents, brother and maternal grandmother are carriers of the defective gene responsible for mevalonate kinase deficiency, consistent with an autosomal recessive mode of inheritance.     

FRET-based screening assay using small-molecule photoluminescent probes in lysate of cells overexpressing RFP-fused protein kinases

An assay was developed for the characterization of protein kinase inhibitors in lysates of mammalian cells based on the measurement of FRET between overexpressed red fluorescent protein (TagRFP)-fused protein kinases (PKs) and luminophore-labeled small-molecule inhibitors (ARC-Photo probes). Two types of the assay, one using TagRFP as the photoluminescence donor together with ARC-Photo probes containing a red fluorophore dye as acceptor, and the other using TagRFP as the acceptor fluorophore in combination with a terbium cryptate-based long-lifetime photoluminescence donor, were used for FRET-based measurements in lysates of the cells overexpressing TagRFP-fused PKs. The second variant of the assay enabled the performance of the measurements under time-resolved conditions that led to substantially higher values of the signal/background ratio and further improved the reliability of the assay.     

Tissue distribution and immunoreactivity of heme-regulated eIF-2 alpha kinase determined by monoclonal antibodies.

A highly purified preparation of heme-regulated inhibitor (HRI), an eIF-2 alpha kinase, from rabbit reticulocyte lysates has been used for generating monoclonal antibodies (mAB). Two hybridoma clones secreting HRI-specific antibodies (mAB A and mAB F) were obtained. Both antibodies immunoprecipitated biosynthetically labeled as well as phosphorylated HRI in reticulocyte lysates and also recognized denatured HRI in a Western blot. In in vitro protein kinase assays, preincubation of HRI with the antibodies significantly diminished both autokinase and eIF-2 alpha kinase activities. HRI from reticulocyte lysates could be quantitatively removed by immunoprecipitation with mAB F, and such HRI-depleted lysates were able to maintain protein synthesis under conditions of heme deficiency. With these monoclonal antibodies, HRI was detected only in the reticulocytes and bone marrow of anemic rabbits, among several rabbit tissues tested. The antibodies did not detect cross-reacting HRI in rat or human reticulocytes or in mouse erythroleukemic cells or human K562 cells even after induction of differentiation, although eIF-2 alpha kinase activity was detected in them. Polyclonal anti-rabbit HRI antibody detected HRI in rat reticulocytes. However, no cross-reacting HRI was detected by polyclonal antibody in human reticulocytes or other cell types tested. These findings suggest that HRI is not ubiquitous, and may be erythroid-specific, and that it is antigenically different in different species.     

The respective roles of the protein kinase and pppA2'' p5'' A2'' p5 A-activated endonuclease in the inhibition of protein synthesis by double stranded RNA in rabbit reticulocyte lysates.

Double-stranded RNA (dsRNA) inhibits protein synthesis in rabbit reticulocyte lysates by activating the synthesis of the endonuclease effector pppA2' p5' A2' p5' A(2-5A) and a protein kinase which phosphorylates the protein synthesis initiation factor eIF-2. Under certain assay conditions, high concentrations of dsRNA are without inhibitory effect in many lysates (high dsRNA "reversible" lysates). In these lysates natural dsRNA at low concentrations stimulated protein kinase activity to a greater extent than did the synthetic dsRNA poly rI.rC. Synthesis of 2--5A was greater when poly rI.rC was used. However, a number of factors, including the salt concentration and messenger RNA used, combine to determine the overall effect of dsRNA on protein synthesis under any given set of experimental conditions.     

Denitration of L-type calcium channel

Tyrosine nitration results in altered function of selective proteins, including human smooth muscle L-type calcium channel, hCa(v)1.2b. We report here that Ca(v)1.2 is also subject to "denitration". Cell lysates from activated macrophage-like cell line, RAW264.7 cells, reversed peroxynitrite-induced nitration of the carboxy terminus of Ca(v)1.2 in a 1D gel assay. Tyrosine phosphorylation of the calcium channel by c-src kinase was blocked by nitration but reversed by pretreatment with RAW264.7 cell lysates. These findings indicate that denitration may be a physiological mechanism to restore cellular excitability during inflammation.     

Identification of SRPK1 and SRPK2 as the major cellular protein kinases phosphorylating hepatitis B virus core protein

Phosphorylation of hepatitis B virus (HBV) core protein has recently been shown to be a prerequisite for pregenomic RNA encapsidation into viral capsids, but the host cell kinases mediating this essential step of the HBV replication cycle have not been identified. We detected two kinases of 95 and 115 kDa in HuH-7 total cell lysates which interacted specifically with the HBV core protein and phosphorylated its arginine-rich C-terminal domain. The 95-kDa kinase was purified and characterized as SR protein-specific kinase 1 (SRPK1) by mass spectrometry. Based on this finding, the 115-kDa kinase could be identified as the related kinase SRPK2 by immunoblot analysis. In vitro, both SRPKs phosphorylated HBV core protein on the same serine residues which are found to be phosphorylated in vivo. Moreover, the major cellular HBV core kinase activity detected in the total cell lysate showed biochemical properties identical to those of SRPK1 and SRPK2, as examined by measuring binding to a panel of chromatography media. We also clearly demonstrate that neither the cyclin-dependent kinases Cdc2 and Cdk2 nor protein kinase C, previously implicated in HBV core protein phosphorylation, can account for the HBV core protein kinase activity. We conclude that both SRPK1 and SRPK2 are most likely the cellular protein kinases mediating HBV core protein phosphorylation during viral infection and therefore represent important host cell targets for therapeutic intervention in HBV infection.     

Identification of a novel substrate for TNFalpha-induced kinase NUAK2

TNFα has multiple important cellular functions both in normal cells and in tumor cells. To explore the role of TNFα, we identified NUAK family, SNF1-like kinase 2 (NUAK2), as a TNFα-induced kinase by gene chip analysis. NUAK2 is known to be induced by various cellular stresses and involved in cell mortality, however, its substrate has never been identified. We developed original protocol of de novo screening for kinase substrates using an in vitro kinase assay and high performance liquid chromatography (HPLC). Using this procedure, we identified myosin phosphatase target subunit 1 (MYPT1) as a specific substrate for NUAK2. MYPT1 was phosphorylated at another site(s) by NUAK2, other than known Rho-kinase phosphorylation sites (Thr696 or Thr853) responsible for inhibition of myosin phosphatase activity. These data suggests different phosphorylation and regulation of MYPT1 activity by NUAK2.