An Adaptor Domain‐Mediated Autocatalytic Interfacial Kinase Reaction

This paper describes a model system for studying the autocatalytic phosphorylation of an immobilized substrate by a kinase enzyme. This work uses self‐assembled monolayers (SAMs) of alkanethiolates on gold to present the peptide substrate on a planar surface. Treatment of the monolayer with Abl kinase results in phosphorylation of the substrate. The phosphorylated peptide then serves as a ligand for the SH2 adaptor domain of the kinase and thereby directs the kinase activity to nearby peptide substrates. This directed reaction is intramolecular and proceeds with a faster rate than does the initial, intermolecular reaction, making this an autocatalytic process. The kinetic non‐linearity gives rise to properties that have no counterpart in the corresponding homogeneous phase reaction: in one example, the rate for phosphorylation of a mixture of two peptides is faster than the sum of the rates for phosphorylation of each peptide when presented alone. This work highlights the use of an adaptor domain in modulating the activity of a kinase enzyme for an immobilized substrate and offers a new approach for studying biochemical reactions in spatially inhomogeneous settings.     

Multiplexed tyrosine kinase activity detection in cancer cells using a hydrogel immobilized substrate

Kinases play a key role in cellular signaling, and the overactivation or overexpression of these kinases has been linked to a variety of cancers. Tyrosine kinase inhibitors treat the mechanism of these cancers by targeting the specific kinases that are overactive. Some patients, however, do not respond to these inhibitors or develop resistance to these inhibitors during treatment. Additionally, even within cancers of the same tissue type, different kinases may be overactive in different patients. For example, some lung cancers overexpress epidermal growth factor receptor (EGFR) and respond to EGFR inhibitors, whereas other lung cancers do not overexpress EGFR and receive no benefit from this treatment. Even among patients exhibiting EGFR overexpression, some do not respond to EGFR kinase inhibitors because other kinases, such as Met kinase, are also overactivated. Here we describe a quantitative and specific multiplexed microfluidic assay using a hydrogel immobilized substrate for measuring the kinase activity of Met and Abl kinase from cancer cells. We immobilized kinase-specific substrates on macroporous hydrogel micropillars in microchannels. These microchannels were incubated with 6 μl of a kinase reaction solution containing cancer cell lysate, and we measured kinase activity via fluorescence detection of a phosphotyrosine antibody. We showed that the assay can specifically measure the activity of both Met and Abl kinase within one microchannel and has the potential to measure the activity of as many as five kinases within one microchannel. The assay also detected Met kinase inhibition from lysates of cancer cells grown in the Met kinase inhibitor PHA665752.

Mass-tag technology responding to intracellular signals as a novel assay system for the diagnosis of tumor

A novel mass spectrometry-based assay system for determining protein kinase activity employing mass-tagged substrate peptide probes was used for the diagnosis of tumors. Two peptide probes (H-type and D-type) were synthesized containing the same substrate peptide sequence for protein kinase C (PKC). The molecular weights of the two probes differ because of the incorporation of deuterium into the acetyl groups of the D-type probe. The lysates of the normal and tumor tissue were prepared and reacted with the H- and D-type peptide probes, respectively. The PKC activities of the normal and tumor tissues can be compared simply and directly by calculating the phosphorylated ratio to each peptide probe, obtained from the peak intensity of the mass spectrum after mixing of the two reaction solutions. The phosphorylation ratio for the reaction of the H-type peptide probe with the tumor tissue lysate (B16 melanoma) was more than three times higher than that of the D type peptide probe with the normal skin tissue lysate. These results show that the novel assay system for detecting protein kinase activity using mass-tag technology can be a simple and useful means to profile protein kinase activity for cell or tissue lysate samples, and can be applied to the diagnosis of tumors.     

