NSF 2010 Project MCB 0114769
PI: Alice Harmon, University of Florida
Co-PI: John Cushman, University of Nevada, Reno
Co-PI: Jeffery Harper, The Scripps Research Institiute
Co-PI: Estelle Hrabak, University of New Hampshire
Co-PI: Michael Sussman, Universtiy of Wisconsin, Madison
Abstract
Protein phosphorylation is a major mode of regulation of metabolism, gene expression, and cellular architecture in eukaryotic cells, and defining phosphorylation-based regulatory networks is essential for understanding the function of the Arabidopsis genome. The goal of this project is to define phosphorylation networks that are related to the function of calcium-dependent protein kinases (CDPKs) and four closely related families; CDPK-related kinases (CRKs), phosphoenolpyruvate carboxylase kinases (PPCKs), PPCK-related kinases (PEPRKs), and SNF-1 related kinases (SnRKs). These 84 kinases, which represent about 9% of all the protein kinases in the Arabidopsis genome, are involved in all aspects of plant development and physiology and participate in the coupling of cellular responses to environmental and developmental signals. A list of these protein kinases including gene identification numbers is available below and at http://www.arabidopsis.org. Further information on the genes including links to database records is available at http://plantsp.genomics.purdue.edu. This research will investigate the function of 64 members of these families through determination of the subcellular location of each kinase and identification of downstream targets and other proteins with which the kinases associate. This information will give insight into the physiological roles of each kinase by identifying signaling networks in which each participates. This research will also determine the target sequences in substrate proteins that are phosphorylated by each kinase, and these results will contribute to understanding the overlap in kinase function and cross-talk between signaling pathways. The results of this work will be made available on a yearly basis at the two URLs given above.
For educational outreach, we will develop lectures, class materials, and virtual laboratory exercises on the subjects of plant protein phosphorylation and genomics, for use in junior colleges. We will conduct a pilot study in which the effectiveness of these materials is evaluated.
Return to indexSpecific Goals
1. Identify kinase substrates using substrate traps. Three major approaches will be pursued: a) Use a proven CDPK substrate trap design to identify potential substrates in a yeast two-hybrid system; b) Develop and utilize alternative substrate trap designs of general application to all serine/threonine (Ser/Thr) kinases: c) Evaluate alternative interaction screens..
Deliverables
A. Identification of substrates and other proteins that interact with 64 CDPK superfamily members and with general applicability to the entire Arabidopsis proteome.
B. Development and evaluation of substrate trap designs of general applicability to all Ser/Thr kinases for identification of substrates/interacting proteins in planta.
C. Evaluation of alternative bacterial and yeast two-hybrid systems for high-throughput implementation.
2. Determine the subcellular locations of membrane-associated or compartmentalized kinases. Our approach will be to co-express and visualize by confocal microscopy both kinases tagged with yellow or green fluorescent protein and markers specific for different membrane or cytoskeletal proteins tagged with cyan or red fluorescent protein.
Deliverables
A. A set of targeted reporters for co-visualization controls tagged with DsRFP and CFP.
B. 64 YFP or GFP tagged kinases.
C. Subcellular locations determined by cytology for 64 kinases.
3. Use mass spectrometry to identify substrates phosphorylated by kinases in vitro and map the phosphorylation sites in substrates and kinases.
Deliverables.
C. Establishment of mass spectrometry facilities that, in the future, will be available to the plant science community for the analysis of phosphoproteins.
4. Develop lectures, class materials, and web-based "virtual laboratory" exercises related to the subject of plant protein kinases and genomics and targeted for community colleges.
Deliverables.
A. Class materials for a 6 week module to be incorporated into introductory biology or botany courses.
B. Pilot study on the use of the teaching materials in community colleges and evaluation of their effectiveness.
Gene Tables for the Arabidopsis CDPK/SnRK Gene Family
Accession numbers are gi numbers for protein records derived from genomic sequences except for those marked with #, which are protein records derived from cDNA sequences. Sequences that are incorrectly annotated in GenBank are marked with asterisks. The correct annotation for these sequences is available at http://plantsp.genomics.purdue.edu.
Table 1. Calcium-Dependent Protein Kinases (CPKs)
Table 2. CPK-Related Kinases (CRKs)
| Gene Family Name | PlantsP ID | Genbank accession | Standard gene name |
| CRK1 | 21623 | 3402722 | At2g41140 |
| CRK2 | 21712 | 9294612 | At3g19100 |
| CRK3 | 21571 | 3831444 | At2g46700 |
| CRK4 | 21984 | 10177904 | AT5g24430 |
| CRK5 | 21757 | 6561993 | At3g50530 |
| CRK6 | 21752 | 6553918 | At3g49370 |
| CRK7 | 21790 | 9662995 | At3g56760 |
| CRK8 | 21393 | 10120419 | At1g49580 |
Table 3. PEP Carboxylase Kinases (PPCKs)
| Gene Family Name | PlantsP ID | Genbank accession | Standard gene name |
| PPCK1 | 21409 | 6318613 | At1g08650 |
| PPCK2 | 21203 | 6175181 | At3g04530 |
Table 4. PPCK-Related Kinases (PEPRKs)
| Gene Family Name | PlantsP ID | Genbank accession | Standard gene name |
| PEPRK1 | 21496 | 8778638 | At1g12580 |
| PEPRK2 | 21141 | none | At1g12680 |
Table 5. SnF1-Related Kinases (SnRKs)
Naming convention: SnRKx.y, where x is the subgroup and y is the gene number.