Calcium-Dependent Protein Kinases from Arabidopsis show substrate specificity differences in an analysis of 103 substrates
The identification of substrates represents a critical challenge for understanding any protein kinase-based signal transduction pathway. In Arabidopsis, there are more than 1000 different protein kinases, 34 of which belong to a family of Ca2+-dependent protein kinases (CPKs). While CPKs are impl...
| Main Authors: | , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2011-08-01
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| Series: | Frontiers in Plant Science |
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| Online Access: | http://journal.frontiersin.org/Journal/10.3389/fpls.2011.00036/full |
| _version_ | 1818570881287323648 |
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| author | Amy eCurran Ing-Feng eChang Chia-Lun eChang Shilpi eGarg Rodriguez Milla Miguel Yoshimi D Barron Ying eLi Shawn eRomanowsky John Chandler Cushman Michael eGribskov Alice eHarmon Jeffrey F eHarper |
| author_facet | Amy eCurran Ing-Feng eChang Chia-Lun eChang Shilpi eGarg Rodriguez Milla Miguel Yoshimi D Barron Ying eLi Shawn eRomanowsky John Chandler Cushman Michael eGribskov Alice eHarmon Jeffrey F eHarper |
| author_sort | Amy eCurran |
| collection | DOAJ |
| description | The identification of substrates represents a critical challenge for understanding any protein kinase-based signal transduction pathway. In Arabidopsis, there are more than 1000 different protein kinases, 34 of which belong to a family of Ca2+-dependent protein kinases (CPKs). While CPKs are implicated in regulating diverse aspects of plant biology, from ion transport to transcription, relatively little is known about isoform-specific differences in substrate specificity, or the number of phosphorylation targets. Here, in vitro kinase assays were used to compare phosphorylation targets of four CPKs from Arabidopsis (CPK1, 10, 16 and 34). Significant differences in substrate specificity for each kinase were revealed by assays using 103 different substrates. For example CPK16 phosphorylated Serine 109 in a peptide from the stress-regulated protein, Di19-2 with KM ~70 µM, but this site was not phosphorylated significantly by CPKs 1, 10, or 34. In contrast, CPKs 1, 10, and 34 phosphorylated 93 other peptide substrates not recognized by CPK16. Examples of substrate specificity differences among all four CPKs were verified by kinetic analyses. To test the correlation between in vivo phosphorylation events and in vitro kinase activities, assays were performed with 274 synthetic peptides that contained phosphorylation sites previously mapped in proteins isolated from plants (in vivo-mapped sites). Of these, 74 (27%) were found to be phosphorylated by at least one of the four CPKs tested. This 27% success rate validates a robust strategy for linking the activities of specific kinases, such as CPKs, to the thousands of in planta phosphorylation sites that are being uncovered by emerging technologies. |
| first_indexed | 2024-12-14T13:47:46Z |
| format | Article |
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| institution | Directory Open Access Journal |
| issn | 1664-462X |
| language | English |
| last_indexed | 2024-12-14T13:47:46Z |
| publishDate | 2011-08-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Plant Science |
| spelling | doaj.art-2e48043c6d45488f97480ba2d198e7732022-12-21T22:59:15ZengFrontiers Media S.A.Frontiers in Plant Science1664-462X2011-08-01210.3389/fpls.2011.0003610775Calcium-Dependent Protein Kinases from Arabidopsis show substrate specificity differences in an analysis of 103 substratesAmy eCurran0Ing-Feng eChang1Chia-Lun eChang2Shilpi eGarg3Rodriguez Milla Miguel4Yoshimi D Barron5Ying eLi6Shawn eRomanowsky7John Chandler Cushman8Michael eGribskov9Alice eHarmon10Jeffrey F eHarper11University of Nevada, RenoNational Taiwan UniversityNational Taiwan UniversityUniversity of Nevada, RenoCentro de Investigaciones Biológicas–CSICUniversity of Nevada, RenoUniversity of Nevada, RenoUniversity of Nevada, RenoUniversity of Nevada, RenoPurdue UniversityU of FloridaUniversity of Nevada, RenoThe identification of substrates represents a critical challenge for understanding any protein kinase-based signal transduction pathway. In Arabidopsis, there are more than 1000 different protein kinases, 34 of which belong to a family of Ca2+-dependent protein kinases (CPKs). While CPKs are implicated in regulating diverse aspects of plant biology, from ion transport to transcription, relatively little is known about isoform-specific differences in substrate specificity, or the number of phosphorylation targets. Here, in vitro kinase assays were used to compare phosphorylation targets of four CPKs from Arabidopsis (CPK1, 10, 16 and 34). Significant differences in substrate specificity for each kinase were revealed by assays using 103 different substrates. For example CPK16 phosphorylated Serine 109 in a peptide from the stress-regulated protein, Di19-2 with KM ~70 µM, but this site was not phosphorylated significantly by CPKs 1, 10, or 34. In contrast, CPKs 1, 10, and 34 phosphorylated 93 other peptide substrates not recognized by CPK16. Examples of substrate specificity differences among all four CPKs were verified by kinetic analyses. To test the correlation between in vivo phosphorylation events and in vitro kinase activities, assays were performed with 274 synthetic peptides that contained phosphorylation sites previously mapped in proteins isolated from plants (in vivo-mapped sites). Of these, 74 (27%) were found to be phosphorylated by at least one of the four CPKs tested. This 27% success rate validates a robust strategy for linking the activities of specific kinases, such as CPKs, to the thousands of in planta phosphorylation sites that are being uncovered by emerging technologies.http://journal.frontiersin.org/Journal/10.3389/fpls.2011.00036/fullPhosphorylationSubstrate SpecificityCalcium Dependent Protein KinaseCPK16phospho-proteomics |
| spellingShingle | Amy eCurran Ing-Feng eChang Chia-Lun eChang Shilpi eGarg Rodriguez Milla Miguel Yoshimi D Barron Ying eLi Shawn eRomanowsky John Chandler Cushman Michael eGribskov Alice eHarmon Jeffrey F eHarper Calcium-Dependent Protein Kinases from Arabidopsis show substrate specificity differences in an analysis of 103 substrates Frontiers in Plant Science Phosphorylation Substrate Specificity Calcium Dependent Protein Kinase CPK16 phospho-proteomics |
| title | Calcium-Dependent Protein Kinases from Arabidopsis show substrate specificity differences in an analysis of 103 substrates |
| title_full | Calcium-Dependent Protein Kinases from Arabidopsis show substrate specificity differences in an analysis of 103 substrates |
| title_fullStr | Calcium-Dependent Protein Kinases from Arabidopsis show substrate specificity differences in an analysis of 103 substrates |
| title_full_unstemmed | Calcium-Dependent Protein Kinases from Arabidopsis show substrate specificity differences in an analysis of 103 substrates |
| title_short | Calcium-Dependent Protein Kinases from Arabidopsis show substrate specificity differences in an analysis of 103 substrates |
| title_sort | calcium dependent protein kinases from arabidopsis show substrate specificity differences in an analysis of 103 substrates |
| topic | Phosphorylation Substrate Specificity Calcium Dependent Protein Kinase CPK16 phospho-proteomics |
| url | http://journal.frontiersin.org/Journal/10.3389/fpls.2011.00036/full |
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