MALDI‐TOF Mass‐Spectrometry‐Based Versatile Method for the Characterization of Protein Kinases

We describe a MALDI‐TOF mass‐spectrometry‐based method that is rapid and versatile for the characterization of protein kinases and their inhibitors. We have designed new kinase substrates by the modification of common synthetic peptides, such as kemptide (LRRALS G), CaMKII substrate (KRQQS FDLF), erktide (ATGPLS PGPFGRR), abltide (EAIY AAPFAKKK), srctide (AEEEIY GEFEAKKKK), neurogranin (AAAKIQAS FRGHMARKK), and casein kinase I (CKI) substrate (RRKDLHDDEEDEAMS ITA). There are two fundamental points on which the proposed method is based to improve the mass‐spectrometric response: 1) mass tag technology by N‐derivatization through stable isotope labeling and 2) C‐terminal conjugation with tryptophanylarginine (WR). It was suggested that C‐terminal conjugation with the WR moiety enhances the ionization potency of these new substrates 1.5–13.7 times as much as those of the original peptides. We demonstrated, by using modified abltide (Ac‐EAIY AAPFAKKKWR‐NH₂), that WR conjugation at the C‐terminus in combination with stable‐isotope labeling at the N‐terminus allowed the quantitative assay of recombinant c‐Abl kinase in the presence of adenosine 5′‐triphosphate (ATP; K_M,ATP=18.6 μM and V_max=642 pmol min^?1 μg^?1). The present protocol made a simple and reliable inhibition assay of recombinant c‐Abl kinase by imatinib possible (IC_{50(recombinant)}=291 nM ; STI571, Gleevec; Novartis Pharma). Moreover, it was also demonstrated that this ATP noncompetitive inhibitor differentiates between two conformers of c‐Abl kinases: the phosphorylated active and dephosphorylated inactive forms (IC_{50(active form)}=1049 nM and IC_{50(inactive form)}=54 nM ). The merit of this approach is evident because the present protocol can be applied to the direct monitoring of the activities of living cell kinases by using cancer‐cell lines, such as mouse B16 melanoma cells and human lung cancer K562 cells. A multiple‐kinase assay that uses K562 cell lysate in the presence of seven new synthetic substrates made high‐throughput inhibitor profiling possible. It should be emphasized that this radioactive isotope‐free quantitative kinase assay will greatly accelerate the discovery of a new generation of potential kinase inhibitors that exhibit highly selective or unique inhibitory profiles.     

Analysis of protein phosphorylation combining capillary electrophoresis with ATP analog labeling technique

Protein phosphorylation is one of the most basic mechanisms for regulating and controlling protein biological activity and function, and it is also a very important posttranslational modification process. Protein phosphorylation participates in and regulates many life activities such as signal transduction, gene expression, cell cycle, and so on. In this paper, we propose a method for the determination of the protein phosphorylation combining capillary electrophoresis (CE) with ATP analog labeling technique. We synthesized two new ATP analogs (ATP-NB and ATP-TATD-NB) functionalized by norbornene. Using Abl kinase as a model, we established a method for the determination of the kinase activity in solution and lysate by CE with laser-induced fluorescence detection (CE-LIF). This method was used to evaluate the efficiencies of kinase inhibitors. The IC₅₀ values obtained are basically consistent with the reports. By D–A reaction (inverse electron demand Diels–Alder reaction) to label TZ-BODIPY fluorescence, we also realized the phosphorylation fluorescence detection of substrate peptide. Then, we used fluorescence confocal microscopy imaging technology to study the phosphorylation of proteins in vivo by the D–A reaction of ATP-NB and TZ-BODIPY. Our preliminary results documented that the combination of CE-LIF with analog ATP-NB labeling technique is an effective strategy for the determination of the protein phosphorylation and the kinase activity and for screening of kinase inhibitors. The D–A reaction of ATP-NB and TZ-BODIPY also laid the foundation for the subsequent in situ study of protein phosphorylation.     

Antibody-free lanthanide-based fluorescent probe for determination of protein tyrosine kinase and phosphatase activities

A single-labeled peptide probe for measuring peptide phosphorylation status was developed by using a phosphate sensitive terbium chelate. The activity of Abl protein tyrosine kinase and T-cell protein Tyrosine phosphatase (TC PTP) was monitored in real time. To study the probe design in detail, variable substrate peptide sequences, where the enzyme target site was located from two to five amino acids apart from the nearest tyrosine residue, were synthesized. The maximum change observed in fluorescence intensity after phosphorylation was up to 320%, when the phosphorylated tyrosine was located two amino acids from the lysine coupled to the phosphate sensitive terbium chelate, demonstrating an excellent performance for a homogeneous assay. Also the longer distance of five amino acids between the phosphorylated tyrosine residue and terbium chelate resulted up to 260% change in fluorescence intensity.

