Development of a Protein Chip to Measure PKC beta Activity

作者：Lee Dong Woo; Kim Hae Jong; Choi Chi Ho; Shin Jeong Hyun; Kim Eun Ki^*

来源：Applied Biochemistry and Biotechnology, 2011, 163(6): 803-812.

DOI：10.1007/s12010-010-9084-z

• PKC beta
• Tyrosinase
• Protein chip
• HTS
• Depigmenting agents

Phosphorylation of proteins by kinases plays an important role in regulating cellular processes including melanin production in the skin cells. Protein kinase C beta (PKC beta) is known to be involved in phosphorylating tyrosinase, the key enzyme of melanin production, regulating the skin pigmentation process. In melanogenesis, PKC beta activates the tyrosinase by phosphorylation of its two serine residues. In this study, phosphorylation activity by PKC beta 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 PKC beta substrate peptide on epoxy-treated slide surface, PKC beta reaction mix was applied over the immobilized MBP-fused PKC beta 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 PKC beta protein chip as a high-throughput screening tool in the screening of depigmenting agents.

• 出版日期2011-3