TY - JOUR
T1 - The synthetic peptide RPRAATF allows specific assay of akt activity in cell lysates
AU - Bozinovski, Steven
AU - Cristiano, Briony E.
AU - Marmy-Conus, Nelly
AU - Pearson, Richard B.
PY - 2002/6/1
Y1 - 2002/6/1
N2 - The Akt protein kinase is a critical signaling molecule in a range of cellular processes. A key to identifying the role of this pleiotropic kinase in any particular process is the ability to quantitate its activity. In this study we show that the synthetic peptide RPRAATF is a specific substrate for the kinase in crude cell extracts, thus enabling rapid, convenient, and sensitive assay of Akt activity. Peptide kinase activity was confined to a single peak upon sequential ion-exchange chromatography of whole-cell extracts of Balb/c 3T3 fibroblasts. This activity was stimulated by both platelet-derived growth factor and pervanadate, phosphatidyl inositol 3-kinase dependent, and inhibited by specific immunodepletion with anti-Akt antisera. Furthermore, direct assays of crude extracts from a range of cell types using this peptide were consistent with the results obtained using specific immunoprecipitation assays.
AB - The Akt protein kinase is a critical signaling molecule in a range of cellular processes. A key to identifying the role of this pleiotropic kinase in any particular process is the ability to quantitate its activity. In this study we show that the synthetic peptide RPRAATF is a specific substrate for the kinase in crude cell extracts, thus enabling rapid, convenient, and sensitive assay of Akt activity. Peptide kinase activity was confined to a single peak upon sequential ion-exchange chromatography of whole-cell extracts of Balb/c 3T3 fibroblasts. This activity was stimulated by both platelet-derived growth factor and pervanadate, phosphatidyl inositol 3-kinase dependent, and inhibited by specific immunodepletion with anti-Akt antisera. Furthermore, direct assays of crude extracts from a range of cell types using this peptide were consistent with the results obtained using specific immunoprecipitation assays.
KW - Akt
KW - Peptide phosphorylation
KW - Protein kinase B
KW - Substrate specificity
UR - http://www.scopus.com/inward/record.url?scp=0036605838&partnerID=8YFLogxK
U2 - 10.1006/abio.2002.5659
DO - 10.1006/abio.2002.5659
M3 - Article
C2 - 12018943
AN - SCOPUS:0036605838
SN - 0003-2697
VL - 305
SP - 32
EP - 39
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 1
ER -