| NSF Org: |
MCB Division of Molecular and Cellular Biosciences |
| Recipient: |
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| Initial Amendment Date: | August 1, 1997 |
| Latest Amendment Date: | November 8, 1999 |
| Award Number: | 9723539 |
| Award Instrument: | Continuing Grant |
| Program Manager: |
Randolph Addison
MCB Division of Molecular and Cellular Biosciences BIO Directorate for Biological Sciences |
| Start Date: | August 1, 1997 |
| End Date: | July 31, 2001 (Estimated) |
| Total Intended Award Amount: | $371,850.00 |
| Total Awarded Amount to Date: | $371,850.00 |
| Funds Obligated to Date: |
FY 1998 = $120,000.00 FY 1999 = $120,000.00 FY 2000 = $5,000.00 |
| History of Investigator: |
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| Recipient Sponsored Research Office: |
10550 N TORREY PINES RD LA JOLLA CA US 92037-1000 (858)784-8653 |
| Sponsor Congressional District: |
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| Primary Place of Performance: |
10550 N TORREY PINES RD LA JOLLA CA US 92037-1000 |
| Primary Place of
Performance Congressional District: |
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| Unique Entity Identifier (UEI): |
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| Parent UEI: |
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| NSF Program(s): | SIGNAL TRANSDCTN/CELL REGULATN |
| Primary Program Source: |
app-0197 app-0198 app-0199 |
| Program Reference Code(s): |
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| Program Element Code(s): |
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| Award Agency Code: | 4900 |
| Fund Agency Code: | 4900 |
| Assistance Listing Number(s): | 47.074 |
ABSTRACT
9723539 Harper The long range goal of the proposed research is to understand the role of Ca2+-Dependent Protein Kinases (CDPKs) in plant growth and development. Our approach is to first understand the structural basis for how CDPKs decode and transduce Ca2+ signals, and to use that information to develop tools for investigating their in vivo functions. Isoform CPK-1 from Arabidopsis is used here as a model CDPK. CDPKs are unique because of their structural arrangement: Within a single polypeptide, a kinase is fused to a C-terminal calmodulin-like regulatory domain (CaM-LD). The CaM-LD contains four Ca2+ -binding EF-hands, which makes CDPKs a direct target (decoder) of Ca2+ signals. Prior NSF supported research indicated that kinase activation involves intramolecular binding between the CaM-LD and the adjacent junction (autoinhibitory) domain. The current research builds on that observation and is divided into the following 3 objectives: 1. Test the hypothesis that the CaM-LD is always bound to the junction (even in the absence of Ca2+), and that Ca2+ triggers a change in this complex which functions to disengage the autoinhibitor (i.e. activate the kinase). The approach is to use multi-dimensional NMR to solve the structure of an isolated junction/CaM-LD protein in the presence and absence of Ca2+. 2. Test the hypothesis that the sequence (tether) which connects the CaM-LD to its upstream binding sequence provides an important structural constraint, (i.e. the tether is not just a simple flexible linker between two domains). The approach is to increase the length and flexibility of the tether by site specific mutagenesis and evaluate the impact of these changes on the Ca2+ activation mechanism. 3. Screen for a temperature sensitive kinase mutant which becomes Ca2+ independent at high temperatures (e.g. 25 oC). The approach is to introduce mutations which weaken the binding of the pseudosubstrate autoinhibitor to the kinase, thereby making the autoinhibitor more sensitive to therma l destabilization. The purpose is to obtain a temperature sensitive kinase which can be introduced into transgenic plants and used to conditionally activate a CDPK pathway. The primary significance of the proposed research should come from insights obtained from the 3 dimensional structures of an apo and Ca2+ loaded Junction/CaM-LD complex. The short term goal is to use these structures to propose detailed models on how Ca2+ activates a CDPK, and to test alternative models using a molecular genetic and biochemical approach. Many extrinsic signals that are perceived by cells at the cell surface signal that induce physiological and developmental events. One of most widely used signaling systems in living cells is the calcium-dependent-protein kinase CDPK in which a calcium signal results in the CDPK attaching a phosphate to a target protein transmitting the calcium signal into a modification of a protein that usually alters its activity. How the CDPK functions is of fundamental importance in elucidating the perception of signals by cells. This award will fund the collaborative research by experts in the protein kinase field and the nuclear magnetic resonance field (NMR). NMR is useful to determine the exact structure of proteins. The research to be conducted will elucidate the changes in structure of CDPK that occur during its function.
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