Activity profile of dust mite allergen extract using substrate libraries and functional proteomic microarrays

Enzymatic activity in the fecal droppings from the house dust mite has been postulated to contribute to the elicited allergic response. Screening dust mite extracts through 137,180 tetrapeptide fluorogenic substrates allowed for the characterization of proteolytic substrate specificity from the potential cysteine and serine proteases in the extract. The extract was further screened against a 4000 member peptide nucleic acid (PNA) encoded inhibitor library designed to target cysteine proteases using microarray detection. Affinity chromatography coupled with mass spectrometry identified Der p 1 as one of the proteases targeted by the PNA inhibitors in the dust mite lysate. A phenotypic readout of Der p 1 function in allergy progression was demonstrated by the inhibition of CD25 cleavage from T cells by dust mite extract that had been treated with the Der p 1 inhibitor identified from the PNA-encoded inhibitor library.     

蛋白质组衍生肽段文库用于鉴定酪蛋白激酶2的底物模体

模体是蛋白质二级结构上的一种特征序列,激酶底物通常具有一类特征性的模体,识别底物模体对鉴定激酶底物具有重要意义。为了快速鉴定激酶底物模体,将全细胞蛋白质酶解液作为肽段文库的来源,采用碱性磷酸酶去除肽段的固有磷酸化后构建了用于筛选激酶底物模体的肽段文库。该肽段文库是大量非磷酸化肽段的混合物,将此混合肽段与酪蛋白激酶2和三磷酸腺苷作用30 min后,通过固定化金属离子亲和色谱法富集磷酸化肽段,采用反相液相色谱-串联质谱分析,成功地鉴定到472条非冗余底物肽段,包含451个非冗余磷酸化位点,并由此得到底物模体S/T-D/E-x-D/E。该法能够快速准确地筛选出激酶底物模体,对研究激酶-底物识别以及信号转导过程具有重要意义。     

Extended peptide-based inhibitors efficiently target the proteasome and reveal overlapping specificities of the catalytic beta-subunits

BACKGROUND: The 26S proteasome is responsible for most cytosolic proteolysis, and is an important protease in major histocompatibility complex class I-mediated antigen presentation. Constitutively expressed proteasomes from mammalian sources possess three distinct catalytically active species, beta1, beta2 and beta5, which are replaced in the gamma-interferon-inducible immunoproteasome by a different set of catalytic subunits, beta1i, beta2i and beta5i, respectively. Based on preferred cleavage of short fluorogenic peptide substrates, activities of the proteasome have been assigned to individual subunits and classified as 'chymotryptic-like' (beta5), 'tryptic-like' (beta2) and 'peptidyl-glutamyl peptide hydrolyzing' (beta1). Studies with protein substrates indicate a far more complicated, less strict cleavage preference. We reasoned that inhibitors of extended size would give insight into the extent of overlapping substrate specificity of the individual activities and subunits. RESULTS: A new class of proteasome inhibitors, considerably extended in comparison with the commonly used fluorescent substrates and peptide-based inhibitors, has been prepared. Application of the safety catch resin allowed the generation of the target compounds using a solid phase protocol. Evaluation of the new compounds revealed a set of highly potent proteasome inhibitors that target all individual active subunits with comparable affinity, unlike the other inhibitors described to date. Modification of the most active compound, adamantane-acetyl-(6-aminohexanoyl)(3)-(leucinyl)(3)-vinyl-(methyl)-sulfone (AdaAhx(3)L(3)VS), itself capable of proteasome inhibition in living cells, afforded a new set of radio- and affinity labels. CONCLUSIONS: N-terminal extension of peptide vinyl sulfones has a profound influence on both their efficiency and selectivity as proteasome inhibitors. Such extensions greatly enhance inhibition and largely obliterate selectivity towards the individual catalytic activities. We conclude that for the interaction with larger substrates, there appears to be less discrimination of different substrate sequences for the catalytic activities than is normally assumed based on the use of small peptide-based substrates and inhibitors. The compounds described here are readily accessible synthetically, and are more potent inhibitors in living cells than their shorter peptide vinyl sulfone counterparts.     

