Development of a peptide inhibitor-based cantilever sensor assay for cyclic adenosine monophosphate-dependent protein kinase

A highly sensitive nanomechanical cantilever sensor assay based on an electrical measurement has been developed for detecting activated cyclic adenosine monophosphate (cyclic AMP)-dependent protein kinase (PKA). Employing a peptide derived from the heat-stable protein kinase inhibitor (PKI), a magnetic bead system was first selected as a vehicle to immobilize the PKI-(5-24) peptide for capturing PKA catalytic subunit and the activity assay was applied for indirectly assessing the binding. Synergistic interactions of adenosine triphosphate (ATP) and the peptide inhibitor with the kinase were then investigated by a solution phase capillary electrophoretic assay, and by surface plasmon resonance technology which involved immobilization of the peptide inhibitor. After systemically evaluated by a homogeneous direct binding assay, the ATP-dependent recognition of the catalytic subunit of PKA by PKI-(5-24) was successfully transferred on to the nanomechanical cantilevers at protein concentrations of 6.6 pM-66 nM, exhibiting much higher sensitivity and wider dynamic range than the conventional activity assay. Thus, direct assessment of activated kinases using the cantilever sensor system functionalized with specific peptide inhibitors holds great promise in analytical applications and clinical medicine.     

Systematic development of an enzymatic phosphorylation assay compatible with mass spectrometric detection

Abstract The enzymatic peptide phosphorylation by cAMP-dependent protein kinase A (PKA) was optimized and monitored by means of electrospray ionization mass spectrometry (ESI-MS). The direct detection of phosphorylated peptides by MS renders labeling unnecessary, reduces time and labor, due to less initial sample pretreatment. In this study the phosphorylation of the peptide malantide by PKA was performed in batch and reaction compounds were detected by ESI-MS after the incubation time. The subsequent product quantitation was accomplished by using one-point normalization. Applying this set-up, optimum solvent conditions (such as salt and modifier content), concentrations of essential reaction compounds (such as cAMP, Mg²⁺ and ATP), and the influence of reaction properties (such as pH and reaction time) were determined. The reaction milieu has to be suitable for both, the enzymatic reaction and the mass spectrometric detection. We found that the modifier content and the pH value had to be changed after the enzymatic reaction occurred. Through the addition of methanol and acetic acid, the reaction stopped immediately and a more sensitive mass spectrometric detection could be obtained simultaneously. Furthermore, an inhibitor study was performed, testing the inhibition potency of three protein kinase A inhibitors (PKIs). IC₅₀ values were determined and used to calculate the K _i values, that were 7.4, 19.0 and 340.0 nmol/L for PKI(6-22)amide, PKI(5-24)amide, and PKI(14-24)amide, respectively. These data vary between factor 4.4 (for PKI(6-22)amide) and 8.3 (for PKI(5-24)amide) compared to the K _i values described in literature. However, the K _i values are in good agreement with the data mainly obtained by fluorescence- or radioactivity-based methods. Nevertheless, our results indicate that ESI-MS is a realistic alternative to radioactivity and fluorescence detection in determining enzymatic activity. Furthermore we were able to illustrate its high potential as a quantitative detection method.     

Isoform-specific PKA dynamics revealed by dye-triggered aggregation and DAKAP1alpha-mediated localization in living cells

The tetracysteine sequence YRECCPGCCMWR fused to the N terminus of green fluorescent protein (GFP) self-aggregates upon biarsenical labeling in living cells or in vitro. Such dye-triggered aggregates form temperature-dependent morphologies and are dispersed by photobleaching. Fusion of the biarsenical aggregating GFP to the regulatory (R) or catalytic (C) subunit of PKA traps intact holoenzyme in compact fluorescent puncta upon biarsenical labeling. Contrary to the classical model of PKA activation, elevated cAMP does not allow RIalpha and Calpha to diffuse far apart unless the pseudosubstrate inhibitor PKI or locally concentrated substrate is coexpressed. However, RIIalpha releases Calpha upon elevated cAMP alone, dependent on autophosphorylation of the RIIalpha inhibitory domain. DAKAP1alpha overexpression induced R and C outer mitochondrial colocalization and showed similar regulation. Overall, effective separation of type I PKA is substrate dependent, whereas type II PKA dissociation relies on autophosphorylation.     

