Development of a protein chip to measure PKCβ activity.
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.
Lee
Dong-Woo
DW
National Research Lab of Skin-Bioactive Materials, Department of Biological Engineering, Inha University, Incheon, South Korea.
Kim
Hae Jong
HJ
Choi
Chi-Ho
CH
Shin
Jeong-Hyun
JH
Kim
Eun-Ki
EK
D000255:Adenosine Triphosphate / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D057075:Enzyme Assays; D057166:High-Throughput Screening Assays / Q000379:methods*; D006657:Histones / Q000378:metabolism; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D058619:Maltose-Binding Proteins / Q000378:metabolism; D010455:Peptides / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D040081:Protein Array Analysis / Q000379:methods*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D064546:Protein Kinase C beta; D013379:Substrate Specificity / Q000187:drug effects
D006657:Histones; D007211:Indoles; D008301:Maleimides; D058619:Maltose-Binding Proteins; D010455:Peptides; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D064546:Protein Kinase C beta; C070515:bisindolylmaleimide I