Use of capillary electrophoresis and endogenous fluorescent substrate to monitor intracellular activation of protein kinase A.
Here we demonstrate for the first time the use of an endogenous multiphosphorylatable substrate for monitoring the intracellular activation of kinase with capillary electrophoresis. First, we devised a novel PCR-based strategy for controlled generation of short multirepeat DNA sequences and applied this method to generate a green fluorescence protein (GFP)-tagged protein substrate containing eight phosphorylation sites for protein kinase A (PKA). The protein substrate was transiently expressed in C2C12 rat myoblast cells, and intracellular PKA was then activated by adding [8]-bromo-cyclic AMP to the cell culture medium. Phosphorylated product and nonphosphorylated substrate present in the crude cell extract were separated by capillary zone electrophoresis and detected with laser-induced fluorescence of the GFP tag. The identities of two electrophoretic peaks were confirmed by both phosphorylation of the substrate and dephosphorylation of the product in vitro. The proposed method was applied to monitoring the activation of PKA in single myoblast cells. It advantageously allowed us to avoid microinjection of the substrate, the procedure that is both hard to perform and excessively invasive when applied to small mammalian cells.
Department of Chemistry, York University, Toronto, Ontario, M3J-1P3 Canada.
Arash
A
Zarrine-Afsar
Sergey N
SN
Krylov
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D019075:Electrophoresis, Capillary / Q000379:methods*; D004789:Enzyme Activation; D005453:Fluorescence; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D011993:Recombinant Fusion Proteins; D015124:8-Bromo Cyclic Adenosine Monophosphate; D017868:Cyclic AMP-Dependent Protein Kinases