An enzyme-linked immunosorbent assay for protein kinase D activity using phosphorylation site-specific antibodies.
The protein kinase D (PKD) family is a novel group of kinases that are involved in the regulation of cell proliferation and apoptosis, and several other physiological processes. Hence, these enzymes are attractive targets for pharmacological intervention, but no specific PKD inhibitors are known. With this in mind, we have developed a high-throughput, non-radioactive enzyme-linked immunosorbent assay (ELISA) method to monitor the PKD activity with myelin basic protein (MBP) as substrate. We determined that MBP is phosphorylated by PKD on Ser-160 and that this phosphorylation can be quantified in ELISAs, by the use of phosphorylation site-specific antibodies. Antibodies were developed that are highly specific for the MBP peptide sequence surrounding the phosphorylated Ser-160. We show that our high-throughput kinase assay is useful not only for determining the cellular PKD activity but also to screen for PKD-inhibitory compounds. Our ELISA has advantages over the current radioisotope kinase assay in terms of simplicity and environmental safety.
Rykx
An
A
Molecular Medicine of Protein Kinases, Division of Biochemistry, Department of Molecular Cell Biology, Faculty of Medicine, Katholieke Universiteit Leuven, Leuven, Belgium.
Vancauwenbergh
Sadia
S
De Kimpe
Line
L
Janssens
Katrien
K
Vandoninck
Sandy
S
Waelkens
Etienne
E
Vandenheede
Jackie R
JR
Van Lint
Johan
J
D000255:Adenosine Triphosphate / Q000378:metabolism; D000906:Antibodies / Q000032:analysis / Q000737:chemistry*; D000918:Antibody Specificity; D049109:Cell Proliferation / Q000187:drug effects; D003101:Collodion; D004247:DNA / Q000096:biosynthesis; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay; D013058:Mass Spectrometry; D008567:Membranes, Artificial; D004676:Myelin Basic Protein / Q000737:chemistry / Q000276:immunology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000302:isolation & purification; D013379:Substrate Specificity
D000906:Antibodies; D004791:Enzyme Inhibitors; D008567:Membranes, Artificial; D004676:Myelin Basic Protein; D000255:Adenosine Triphosphate; D003101:Collodion; D004247:DNA; C089212:protein kinase D; D011493:Protein Kinase C