| Autor: |
Ahsan N; Division of Biochemistry, Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA.; Department of Chemistry and Biochemistry, Mass Spectrometry, Proteomics and Metabolomics Core Facility, Stephenson Life Sciences Research Center, The University of Oklahoma, Norman, OK 73019, USA., Kataya ARA; Division of Biochemistry, Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA., Rao RSP; Division of Biochemistry, Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA.; Center for Bioinformatics, NITTE Deemed to be University, Mangaluru 575018, India., Swatek KN; Division of Biochemistry, Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA.; Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, UK., Wilson RS; Division of Biochemistry, Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA.; Arvinas, Inc., New Haven, CT 06511, USA., Meyer LJ; Division of Biochemistry, Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA.; Bayer Crop Science, St. Louis, MO 63141, USA., Tovar-Mendez A; Division of Biochemistry, Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA.; Elemental Enzymes, St. Louis, MO 63132, USA., Stevenson S; Division of Biochemistry, Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA., Maszkowska J; Institute of Biochemistry and Biophysics, Polish Academy of Sciences, ul. Pawińskiego 5a, 02-106 Warsaw, Poland., Dobrowolska G; Institute of Biochemistry and Biophysics, Polish Academy of Sciences, ul. Pawińskiego 5a, 02-106 Warsaw, Poland., Yao Q; Department of Electrical Engineering & Computer Science, Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA., Xu D; Department of Electrical Engineering & Computer Science, Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA., Thelen JJ; Division of Biochemistry, Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA. |
| Abstrakt: |
Members of the calcium-dependent protein kinase (CDPK/CPK) and SNF-related protein kinase (SnRK) superfamilies are commonly found in plants and some protists. Our knowledge of client specificity of the members of this superfamily is fragmentary. As this family is represented by over 30 members in Arabidopsis thaliana , the identification of kinase-specific and overlapping client relationships is crucial to our understanding the nuances of this large family of kinases as directed towards signal transduction pathways. Herein, we used the kinase client (KiC) assay-a relative, quantitative, high-throughput mass spectrometry-based in vitro phosphorylation assay-to identify and characterize potential CPK/SnRK targets of Arabidopsis. Eight CPKs (1, 3, 6, 8, 17, 24, 28, and 32), four SnRKs (subclass 1 and 2), and PPCK1 and PPCK2 were screened against a synthetic peptide library that contains 2095 peptides and 2661 known phosphorylation sites. A total of 625 in vitro phosphorylation sites corresponding to 203 non-redundant proteins were identified. The most promiscuous kinase, CPK17, had 105 candidate target proteins, many of which had already been discovered. Sequence analysis of the identified phosphopeptides revealed four motifs: LxRxxS, RxxSxxR, RxxS, and LxxxxS, that were significantly enriched among CPK/SnRK clients. The results provide insight into both CPK- and SnRK-specific and overlapping signaling network architectures and recapitulate many known in vivo relationships validating this large-scale approach towards discovering kinase targets. |