Recognition-domain focused chemosensors: versatile and efficient reporters of protein kinase activity

Catalyzed by kinases, serine/threonine and tyrosine phosphorylation is a vital mechanism of intracellular regulation. Thus, assays that easily monitor kinase activity are critical in both academic and pharmaceutical settings. We previously developed sulfonamido-oxine (Sox)-based fluorescent peptides following a beta-turn focused (BTF) design for the continuous assay of kinase activity in vitro and in cell lysates. Upon phosphorylation of the Sox-containing peptide, the chromophore binds Mg (2+) and undergoes chelation-enhanced fluorescence (CHEF). Although the design was applied successfully to the development of several kinase sensors, an intrinsic limitation was that only residues C- or N-terminal to the phosphorylated residue could be used to derive specificity for the target kinase. To address this limitation, a new, recognition-domain focused (RDF) strategy was developed that also relies on CHEF. In this approach, the requirement for the constrained beta-turn motif is obviated by alkylation of a cysteine residue with a Sox-based derivative to afford an amino acid termed C-Sox. The RDF design allows inclusion of extended binding determinants to maximize recognition by the cognate kinase, which has now permitted the construction of chemosensors for a variety of representative Ser/Thr (PKC alpha, PKC betaIota, PKC delta, Pim2, Akt1, MK2, and PKA) as well as receptor (IRK) and nonreceptor (Src, Abl) Tyr kinases with greatly enhanced selectivity. The new sensors have up to 28-fold improved catalytic efficiency and up to 66-fold lower K M when compared to the corresponding BTF probes. The improved generality of the strategy is exemplified with the synthesis and analysis of Sox-based probes for PKC betaIota and PKC delta, which were previously unattainable using the BTF approach.     

Macroporous hydrogel micropillars for quantifying Met kinase activity in cancer cell lysates

Overactive and overexpressed kinases have been implicated in the cause and progression of many cancers. Kinase inhibitors offer a targeted approach for treating cancers associated with increased or deregulated kinase activity. Often, however, cancer cells exhibit initial resistance to these inhibitors or evolve to develop resistance during treatment. Additionally, cancers of any one tissue type are typically heterogeneous in their oncogenesis mechanisms, and thus diagnosis of a particular type of cancer does not necessarily provide insight into what kinase therapies may be effective. For example, while some lung cancer cells that overexpress the epidermal growth factor receptor (EFGR) respond to treatment with EGFR kinase inhibitors, overexpression or hyperactivity of Met kinase correlates with resistance to EGFR kinase inhibitors. Here we describe a microfluidic-based assay for quantifying Met kinase activity in cancer cell lysates with the eventual goals of predicting cancer cell responsiveness to kinase inhibitors and monitoring development of resistance to these inhibitors. In this assay, we immobilized a phosphorylation substrate for Met kinase into macroporous hydrogel micropillars. We then exposed the micropillars to a cancer cell lysate and detected substrate phosphorylation using a fluorescently conjugated antibody. This assay is able to quantify Met kinase activity in whole cell lysate from as few as 150 cancer cells. It can also detect cells expressing overactive Met kinase in a background of up to 75% non-cancerous cells. Additionally, the assay can quantify kinase inhibition by the Met-specific kinase inhibitors SU11274 and PHA665752, suggesting predictive capability for cellular response to kinase inhibitors.     

A Dual‐Response DNA Probe for Simultaneously Monitoring Enzymatic Activity and Environmental pH Using a Nanopore

Both protease overexpression and local pH changes are key signatures of cancer. However, the sensitive detection of protease activities and the accurate measurement of pH in a tumor environment remain challenging. Here, we develop a dual‐response DNA probe that can simultaneously monitor protease activities and measure the local pH by translocation through α‐hemolysin (αHL). The DNA probe bears a short peptide containing phenylalanine at a pre‐designed position. Enzymatic cleavage of the peptide either exposes or removes the N‐terminal phenylalanine that can form a complex with cucurbit[7]uril. Translocation of the DNA hybrid through αHL generates current signatures that can be used to quantify protease activities. Furthermore, the current signatures possess a pH‐dependent pattern that reflects the local pH. Our results demonstrate that the versatile DNA probe may be further explored for simultaneously measuring multiple parameters of a complex system such as single cells in the future.     

