PMID- 15927069
OWN - NLM
STAT- MEDLINE
DCOM- 20060222
LR  - 20211203
IS  - 1472-6750 (Electronic)
IS  - 1472-6750 (Linking)
VI  - 5
DP  - 2005 May 31
TI  - High-throughput kinase assays with protein substrates using fluorescent polymer 
      superquenching.
PG  - 16
AB  - BACKGROUND: High-throughput screening is used by the pharmaceutical industry for 
      identifying lead compounds that interact with targets of pharmacological 
      interest. Because of the key role that aberrant regulation of protein 
      phosphorylation plays in diseases such as cancer, diabetes and hypertension, 
      kinases have become one of the main drug targets. With the exception of 
      antibody-based assays, methods to screen for specific kinase activity are 
      generally restricted to the use of small synthetic peptides as substrates. 
      However, the use of natural protein substrates has the advantage that potential 
      inhibitors can be detected that affect enzyme activity by binding to a site other 
      than the catalytic site. We have previously reported a non-radioactive and 
      non-antibody-based fluorescence quench assay for detection of phosphorylation or 
      dephosphorylation using synthetic peptide substrates. The aim of this work is to 
      develop an assay for detection of phosphorylation of chemically unmodified 
      proteins based on this polymer superquenching platform. RESULTS: Using a modified 
      QTL Lightspeed assay, phosphorylation of native protein was quantified by the 
      interaction of the phosphorylated proteins with metal-ion coordinating groups 
      co-located with fluorescent polymer deposited onto microspheres. The binding of 
      phospho-protein inhibits a dye-labeled "tracer" peptide from associating to the 
      phosphate-binding sites present on the fluorescent microspheres. The resulting 
      inhibition of quench generates a "turn on" assay, in which the signal correlates 
      with the phosphorylation of the substrate. The assay was tested on three 
      different proteins: Myelin Basic Protein (MBP), Histone H1 and Phosphorylated 
      heat- and acid-stable protein (PHAS-1). Phosphorylation of the proteins was 
      detected by Protein Kinase Calpha (PKCalpha) and by the Interleukin -1 
      Receptor-associated Kinase 4 (IRAK4). Enzyme inhibition yielded IC50 values that 
      were comparable to those obtained using peptide substrates. Statistical 
      parameters that are used in the high-throughput community to determine assay 
      robustness (Z'-value) demonstrate the suitability of this format for 
      high-throughput screening applications for detection of inhibitors of enzyme 
      activity. CONCLUSION: The QTL Lightspeed protein detection system provides a 
      simple mix and measure "turn on" assay for the detection of kinase activity using 
      natural protein substrates. The platform is robust and allows for identification 
      of inhibitors of kinase activity.
FAU - Rininsland, Frauke
AU  - Rininsland F
AD  - QTL Biosystems, 2778 Agua Fria Street, Santa Fe, NM 87507, USA. frauke@qtlbio.com
FAU - Stankewicz, Casey
AU  - Stankewicz C
FAU - Weatherford, Wendy
AU  - Weatherford W
FAU - McBranch, Duncan
AU  - McBranch D
LA  - eng
PT  - Journal Article
DEP - 20050531
PL  - England
TA  - BMC Biotechnol
JT  - BMC biotechnology
JID - 101088663
RN  - 0 (Adaptor Proteins, Signal Transducing)
RN  - 0 (Carrier Proteins)
RN  - 0 (Cell Cycle Proteins)
RN  - 0 (EIF4EBP1 protein, human)
RN  - 0 (Fluorescent Dyes)
RN  - 0 (Histones)
RN  - 0 (Intracellular Signaling Peptides and Proteins)
RN  - 0 (Ions)
RN  - 0 (Myelin Basic Protein)
RN  - 0 (Peptide Library)
RN  - 0 (Peptides)
RN  - 0 (Phosphoproteins)
RN  - 0 (Polymers)
RN  - 0 (Proteins)
RN  - EC 2.7.- (Phosphotransferases)
RN  - EC 2.7.11.1 (IRAK4 protein, human)
RN  - EC 2.7.11.1 (Interleukin-1 Receptor-Associated Kinases)
RN  - EC 2.7.11.1 (Protein Serine-Threonine Kinases)
RN  - EC 2.7.11.13 (Protein Kinase C-alpha)
SB  - IM
MH  - Adaptor Proteins, Signal Transducing
MH  - Animals
MH  - Binding Sites
MH  - Carrier Proteins/analysis
MH  - Cattle
MH  - Cell Cycle Proteins
MH  - Fluorescence Polarization
MH  - Fluorescent Dyes/*pharmacology
MH  - Histones/analysis
MH  - Humans
MH  - Inhibitory Concentration 50
MH  - Interleukin-1 Receptor-Associated Kinases
MH  - Intracellular Signaling Peptides and Proteins/analysis
MH  - Ions
MH  - Myelin Basic Protein/analysis
MH  - Peptide Library
MH  - Peptides/chemistry
MH  - Phosphoproteins/analysis
MH  - Phosphorylation
MH  - Phosphotransferases/*chemistry
MH  - Polymers/*chemistry
MH  - Protein Array Analysis/methods
MH  - Protein Kinase C-alpha/analysis
MH  - Protein Serine-Threonine Kinases/analysis
MH  - Proteins/*chemistry
MH  - Quantitative Trait Loci
MH  - Sequence Analysis, Protein/*methods
PMC - PMC1166542
EDAT- 2005/06/02 09:00
MHDA- 2006/02/24 09:00
PMCR- 2005/05/31
CRDT- 2005/06/02 09:00
PHST- 2005/03/01 00:00 [received]
PHST- 2005/05/31 00:00 [accepted]
PHST- 2005/06/02 09:00 [pubmed]
PHST- 2006/02/24 09:00 [medline]
PHST- 2005/06/02 09:00 [entrez]
PHST- 2005/05/31 00:00 [pmc-release]
AID - 1472-6750-5-16 [pii]
AID - 10.1186/1472-6750-5-16 [doi]
PST - epublish
SO  - BMC Biotechnol. 2005 May 31;5:16. doi: 10.1186/1472-6750-5-16.