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dc.contributor.authorLukovic, Elvedin
dc.contributor.authorTaylor, Elizabeth Vogel
dc.contributor.authorImperiali, Barbara
dc.date.accessioned2012-03-02T20:15:55Z
dc.date.available2012-03-02T20:15:55Z
dc.date.issued2009-08
dc.identifier.issn0044-8249
dc.identifier.issn1521-3757
dc.identifier.urihttp://hdl.handle.net/1721.1/69585
dc.description.abstractProtein kinases are important regulators of cellular function, and the dynamics of their activities are critical indicators of the health or pathology of living systems.[1, 2] In particular, extracellular-signal regulated kinases 1 and 2 (ERK1/2) play a pivotal role in the mitogen-activated protein kinase (MAPK) signaling pathway responsible for regulated cell survival and proliferation.[3] The centrality of these enzymes in normal and diseased cell states underscores the need for high throughput, selective, and sensitive methods that accurately and directly diagnose kinase activities. The benchmark phosphorylation assays for ERK1/2 rely on transfer of radioactive γ-phosphate of [γ-32P]ATP to peptide or protein substrates.[4] While broadly employed, this approach has limitations, including the discontinuous nature of the radioactive assay and the non-native ATP concentrations that are utilized. Alternatively, for cellular imaging, genetically-encoded sensors that rely on phosphorylation-based changes in fluorescence resonance energy transfer (FRET) between fluorescent protein pairs[5, 6] have been constructed for several kinases, including ERK1/2.[7-10] These sensors are powerful because they can be expressed in cells, however, they cannot be used for high throuput screening of recombinant enzymes and unfractionated cell lysates due to the very limited fluorescence changes that accompany phosphorylation. As a complementary approach, probes based on small, organic fluorophores with direct readouts[6, 11] can give sensitive and robust signals under physiogical conditions and are thus amenable to high throughput applications. For example, we have incorporated a sulfonamido-oxine (Sox) chromophore into peptides[12, 13] to report phosphorylation via chelation-enhanced fluorescence (CHEF) (Figure 1a). The weak binding affinity of the unphosphorylated substrate for Mg2+ increases significantly upon phosphorylation, resulting in robust (2- to 12-fold) fluorescence enhancements. This versatile peptide-based sensor design has been applied to monitor the activity of numerous Ser/Thr and Tyr kinases both in vitro[13] and in cell lysates.en_US
dc.description.sponsorshipNational Institutes of Health (U.S.) (NIH Cell Migration Consortium (GM064346))en_US
dc.language.isoen_US
dc.publisherWiley-Blackwell Pubishersen_US
dc.relation.isversionofhttp://dx.doi.org/10.1002/ange.200902374en_US
dc.rightsCreative Commons Attribution-Noncommercial-Share Alike 3.0en_US
dc.rights.urihttp://creativecommons.org/licenses/by-nc-sa/3.0/en_US
dc.sourceProf. Imperiali via Erja Kajosaloen_US
dc.titleMonitoring Protein Kinases in Cellular Media with Highly Selective Chimeric Reportersen_US
dc.typeArticleen_US
dc.identifier.citationLuković, Elvedin, Elizabeth Vogel Taylor, and Barbara Imperiali. “Monitoring Protein Kinases in Cellular Media with Highly Selective Chimeric Reporters.” Angewandte Chemie 121.37 (2009): 6960–6963.en_US
dc.contributor.departmentMassachusetts Institute of Technology. Department of Biologyen_US
dc.contributor.departmentMassachusetts Institute of Technology. Department of Chemistryen_US
dc.contributor.approverImperiali, Barbara
dc.contributor.mitauthorImperiali, Barbara
dc.contributor.mitauthorLukovic, Elvedin
dc.contributor.mitauthorTaylor, Elizabeth Vogel
dc.relation.journalAngewandte Chemieen_US
dc.eprint.versionAuthor's final manuscripten_US
dc.type.urihttp://purl.org/eprint/type/JournalArticleen_US
eprint.statushttp://purl.org/eprint/status/PeerRevieweden_US
dspace.orderedauthorsLuković, Elvedin; Vogel Taylor, Elizabeth; Imperiali, Barbaraen
dc.identifier.orcidhttps://orcid.org/0000-0002-5749-7869
dc.identifier.orcidhttps://orcid.org/0000-0002-8121-9519
mit.licenseOPEN_ACCESS_POLICYen_US


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