A highly potent and selective PKCalpha inhibitor generated via combinatorial modification of a peptide scaffold

A potent and highly selective inhibitor of protein kinase C alpha has been generated via the combinatorial modification of a consensus sequence peptide. The inhibitor displays a Ki of 800 pM versus variable peptide substrate and good selectivity versus other members of the PKC family, including PKCbeta (385-fold), PKCgamma (580-fold), PKCdelta (2730-fold), PKCepsilon (600-fold), PKCeta (1310-fold), PKCtheta (1210-fold), PKCiota (940-fold), and PKCzeta (640-fold). The parallel synthesis strategy employed is easily automated and straightforward to implement.     

Nanomolar binding of peptides containing noncanonical amino acids by a synthetic receptor

This paper describes the molecular recognition of phenylalanine derivatives and their peptides by the synthetic receptor cucurbit[7]uril (Q7). The 4-tert-butyl and 4-aminomethyl derivatives of phenylalanine (tBuPhe and AMPhe) were identified from a screen to have 20-30-fold higher affinity than phenylalanine for Q7. Placement of these residues at the N-terminus of model tripeptides (X-Gly-Gly), resulted in no change in affinity for tBuPhe-Gly-Gly, but a remarkable 500-fold increase in affinity for AMPhe-Gly-Gly, which bound to Q7 with an equilibrium dissociation constant (K(d)) value of 0.95 nM in neutral phosphate buffer. Structure-activity studies revealed that three functional groups work in a positively cooperative manner to achieve this extraordinary stability (1) the N-terminal ammonium group, (2) the side chain ammonium group, and (3) the peptide backbone. Addition of the aminomethyl group to Phe substantially improved the selectivity for peptide versus amino acid and for an N-terminal vs nonterminal position. Importantly, Q7 binds to N-terminal AMPhe several orders of magnitude more tightly than any of the canonical amino acid residues. The high affinity, single-site selectivity, and small modification in this system make it attractive for the development of minimal affinity tags.     

Importance of extended protease substrate recognition motifs in steering BNIP-2 cleavage by human and mouse granzymes B

Background

Previous screening of the substrate repertoires and substrate specificity profiles of granzymes resulted in long substrate lists highly likely containing bystander substrates. Here, a recently developed degradomics technology that allows distinguishing efficiently from less efficiently cleaved substrates was applied to study the degradome of mouse granzyme B (mGrB).

Results

In vitro kinetic degradome analysis resulted in the identification of 37 mGrB cleavage events, 9 of which could be assigned as efficiently targeted ones. Previously, cleavage at the IEAD₇₅ tetrapeptide motif of Bid was shown to be efficiently and exclusively targeted by human granzyme B (hGrB) and thus not by mGrB. Strikingly, and despite holding an identical P4-P1 human Bid (hBid) cleavage motif, mGrB was shown to efficiently cleave the BCL2/adenovirus E1B 19 kDa protein-interacting protein 2 or BNIP-2 at IEAD₂₈. Like Bid, BNIP-2 represents a pro-apoptotic Bcl-2 protein family member and a potential regulator of GrB induced cell death. Next, in vitro analyses demonstrated the increased efficiency of human and mouse BNIP-2 cleavage by mGrB as compared to hGrB indicative for differing Bid/BNIP-2 substrate traits beyond the P4-P1 IEAD cleavage motif influencing cleavage efficiency. Murinisation of differential primed site residues in hBNIP-2 revealed that, although all contributing, a single mutation at the P3′ position was found to significantly increase the mGrB/hGrB cleavage ratio, whereas mutating the P1′ position from I₂₉?>?T yielded a 4-fold increase in mGrB cleavage efficiency. Finally, mutagenesis analyses revealed the composite BNIP-2 precursor patterns to be the result of alternative translation initiation at near-cognate start sites within the 5′ leader sequence (5′UTR) of BNIP-2.