A dual parameter FRET probe for measuring PKC and PKA activity in living cells

Cell function is regulated by complex and often interdependent networks of signaling molecules. To accurately describe these networks, it is important to monitor multiple signals in parallel. To this end, we have developed a genetically encoded, FRET-based probe that independently monitors both protein kinase A (PKA) and protein kinase C (PKC) activity in vivo. Artificial as well as physiological stimulants produced a negative or positive change in FRET efficiency following PKA or PKC activation, respectively. Mutations of the phosphate-accepting amino acids of the PKC substrate yielded a probe that was sensitive to PKA activation alone.     

Singlet oxygen-induced activation of Akt/protein kinase B is independent of growth factor receptors

Singlet oxygen (1O2)-induced cytotoxicity is believed to be responsible for responses to photodynamic therapy and for apoptosis of T helper cells after UV-A treatment. Other cytotoxic oxidants, such as hydrogen peroxide and peroxynitrite have been shown to stimulate cell survival signaling pathways in addition to causing cell death. Both these oxidants stimulate the Akt/protein kinase B survival signaling pathway through activation of membrane tyrosine kinase growth factor receptors. We evaluated the ability of 1O2 to activate the Akt/protein kinase B pathway in NIH 3T3 cells and examined potential activation pathways. Exposure of fibroblasts to 1O2 elicited a strong and sustained phosphorylation of Akt, which occurred concurrently with phosphorylation of p38 kinase, a proapoptotic signal. Inhibition of phosphatidylinositol-3-OH kinase (PI3-K) completely blocked Akt phosphorylation. Significantly, cell death induced by 1O2 was enhanced by inhibition of PI3-K, suggesting that activation of Akt by 1O2 may contribute to fibroblast survival under this form of oxidative stress. 1O2 treatment did not induce phosphorylation of platelet-derived growth factor receptor (PDGFR) or activate SH-PTP2, a substrate of growth factor receptors, suggesting that PDGFR was not activated. In addition, specific inhibition of PDGFR did not affect Akt phosphorylation elicited by 1O2. Activation of neither focal adhesion kinase (FAK) nor Ras protein, both of which mediate responses to reactive oxygen species, appeared to be pathways for the 1O2-induced activation of the PI3-K-Akt survival pathway. Thus, activation of Akt by 1O2 is mediated by PI3-K and contributes to a survival response that counteracts cell death after 1O2-induced injury. However, unlike the response to other oxidants, activation of the PI3-K-Akt by 1O2 does not involve activation of growth factor receptors, FAK or Ras protein.     

Electrochemical sensing for the determination of activated cyclic AMP-dependent protein kinase.

A novel electrochemical system has been developed for monitoring the cyclic AMP-dependent protein kinase (PKA) activity. In this method, PKA activity was monitored as the change in the redox current of a ferrocene-pendant PKA substrate peptide (Fe-LRRASLG) on a gold electrode, which had been modified with thioctic acid, using cyclic voltammetry. The phosphrylation of the ferrocene-pendant substrate with PKA changed the net charge from +1 to -1. This caused a decrease in the redox current of the ferrocene unit due to an electrostatic repulsion between the substrate and the anionic surface of the electrode. We expect that this method is potentially useful for monitoring the enzyme activity in medical or pharmacological fields.     

Shear stress stimulates phosphorylation of protein kinase A substrate proteins including endothelial nitric oxide synthase in endothelial cells

Fluid shear stress plays a critical role in vascular health and disease. While protein kinase A (PKA) has been implicated in shear-stimulated signaling events in endothelial cells, it remains unclear whether and how PKA is stimulated in response to shear stress. This issue was addressed in the present study by monitoring the phosphorylation of endogenous substrates of PKA. Shear stress stimulated the phosphorylation of cAMP responsive element binding protein (CREB) in a PKA-dependent manner. Western blot analysis using the antibody reactive against the consensus motif of PKA substrates detected two proteins, P135 and P50, whose phosphorylation was increased by shear stress. The phosphorylation of P135 was blocked by a PKA inhibitor, H89, but not by a phosphoinositide 3-kinase inhibitor, wortmannin. Expression of a constitutively active PKA subunit stimulated P135 phosphorylation, supporting the potential of P135 as a PKA substrate. P135 was identified as endothelial nitric oxide synthase (eNOS) by immunoprecipitation study. PKA appeared to mediate shear stress-stimulated eNOS activation. Shear stress stimulated intracellular translocation of PKA activity from 'soluble' to 'particulate' fractions without involving cellular cAMP increase. Taken together, this study suggests that shear stress stimulates PKA-dependent phosphorylation of target proteins including eNOS, probably by enhancing intracellular site-specific interactions between protein kinase and substrates.     

