Development of a Protein Chip to Measure PKCβ Activity

Lee, Dong Hoon; Kim, Hae Jong; Choi, Chul-Hyun; Shin, Jeonghyun; Kim, Eun-Ki

doi:10.1007/s12010-010-9084-z

Cited by 5 publications

(4 citation statements)

References 16 publications

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“…Difficulties in applying this strategy to more complex eukaryotes include the availability, maintenance, and use of multiple different cell lines. There has been some success using arrayed-protein chips 10 or bead-immobilized PK 11 to identify PK-clients. Feilner et al .…”

Section: Introductionmentioning

confidence: 99%

“…7 Difficulties in applying this strategy to more complex eukaryotes include the availability, maintenance, and use of multiple different cell lines. There has been some success using arrayed-protein chips 10 or beadimmobilized PK 11 to identify PK-clients. Feilneret al used a chip containing 1690 nonredundant proteins to screen potential clients for two A. thaliana mitogen-activated protein kinases (MAPK).…”

Section: ■ Introductionmentioning

confidence: 99%

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A Versatile Mass Spectrometry-Based Method to Both Identify Kinase Client-Relationships and Characterize Signaling Network Topology

et al. 2013

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While more than a thousand protein kinases (PK) have been identified in the Arabidopsis thaliana genome, relatively little progress has been made towards identifying their individual client proteins. Herein we describe the use of a mass spectrometry-based in vitro phosphorylation strategy, termed Kinase Client assay (KiC assay), to study a targeted-aspect of signaling. A synthetic peptide library comprising 377 in vivo phosphorylation sequences from developing seed was screened using 71 recombinant A. thaliana PK. Among the initial results, we identified 23 proteins as putative clients of 17 PK. In one instance protein phosphatase inhibitor-2 (AtPPI-2) was phosphorylated at multiple-sites by three distinct PK, casein kinase 1-like 10, AME3, and a Ser PK-like protein. To confirm this result, full-length recombinant AtPPI-2 was reconstituted with each of these PK. The results confirmed multiple distinct phosphorylation sites within this protein. Biochemical analyses indicate that AtPPI-2 inhibits type 1 protein phosphatase (TOPP) activity, and that the phosphorylated forms of AtPPI-2 are more potent inhibitors. Structural modeling revealed that phosphorylation of AtPPI-2 induces conformational changes that modulate TOPP binding.

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Section: Introductionmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

A Versatile Mass Spectrometry-Based Method to Both Identify Kinase Client-Relationships and Characterize Signaling Network Topology

et al. 2013

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“…Using this method, sensitivity was increased by three orders of magnitude compared to other microarray approaches with PKA and leptin-kemptide fusions. Lee et al reported analysis of PKCb substrate specificity by microarrays displaying peptide fusion to immobilised maltose binding protein [73]. Another method is the decoration of streptavidin with biotinylated peptides or phosphopeptides.…”

Section: Chemistry Of Peptide Array Preparationmentioning

confidence: 99%

Deciphering Enzyme Function Using Peptide Arrays

2011

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Enzymes are key molecules in signal-transduction pathways. However, only a small fraction of more than 500 human kinases, 300 human proteases and 200 human phosphatases is characterised so far. Peptide microarray based technologies for extremely efficient profiling of enzyme substrate specificity emerged in the last years. This technology reduces set-up time for HTS assays and allows the identification of downstream targets. Moreover, peptide microarrays enable optimisation of enzyme substrates. Focus of this review is on assay principles for measuring activities of kinases, phosphatases or proteases and on substrate identification/optimisation for kinases. Additionally, several examples for reliable identification of substrates for lysine methyl-transferases, histone deacetylases and SUMO-transferases are given. Finally, use of high-density peptide microarrays for the simultaneous profiling of kinase activities in complex biological samples like cell lysates or lysates of complete organisms is described. All published examples of peptide arrays used for enzyme profiling are summarised comprehensively.

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“…PKC widely exists in animal tissues and cells, is the main mediator of signal transduction pathway, and also plays a role that cannot be underestimated in physiological processes such as cell proliferation and differentiation [ 14 ]. In the melanin metabolism pathway, PKC activates the tyrosinase by phosphorylation of its two serine residues [ 15 ]. Activated PKC is bound to the melanosome membrane, and unactivated PKC is free in the cytoplasm of melanocytes [ 16 ].…”

Section: Introductionmentioning

confidence: 99%

Analysis of protein kinase C (HcPKC) gene expression and single-nucleotide polymorphisms related to inner shell color traits in Hyriopsis cumingii

et al. 2022

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Background Protein kinase C (PKC) is a multifunctional serine and PKC can phosphorylate serine residues in the cytoplasmic domain of tyrosinase, thereby regulating the activity of tyrosinase. Activated PKC is bound to the melanosome membrane, and unactivated PKC is free in the cytoplasm of melanocytes. In this study, we study the role of PKC gene in the melanin synthesis pathway and its effect on the color of the nacre of H. cumingii. Results In this study, a HcPKC gene in H. cumingii was cloned and its effects on melanin synthesis and nacre color were studied. HcPKC was expressed in both purple and white mussels, and the level of mRNA expression was higher in the purple mussels than in white mussels. Strong and specific mRNA signals were detected in the dorsal epithelial cells of the mantle pallial layer, indicating that HcPKC may be involved in nacre formation. After SNP association with inner shell color related traits, according to the principle that 0.25 < PIC < 0.5 is medium polymorphism and PIC < 0.25 is low polymorphism, the A + 332G site on the HcPKC gene was a site of moderate polymorphism, and the other four sites were low polymorphism sex sites. There was strong linkage disequilibrium among the five loci. A haplotype was constructed and it was found that the frequency of T1 (AGGAA)in the white population was significantly higher than that in the purple population (P < 0.05). Conclusion The study found that HcPKC of H. cumingii can be used as a candidate gene related to inner shell color, and some of the SNP sites can be used for molecular-assisted breeding in the spinnaker mussel, providing a reference for cultivating high-quality freshwater pearls.

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Development of a Protein Chip to Measure PKCβ Activity

Cited by 5 publications

References 16 publications

A Versatile Mass Spectrometry-Based Method to Both Identify Kinase Client-Relationships and Characterize Signaling Network Topology

A Versatile Mass Spectrometry-Based Method to Both Identify Kinase Client-Relationships and Characterize Signaling Network Topology

Deciphering Enzyme Function Using Peptide Arrays

Analysis of protein kinase C (HcPKC) gene expression and single-nucleotide polymorphisms related to inner shell color traits in Hyriopsis cumingii

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