Conclusions

Despite their high sequence similarity, and previously explained by their distinct tetrapeptide specificities observed, the substrate repertoires of mouse and human granzymes B only partially overlap. Here, we show that the substrate sequence context beyond the P4-P1 positions can influence orthologous granzyme B cleavage efficiencies to an unmatched extent. More specifically, in BNIP-2, the identical and hGrB optimal IEAD tetrapeptide substrate motif is targeted highly efficiently by mGrB, while this tetrapeptide motif is refractory towards mGrB cleavage in Bid.

     

2-Hydroxyestradiol Overcomes Mesenchymal Stem Cells-Mediated Platinum Chemoresistance in Ovarian Cancer Cells in an ERK-Independent Fashion

Ovarian cancer (OC) is the second most common type of gynecological malignancy. Platinum (Pt)-based chemotherapy is the standard of care for OC, but toxicity and acquired chemoresistance has proven challenging. Recently, we reported that sensitivity to platinum was significantly reduced in a co-culture of OC cells with MSC. To discover compounds capable of restoring platinum sensitivity, we screened a number of candidates and monitored ability to induce PARP cleavage. Moreover, we monitored platinum uptake and expression of ABC transporters in OC cells. Our results showed that 2-hydroxyestradiol (2HE2), a metabolite of estradiol, and dasatinib, an Abl/Src kinase inhibitor, were significantly effective in overcoming MSC-mediated platinum drug resistance. Dasatinib activity was dependent on ERK1/2 activation, whereas 2HE2 was independent of the activation of ERK1/2. MSC-mediated platinum drug resistance was accompanied by reduced intracellular platinum concentrations in OC cells. Moreover, MSC co-cultured with OC cells resulted in downregulation of the expression of cellular transporters required for platinum uptake and efflux. Exposure to 2HE2 and other modulators resulted in an increase in intracellular platinum concentrations. Thus, 2HE2 and dasatinib might act as sensitizers to restore platinum drug sensitivity to OC cells and thus to limit TME-mediated chemoresistance in OC.     

Substrate specificity for catalysis of phosphodiester cleavage by a dinuclear Zn(II) complex

The 2.4 kcal mol(-1) greater stabilization of the transition state for cleavage of the minimal substrate HpPNP compared to the nucleoside substrate UpPNP by the efficient dinuclear metal ion catalyst Zn2(L2O) provides evidence that access to the cationic core of Zn2(L2O) is sterically blocked for the bulkier nucleoside substrates, a flaw that will need to be dealt with in later generations of metal ion catalysts of RNA cleavage.     

In Situ Single‐Cell Western Blot on Adherent Cell Culture

Integrating 2D culture of adherent mammalian cells with single‐cell western blotting (in situ scWB) uses microfluidic design to eliminate the requirement for trypsin release of cells to suspension, prior to single‐cell isolation and protein analysis. To assay HeLa cells from an attached starting state, we culture adherent cells in fibronectin‐functionalized microwells formed in a thin layer of polyacrylamide gel. To integrate the culture, lysis, and assay workflow, we introduce a one‐step copolymerization process that creates protein‐decorated microwells. After single‐cell culture, we lyse each cell in the microwell and perform western blotting on each resultant lysate. We observe cell spreading after overnight microwell‐based culture. scWB reports increased phosphorylation of MAP kinases (ERK1/2, p38) under hypertonic conditions. We validate the in situ scWB with slab‐gel western blot, while revealing cell‐to‐cell heterogeneity in stress responses.     

A peptide microarray for the detection of protein kinase activity in cell lysate.

DNA microarray enables the analysis of DNA or mRNA expression levels, but it has not been possible to completely understand life using obtained information. Consequently, protein or peptide arrays have attracted much interest. Since the development of a practical protein microarray is still far away in light of handling difficulties, the peptide microarray is a promising tool for analyzing protein functions. We have developed a peptide microarray to detect protein kinase activity in cell lysate. All substrate peptides for kinases were immobilized chemoselectively on amino-coated glass slides. After phosphorylation of the immobilized peptides, phosphorylation was detected by fluorescence imaging. We detected the protein kinase activities, including that in cell lysate, in response to drug stimulation. Therefore, this peptide microarray would be useful for a high-throughput kinase assay of intracellular signals and would be applicable to drug screening.