A label-free and sensitive fluorescent assay for one step detection of protein kinase activity and inhibition

In this paper, a label-free, highly sensitive and simple assay for one step detection of protein kinase (PKA) activity and inhibition that avoids the fluorescent dye process has been established. The detection was based on the fluorescence (FL) quenching of peptide-Ag nanoclusters (Ag NCs) caused by antibody modified Au nanoparticles (anti-Au NPs) via fluorescence resonance energy transfer (FRET). With PKA and adenosine 5′-triphosphate (ATP) introduced, the substrate peptide of Ag NCs could react with PKA via targeted phosphorylation, and followed by the linking interactions between peptide-Ag NCs and anti-Au NPs. According to the fluorescence quenching of Ag NCs, the activity of protein kinase can be facilely monitored in the range of 0.1–2000 mU/μL with high sensitivity. The detection limit for PKA is 0.039 mU/μL. We further explored the inhibitory effect of H-89 for protein kinase activity. The developed method was also applied to the investigation of drug-induced PKA activation in HeLa cells, which provides a promising means for screening of kinase-related drugs and the clinical diagnosis of disease.     

Phosphorylation of phospholipase D1 and the modulation of its interaction with RhoA by cAMP-dependent protein kinase

Agents that elevate cellular cAMP are known to inhibit the activation of phospholipase D (PLD). We investigated whether PLD can be phosphorylated by cAMP-dependent protein kinase (PKA) and PKA-mediated phosphorylation affects the interaction between PLD and RhoA, a membrane regulator of PLD. PLD1, but not PLD2 was found to be phosphorylated in vivo by the treatment of dibutyryl cAMP (dbcAMP) and in vitro by PKA. PKA inhibitor (KT5720) abolished the dbcAMP-induced phosphorylation of PLD1, but dibutyryl cGMP (dbcGMP) failed to phosphorylate PLD1. The association between PLD1 and Val14RhoA in an immunoprecipitation assay was abolished by both dbcAMP and dbcGMP. Moreover, RhoA but not PLD1 was dissociated from the membrane to the cytosolic fraction in dbcAMP-treated cells. These results suggest that both PLD1 and RhoA are phosphorylated by PKA and the interaction between PLD1 and RhoA is inhibited by the phosphorylation of RhoA rather than by the phosphorylation of PLD1.     

Charge optimization of the interface between protein kinases and their ligands

Examining the potential for electrostatic complementarity between a ligand and a receptor is a useful technique for rational drug design, and can demonstrate how a system prioritizes interactions when allowed to optimize its charge distribution. In this computational study, we implemented the previously developed, continuum solvent-based charge optimization theory with a simple, quadratic programming algorithm and the UHBD Poisson-Boltzmann solver. This method allows one to compute the best set of point charges for a ligand or ligand region based on the ligand and receptor shape, and the receptor partial charges, by optimizing the binding free energy obtained from a continuum-solvent model. We applied charge optimization to a fragment of the heat-stable protein kinase inhibitor (PKI) of protein kinase A (PKA), to three flavopiridol inhibitors of CDK2, and to cyclin A which interacts with CDK2 to regulate the cell cycle. We found that a combination of global (involving every charge) and local (involving only charges in a local region) optimization can give useful hints for designing better inhibitors. Although some parts of an inhibitor may already contribute significantly to binding, we found that they could still be the most important targets for modifications to obtain stronger binders. In studying the binding of flavopiridol inhibitors to CDK2, comparable binding affinity could be obtained regardless of whether the net charges of the inhibitors were constrained to -2, -1, 0, 1, or 2 during the optimization. This provides flexibility in inhibitor design when a certain net charge of the inhibitor is desired in addition to strong binding affinity. For the study of the PKA-PKI and CDK2-cyclin A interfaces, we identified residues whose charge distributions are already close to optimal and those whose charge distributions could be refined to further improve binding.     

