Monitoring Protein Kinases in Cellular Media with Highly Selective Chimeric Reporters
Protein 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...
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| Format: | Article |
| Language: | en_US |
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Wiley-Blackwell Pubishers
2012
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| Online Access: | http://hdl.handle.net/1721.1/69585 https://orcid.org/0000-0002-5749-7869 https://orcid.org/0000-0002-8121-9519 |
| _version_ | 1811079004607741952 |
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| author | Lukovic, Elvedin Taylor, Elizabeth Vogel Imperiali, Barbara |
| author2 | Massachusetts Institute of Technology. Department of Biology |
| author_facet | Massachusetts Institute of Technology. Department of Biology Lukovic, Elvedin Taylor, Elizabeth Vogel Imperiali, Barbara |
| author_sort | Lukovic, Elvedin |
| collection | MIT |
| description | Protein 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. |
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| id | mit-1721.1/69585 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T11:08:39Z |
| publishDate | 2012 |
| publisher | Wiley-Blackwell Pubishers |
| record_format | dspace |
| spelling | mit-1721.1/695852022-10-01T01:35:03Z Monitoring Protein Kinases in Cellular Media with Highly Selective Chimeric Reporters Lukovic, Elvedin Taylor, Elizabeth Vogel Imperiali, Barbara Massachusetts Institute of Technology. Department of Biology Massachusetts Institute of Technology. Department of Chemistry Imperiali, Barbara Imperiali, Barbara Lukovic, Elvedin Taylor, Elizabeth Vogel Protein 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. National Institutes of Health (U.S.) (NIH Cell Migration Consortium (GM064346)) 2012-03-02T20:15:55Z 2012-03-02T20:15:55Z 2009-08 Article http://purl.org/eprint/type/JournalArticle 0044-8249 1521-3757 http://hdl.handle.net/1721.1/69585 Luković, 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. https://orcid.org/0000-0002-5749-7869 https://orcid.org/0000-0002-8121-9519 en_US http://dx.doi.org/10.1002/ange.200902374 Angewandte Chemie Creative Commons Attribution-Noncommercial-Share Alike 3.0 http://creativecommons.org/licenses/by-nc-sa/3.0/ application/pdf Wiley-Blackwell Pubishers Prof. Imperiali via Erja Kajosalo |
| spellingShingle | Lukovic, Elvedin Taylor, Elizabeth Vogel Imperiali, Barbara Monitoring Protein Kinases in Cellular Media with Highly Selective Chimeric Reporters |
| title | Monitoring Protein Kinases in Cellular Media with Highly Selective Chimeric Reporters |
| title_full | Monitoring Protein Kinases in Cellular Media with Highly Selective Chimeric Reporters |
| title_fullStr | Monitoring Protein Kinases in Cellular Media with Highly Selective Chimeric Reporters |
| title_full_unstemmed | Monitoring Protein Kinases in Cellular Media with Highly Selective Chimeric Reporters |
| title_short | Monitoring Protein Kinases in Cellular Media with Highly Selective Chimeric Reporters |
| title_sort | monitoring protein kinases in cellular media with highly selective chimeric reporters |
| url | http://hdl.handle.net/1721.1/69585 https://orcid.org/0000-0002-5749-7869 https://orcid.org/0000-0002-8121-9519 |
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