Abstract：
:New methods to quantify protein kinase activities directly from complex cellular mixtures are critical for understanding biological regulatory pathways. Herein, a fluorescence-based chemosensor strategy for the direct measurement of kinase activities in crude mammalian cell lysates is described. We first designed a new fluorescent peptide reporter substrate for each target kinase. These kinase chemosensors were readily phosphorylated by recombinant target enzyme and underwent a several-fold fluorescence increase upon phosphorylation. Then, using unfractionated cell lysates, a homogeneous kinase assay was developed that was reproducible, linear and highly preferential for monitoring changes in cellular activity of the target kinase. The general protocol was developed for the kinase Akt and then easily extended to measure protein kinase A (PKA) and mitogen-activated protein kinase-associated protein kinase 2 (MK2) activities. This assay platform is immediately useful for studying protein kinase signaling in crude cellular extracts.
journal_name
Nat Methodsjournal_title
Nature methodsauthors
Shults MD,Janes KA,Lauffenburger DA,Imperiali Bdoi
10.1038/nmeth747keywords:
subject
Has Abstractpub_date
2005-04-01 00:00:00pages
277-83issue
4eissn
1548-7091issn
1548-7105pii
nmeth747journal_volume
2pub_type
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