Protein kinase A mediates microglial activation induced by plasminogen and gangliosides

In the injured brain, microglia is known to be activated and produce proinflammatory mediators such as interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha) and inducible nitric oxide synthase (iNOS). We investigated the role of protein kinase A (PKA) in microglial activation by both plasminogen and gangliosides in rat primary microglia and in the BV2 immortalized murine microglial cell line. Both plasminogen and gangliosides induced IL-1beta, TNF-alpha and iNOS mRNA expression, and that this expression was inhibited by the addition of the PKA inhibitors, KT5720 and H89. Both plasminogen and gangliosides activated PKA and increased the DNA binding activity of the cAMP response element- binding protein (CREB). Furthermore, KT5720 and H89 reduced the DNA binding activities of CREB and NF-kappaB in plasminogen-treated cells. These results suggest that PKA plays an important role in plasminogen and gangliosides- induced microglial activation.     

A fluorescent-labeled oligopeptide for monitoring PKA-mediated phosphorylation

A fluorescent-labeled oligopeptide (DACM-CLRRASLK-fluorescein), containing a consensus amino acid sequence (RRXSL) of cyclic AMP (cAMP) dependent protein kinase A (PKA) substrate-proteins, was designed. The fluorescent peptide was a good substrate of PKA, and the phosphorylation of its serin residue caused an intensive change in fluorescent intensity. We expect that the peptide will be useful as a fluorescent indicator for monitoring PKA activity in living cells.     

Fluorescent detection of protein kinase based on zirconium ions-immobilized magnetic nanoparticles

We report here an affinity separation-based fluorometric method for monitoring the activity and inhibition of protein kinase. In this assay, when the fluorescein isothiocyanate (FITC) labeled substrate peptides (S-peptide) are phosphorylated by kinase, the product peptides (P-peptide) will be adsorbed and concentrated onto the surface of Zr⁴⁺-immobilized nitrilotriacetic acid-coated magnetic nanoparticles (Zr-NTA MNPs) through the chelation of Zr⁴⁺ and phosphate groups. After magnetic separation, the fluorescence intensity of the homogeneous solution changes dramatically. Hence the fluorescence response allows this MNPs-based method to easily probe kinase activity by a spectrometer. The feasibility of the method has been demonstrated by sensitive measurement of the activity of cAMP-dependent protein kinase (PKA) with a low detection limit (0.5 mU μL⁻¹). Moreover, the system is successfully applied to estimate the IC₅₀ value of PKA inhibitor H-89 and detect the Forskolin/3-isobutyl-1-methylxanthine (IBMX) stimulated activation of PKA in cell lysate. Additionally, Zr-NTA MNPs are reusable by stripping Zr⁴⁺ ions from NTA-coated MNPs and rechelating again. This method, which relies on the surface-functionalized MNPs, presents a promising candidate for simple and cost-effective assay of kinase activity and inhibitor screening.     

Computer modeling of the dynamic properties of the cAMP-dependent protein kinase catalytic subunit

The structural dynamics of the cAMP-dependent protein kinase catalytic subunit were modeled using molecular dynamics computational methods. It was shown that the structure of this protein as well as its complexes with ATP and peptide ligand PKI(5-24) consisted of a large number of rapidly inter-converting conformations which could be grouped into subsets proceeding from their similarity. This cluster analysis revealed that conformations which correspond to the “opened” and “closed” structures of the protein were already present in the free enzyme, and most surprisingly co-existed in enzyme–ATP and enzyme–PKI(5-24) complexes as well as in the ternary complex, which included both of these ligands. The results also demonstrated that the most mobile structure segments of the protein were located in the regions of substrate binding sites and that their dynamics were most significantly affected by the binding of the ATP and peptide ligand.     

