Time-resolved Forster resonance energy transfer assays for the binding of nucleotide and protein substrates to p38alpha protein kinase.
We have developed assays for the binding of nucleotide and protein substrates to p38alpha protein kinase based on time-resolved Forster resonance energy transfer. p38alpha was biotinylated by addition of a sequence that targets biotin to a single lysine when coexpressed with biotin ligase in Escherichia coli, allowing formation of a complex between a streptavidin "LANCE" europium chelate conjugate and p38alpha. When this reagent was combined with M39AF, a p38 inhibitor containing a fluorescent moiety whose excitation wavelengths match the emission wavelengths of the europium chelate, a change in ratio of light emitted at 665 nm/615 nm is detected. Less than 100pM complex was detected with a signal/background ratio of >30-fold. The complex exhibits slow, tight binding kinetics where the apparent K(d) decreases with a relaxation time of 21 min at 125 pM biotin-p38alpha. Preincubating inhibitors or ATP with biotin-p38alpha and adding M39AF as a competitor yielded IC(50)s consistent with those measured by enzyme assay for the activated form of biotin-p38alpha. The same technique was also used to measure affinity of inhibitors for the unphosphorylated and catalytically inactive form of biotin-p38alpha. To measure affinity of p38alpha for its protein substrate MK2, we incubated biotin-p38alpha with a glutathione S-transferase MK2 fusion protein. Detection of the complex after incubation with streptavidin-allophycocyanin and a LANCE-conjugated anti-GST allowed measurement of affinity of MK2 for biotin-p38alpha and detection of 0.5 nM p38alpha.MK2 complex with signal/background ratio >5-fold. Competition with unbiotinylated p38alpha yielded an IC(50) value of 5 nM. Activation of either p38alpha or MK2 had no effect on the measured K(d). M39AF was found to bind in a ternary complex with p38alpha.MK2 with lower affinity than that observed in the binary complex with p38alpha alone.
Merck Research Laboratories, 80M127, 126 East Lincoln Avenue, Rahway, NJ 07065, USA.
Wen Xiao
WX
Zhang
Ruixiu
R
Wang
Douglas
D
Wisniewski
Alice I
AI
Marcy
Philip
P
LoGrasso
Jean-Marie
JM
Lisnock
Richard T
RT
Cummings
James E
JE
Thompson
D000818:Animals; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D009711:Nucleotides / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D048051:p38 Mitogen-Activated Protein Kinases / Q000737:chemistry* / Q000235:genetics
D005456:Fluorescent Dyes; D009711:Nucleotides; D048051:p38 Mitogen-Activated Protein Kinases