Optimal Sox-based fluorescent chemosensor design for serine/threonine protein kinases

Shults, Melissa D.; Carrico-Moniz, Dora; Imperiali, Barbara

doi:10.1016/j.ab.2006.03.003

Cited by 81 publications

(81 citation statements)

References 48 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To monitor the enzymatic activity of Syk, a recently developed fluorescence kinase assay was employed. This assay monitors phosphorylation of a peptide substrate, pep7, in real time using the change in fluorescence of a non-natural amino acid (the "Sox" amino acid) incorporated directly into the peptide substrate (19,20). Utilizing Syk that was not treated after purification ("basal Syk"), it was found that product formation was initially linear with time and then plateaued after substrate was completely converted to product (Fig.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Molecular Mechanism of the Syk Activation Switch

Tsang

Giannetti

Shaw

et al. 2008

Journal of Biological Chemistry

139

189

View full text Add to dashboard Cite

Many immune signaling pathways require activation of the Syk tyrosine kinase to link ligation of surface receptors to changes in gene expression. Despite the central role of Syk in these pathways, the Syk activation process remains poorly understood. In this work we quantitatively characterized the molecular mechanism of Syk activation in vitro using a real time fluorescence kinase assay, mutagenesis, and other biochemical techniques. We found that dephosphorylated full-length Syk demonstrates a low initial rate of substrate phosphorylation that increases during the kinase reaction due to autophosphorylation. The initial rate of Syk activity was strongly increased by either pre-autophosphorylation or binding of phosphorylated immune tyrosine activation motif peptides, and each of these factors independently fully activated Syk. Deletion mutagenesis was used to identify regions of Syk important for regulation, and residues 340 -356 of the SH2 kinase linker region were identified to be important for suppression of activity before activation. Comparison of the activation processes of Syk and Zap-70 revealed that Syk is more readily activated by autophosphorylation than Zap-70, although both kinases are rapidly activated by Src family kinases. We also studied Syk activity in B cell lysates and found endogenous Syk is also activated by phosphorylation and immune tyrosine activation motif binding. Together these experiments show that Syk functions as an "OR-gate" type of molecular switch. This mechanism of switch-like activation helps explain how Syk is both rapidly activated after receptor binding but also sustains activity over time to facilitate longer term changes in gene expression.Syk is a tyrosine kinase that functions immediately downstream of antigen receptors in immune cells including B lymphocytes, mast cells, and macrophages (1-3). The central role of Syk in cell types associated with disorders such as rheumatoid arthritis and allergic rhinitis suggests that strategies to block Syk activation may have therapeutic benefit (4 -6). After receptor ligation and phosphorylation, Syk becomes localized to immune receptors and proceeds to phosphorylate downstream targets leading to Ca 2ϩ mobilization, initiation of the extracellular signal-regulated kinase and p38 mitogen-activated protein kinase cascades, and activation of transcription factors such as NF-B (7). Although increased Syk activity is known to be indispensable for each of these cellular events, a molecular-level understanding of the steps leading to Syk activation has not been clearly defined.The Syk domain structure consists of an N-terminal pair of Src homology 2 (SH2) 2 domains separated by an inter-SH2 linker, an SH2-domain-kinase linker, and a C-terminal kinase domain. Recruitment of Syk to immune receptors involves binding of the tandem SH2 domains of Syk to motifs in the receptor known as immune tyrosine activation motifs (ITAMs), which are two YXXL sequences typically separated by 7-12 intervening residues (8, 9). Syk is known to have multiple ...

show abstract

Section: Resultsmentioning

confidence: 99%

“…Briefly, activity was assessed by monitoring the increase in fluorescence after phosphorylation of a commercially available peptide substrate (pep7) that contains the phosphorylation-sensitive amino acid Sox (19,20).…”

