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Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions
Published on: August 2, 2015
Development of a protein chip to measure PKCβ activity
Dong-Woo Lee1, Hae Jong Kim, Chi-Ho Choi
1National Research Lab of Skin-Bioactive Materials, Department of Biological Engineering, Inha University, Incheon, South Korea.
Abstract:
Phosphorylation of proteins by kinases plays an important role in regulating cellular processes including melanin production in the skin cells. Protein kinase C β (PKCβ) is known to be involved in phosphorylating tyrosinase, the key enzyme of melanin production, regulating the skin pigmentation process. In melanogenesis, PKCβ activates the tyrosinase by phosphorylation of its two serine residues. In this study, phosphorylation activity by PKCβ was monitored on a protein chip for the screening of depigmenting agents. As a tyrosinase mimic, 11 or 30 amino acids of the C-terminal of tyrosinase was fused with maltose-binding protein (MBP). After immobilizing the MBP-fused PKCβ substrate peptide on epoxy-treated slide surface, PKCβ reaction mix was applied over the immobilized MBP-fused PKCβ substrate peptide. Phosphorylation was detected with anti-phosphoSer/Thr antibodies, followed by fluorescence-labeled second antibodies. Phosphorylation of MBP-30aa was observed on a protein chip, and this phosphorylation was inhibited by the PKC inhibitor (GF109203X). These results indicate the potential of PKCβ protein chip as a high-throughput screening tool in the screening of depigmenting agents.
Insights
This study developed a protein chip to monitor protein kinase C beta (PKCβ) activity, crucial for skin pigmentation. The chip successfully detected phosphorylation and can be used to screen for depigmenting agents.
Area of Science:
- Biochemistry
- Molecular Biology
- Dermatology
Background:
- Protein phosphorylation regulates cellular processes, including skin melanin production.
- Protein kinase C beta (PKCβ) phosphorylates tyrosinase, a key enzyme in melanogenesis, influencing skin pigmentation.
- Understanding PKCβ's role is vital for developing skin depigmenting agents.
Purpose of the Study:
- To develop and validate a protein chip for monitoring PKCβ phosphorylation activity.
- To assess the potential of this chip as a high-throughput screening tool for depigmenting agents.
Main Methods:
- Tyrosinase C-terminal peptides were fused with maltose-binding protein (MBP).
- MBP-fused peptides were immobilized on an epoxy-treated slide surface.
- PKCβ phosphorylation was detected using anti-phosphoserine/threonine antibodies and fluorescence-labeled secondary antibodies.
Main Results:
- Phosphorylation of the MBP-fused 30-amino acid tyrosinase peptide (MBP-30aa) was successfully detected on the protein chip.
- The observed phosphorylation was inhibited by the specific PKC inhibitor, GF109203X.
- The protein chip demonstrated effective monitoring of PKCβ activity.
Conclusions:
- The developed PKCβ protein chip is a viable tool for high-throughput screening.
- This platform shows promise for identifying novel depigmenting agents for skin pigmentation disorders.
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