Analysis of protein kinase autophosphorylation using expressed protein ligation and computational modeling

Protein kinases represent a family of enzymes that are critical in cell signaling. One mechanism by which protein kinases are regulated is via autophosphorylation. In the studies described here, we have examined the mechanism of autophosphosphorylation at serine 338 in the regulation of protein kinase A (PKA). Expressed protein ligation allowed for the covalent linkage of an ATP moiety to a Ser mimic at this phosphorylation site. Using a combination of size exclusion chromatography, fluorescence nucleotide binding, kinase measurements, and limited proteolysis assays on this semisynthetic ATP-linked protein, we have obtained unique evidence for an intramolecular autophosphorylation mechanism in PKA regulation. Computational analysis provided a plausible model for a PKA conformation consistent with intramolecular phosphoryl transfer. This approach could be applied to other autoprocessing enzymes by exploiting appropriate transition state analogue motifs in the context of protein semisynthesis.     

Luminescent kinase activity biosensors based on a versatile bimolecular switch

Real-time tracking of kinase activity in living systems has revealed new modes of encoding signaling information into spatiotemporal activity patterns and opened new avenues for screening kinase modulators. However, the sensitivity of kinase activity detection, which is commonly coupled to a fluorescence resonance energy transfer (FRET)-based readout, has often been a limiting factor. Here we show that a kinase-inducible bimolecular switch consisting of a substrate for the kinase of interest and a phosphoamino acid binding domain can be designed to sense different kinase activities and coupled to various readouts, thereby allowing for examination of dynamic kinase activity with increased sensitivity and versatility. Specifically, we demonstrate that bimolecular switches designed to sense protein kinase A (PKA) or protein kinase C (PKC) activities can turn on FRET as well as bioluminescence signals. Notably, the FRET-based sensors gain larger dynamic ranges in comparison with their unimolecular counterparts; the novel bioluminescence-based reporters for PKA and PKC show high sensitivity and a unique capability to detect basal kinase activities and should enable new applications in in vivo imaging of kinase activity and high-throughput compound screening. Thus, this generalizable design advances the molecular toolkit of kinase activity detection and provides a means for versatile and sensitive detection of kinase activity in various biological systems.     

Downstream molecular events in the altered profiles of lysophosphatidic acid-induced cAMP in senescent human diploid fibroblasts

Lysophosphatidic acid (LPA) is a phospholipid growth factor that acts through G-protein-coupled receptors. Previously, we demonstrated an altered profile of LPA-dependent cAMP content during the aging process of human diploid fibroblasts (HDFs). In attempts to define the molecular events associated with the age-dependent changes in cAMP profiles, we determined the protein kinase A (PKA) activity, phosphorylation of cAMP-response element binding protein (CREB), and the protein expression of CRE-regulatory genes, c-fos and COX-2 in young and senescent HDFs. We observed in senescent cells, an increase in mRNA levels of the catalytic subunit a of PKA and of the major regulatory subunit Ialpha. Senescence-associated increase of cAMP after LPA treatment correlated well with increased CREB phosphorylation accompanying activation of PKA in senescent cells. In senescent cells, after LPA treatment, the expression of c-fos and COX-2 decreased initially, followed by an increase. In young HDFs, CREB phosphorylation decreased following LPA treatment, and both c-fos and COX-2 protein levels increased rapidly. CRE-luciferase assay revealed higher basal CRE-dependent gene expression in young HDFs compared to senescent HDFs. However, LPA-dependent slope of luciferase increased more rapidly in senescent cells than in young cells, presumably due to an increase of LPA-induced CREB phosphorylation. CRE-dependent luciferase activation was abrogated in the presence of inhibitors of PKC, MEK1, p38MAPK, and PKA, in both young and senescent HDFs. We conclude that these kinase are coactivators of the expression of CRE-responsive genes in LPA-induced HDFs and that their changed activities during the aging process contribute to the final expression level of CRE-responsive genes.     