Section: Methodsmentioning

confidence: 99%

Molecular Mechanism of the Syk Activation Switch

Tsang

Giannetti

Shaw

et al. 2008

Journal of Biological Chemistry

139

189

View full text Add to dashboard Cite

show abstract

“…With different peptide sequences, CHEF sensors have been developed for numerous kinases including PKC, Cdk2, PKA, Akt, MK2, and Pim2. [43] Upon phosphorylation by these kinases, up to an eight-fold increase in fluorescence signals was observed, making these CHEF sensors suitable for studying kinases possessing relatively low abundance and activity. The use of specific sensors for different kinases even allowed the continuous reporting of the activity of multiple kinases from crude homogenates in parallel assays.…”

Section: Abps For Protein Phosphatases and Kinasesmentioning

confidence: 99%

Current Chemical Biology Tools for Studying Protein Phosphorylation and Dephosphorylation

Tan

et al. 2011

Chemistry A European J

View full text Add to dashboard Cite

Amongst different posttranslational events involved in cellular-signaling pathways, phosphorylation and dephosphorylation of proteins are the most prevalent. Aberrant regulations in the cellular phosphoproteome network are implicated in most major human diseases. Consequently, kinases and phosphatases are two of the most important groups of drug targets in medicinal research today. A major challenge in the understanding of protein phosphorylation and dephosphorylation is the sheer complexity of the phosphoproteome network and the lack of tools capable of studying protein phosphorylation and dephosphorylation as they occur in cells. We highlight herein various chemical biology tools that have emerged in the last decade for such studies. First, we discuss the use of small-molecule mimics of phosphoamino acids and their use in elucidating the function of protein phosphorylation and dephosphorylation. We also introduce recent advances in the field of activity-based protein profiling (ABPP) for proteome-wide detection of protein phosphorylation and dephosphorylation. We next discuss the key concepts in the design of peptide- and protein-based biosensors capable of real-time reporting of phosphorylation/dephosphorylation events. Finally, we highlight the application of peptide and small-molecule microarrays (SMMs), and their applications in high-throughput screening and discovery of new compounds related to phosphorylation/dephosphorylation.

show abstract

“…To overcome these drawbacks, a non-radioactive technique was assayed in this work (Omnia® Ser/Thr recombinant kit, Invitrogen). This method utilizes a fluorescent peptide substrate-based technology (Ser/Thr peptide 5) allowing kinase enzymatic characterization and inhibitor screening (Shults et al 2006). In our in vitro kinase assay, PfPKAc activity was detected and characterized using the Ser/Thr peptide 5 (Table 2) and the effect of different kinase inhibitors on the PfPKAc activity was investigated.…”

Section: Kinetic Characteristics Of Overexpressed Pfpkac and Inhibitomentioning

confidence: 99%

Expression and biochemical characterization of the Plasmodium falciparum protein kinase A catalytic subunit

et al. 2009

View full text Add to dashboard Cite

Dissemination of drug-resistant malaria parasites represents one of the most important public health problems; therefore, the development of new antimalarial compounds is required. Cyclic AMP-dependent protein kinase is implicated in numerous cellular processes and an essential role for this enzyme has also been reported in the intraerythrocytic growth of the malaria parasite. The cAMP-dependent protein kinase from Plasmodium falciparum (PfPKA) plays an important role in the parasite life cycle and represents an attractive target for the development of antimalarial drugs. In this work, a recombinant PfPKA catalytic subunit (PfPKAc) was over-expressed in Escherichia coli and successfully purified using a two-step chromatographic process. The enzymatic properties of the recombinant PfPKAc were then determined using a sensitive fluorogenic assay suitable for biochemical characterization and inhibitor screening. This work provides new insights on the study of PfPKAc that will contribute to future investigations of the parasite cAMP signaling pathway and to high-throughput screening of specific malarial PKA inhibitors.

show abstract

Optimal Sox-based fluorescent chemosensor design for serine/threonine protein kinases

Cited by 81 publications

References 48 publications

Molecular Mechanism of the Syk Activation Switch

Molecular Mechanism of the Syk Activation Switch

Current Chemical Biology Tools for Studying Protein Phosphorylation and Dephosphorylation

Expression and biochemical characterization of the Plasmodium falciparum protein kinase A catalytic subunit

Contact Info

Product

Resources

About