Sensitive and versatile electrogenerated chemiluminescence biosensing platform for protein kinase based on Ru(bpy)3 functionalized gold nanoparticles mediated signal transduction

A novel, sensitive and versatile electrogenerated chemiluminescence biosensing platform is developed for monitoring activity and inhibition of protein kinase based on Ru(bpy)₃²⁺ functionalized gold nanoparticles (Ru(bpy)₃²⁺-AuNPs) mediated signal transduction. Ru(bpy)₃²⁺-AuNPs were formed by functionalizing AuNPs with Ru(bpy)₃²⁺ through electrostatic interactions and were used as thiol-versatile signal probe. Casein kinase II (CK2) and cAMP-dependent protein kinase (PKA), two classical protein kinase implicated in disease, were chosen as model protein kinases while a CK2-specific peptide (CRRRADDSDDDDD) and a PKA-specific peptide (CLRRASLG) were employed as molecular substrate for CK2 and PKA, respectively. The specific peptide was self-assembled onto the gold electrode via Au–S bond to form ECL biosensor. Upon thiophosphorylation of the peptide on the electrode in the presence of protein kinase and co-substrate adenosine-5’-(γ-thio)-triphosphate, Ru(bpy)₃²⁺-AuNPs was assembled onto the thiophosphorylated peptides via Au–S bond. The Ru(bpy)₃²⁺-AuNPs attached on electrode surface produce detectable ECL signal in the presence of coreactant tripropylamine. This strategy is promising for multiple protein kinase assay and kinase inhibitor profiling with high sensitivity, good selectivity and versatility. The ECL intensity is proportional to the activity of CK2 in the range of 0.01–0.5 unit/mL with a low detection limit of 0.008 unit/mL and to the activity of PKA in the range of 0.01–0.4 unit/mL with a detection limit of 0.005 unit/mL. Additionally, this assay was applied to the detection of CK2 in serum samples and the inhibition of CK2 and PKA. This work demonstrates that the developed ECL method can provide a sensitive and versatile platform for the detection of kinase activity and drug-screening.     

A CIEF‐LIF method for simultaneous analysis of multiple protein kinases and screening of inhibitors

Here, a CIEF‐LIF method for multiple protein kinase simultaneous analysis and inhibitors throughput screening with fast rate and low cost is presented. Comparing with CZE, CIEF‐LIF exhibited great focusing ability and high separation efficiency for substrate and phosphorylated peptides, and is applicable for multiple kinases simultaneous analysis regardless of their substrate peptides compositions and charge statuses. Thus, highly sensitive analysis for cyclic adenosine 3’, 5’‐monophosphate‐dependent protein kinase (PKA) and cyclin‐dependent kinase 1 (CDK1) was achieved in CIEF‐LIF analysis with detection sensitivity up to 1.25 mU/μL and 0.4 mU/μL, respectively, two magnitudes higher than that of CZE and comparable with that in nanomaterials or green fluorescent protein‐based kinase assay. Moreover, the inhibition effect of inhibitors on multiple kinases could be simultaneously readout in a single electrophoretic run, with half maximal inhibitory concentration of H‐89 for PKA and Ro‐3306 for CDK1 calculated as 37.0 and 35.9 nM, respectively, consistent with literatures reported. The CIEF‐LIF also exhibited strong anti‐interference ability in human breast cancer cell lysates analysis and simulators such as forskolin and 3‐isobutyl‐1‐methylxantine assessment. Therefore, CIEF‐LIF is desirable for future biological application and clinical diagnostics and drug discovery.     

抑制剂BD2对PKA与PKC βII的抑制选择性研究

蛋白激酶A (PKA)和蛋白激酶C (PKC)的过度表达导致细胞生长分化异常, 是治疗肿瘤的潜在靶点. 抑制剂BD2对PKA和PKC抑制作用存在高选择性. 为了探讨BD2高选择性机制, 本工作以PKA与BD2复合物的晶体结构为模板, 通过同源模建结合分子对接的方法构建PKC βII与BD2复合物的结构, 并对PKA-BD2复合物和PKC-BD2复合物进行了2.5 ns的分子动力学模拟, 运用MM-GBSA方法计算了结合自由能, 通过能量分解的方法考察PKA和PKC的主要残基与BD2之间的相互作用和识别机制. 结合能分析结果很好地描述了BD2对PKA抑制活性比其对PKC抑制活性高这一实验现象. 氢键分析和能量分解结果共同说明了BD2的B环和酰胺链部分与PKA和PKC中相应位点的残基之间的相互作用存在差异, 这是BD2存在选择性的内在因素. BD2高选择性作用机制的阐明为进一步基于结构的balanol类抑制剂的结构设计和优化提供了合理的指导.