Characterization of Proteins that Interact with CDPK Gamma

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9604647 Harmon Technical Calmodulin-like domain protein kinases (also called calcium-dependent protein kinases or CDPK) are a new family of protein kinases that are directly regulated by calcium. They contain not only a catalytic domain, but also a calcium-binding domain that is similar to calmodulin. These enzymes are maximally stimulated by micromolar concentrations of Ca2+ and are thought to play a role in cellular responses to diverse stimuli such as light, growth regulators, pathogen attack and environmental stress. A large family of CDPKs is present in plants. Since the activity of these enzymes is regulated by free calcium, they are capable of perceiving and transducing intracellular calcium signals evoked by numerous stimuli. However, information about the specific roles of these kinases in signaling pathways is still lacking. Few protein substrates of CDPKs have been identified, and it is unknown if the enzymes interact with regulatory or anchoring/targeting proteins. To identify proteins that are substrates or regulators of CDPK or serve to target them to specific cellular locations, interaction cloning was undertaken. Screening of a soybean cDNA expression library with three 32P-labeled recombinant CDPKs, identified twelve cDNA clones that encode proteins that interact with CDPKg. Eight of these clones encode known proteins, and several are phosphorylated by CDPKg in vitro. Two of the interacting proteins are serine acetyl transferase (SAT) and glutathione S-transferase (GST), which are involved in cysteine synthesis and glutathione metabolism, respectively. Another is polyubiquitin, which is the precursor of monomeric ubiquitin that is involved in protein degradation pathways. Current evidence points to roles for glutathione (a cysteine-containing tripeptide) metabolism and protein degradation in cellular responses to stress or pathogen attack. The objective of this proposal is to characterize the interaction between the proteins identified in our screen and CDPKg in vitro and t o use a soybean cell culture model system to study their interactions and roles in vivo. We will test the hypothesis that CDPKg activity and its interaction with ubiquitin, SAT, GST and other proteins play roles in the response of soybean cells to stress or pathogen attack. These studies are an important step towards identifying specific roles of CDPKg in vivo and may provide insight into cellular components involved in plant defense responses. The objectives of this project are to first characterize the interaction of CDPKg with the proteins identified by interaction cloning. Clones encoding known proteins will be expressed as a fusion proteins with maltose binding protein or another affinity tag. Antibodies to interacting proteins will be made and the subcellular location of the proteins determined. Binding of the interacting proteins to recombinant CDPKg on protein blots and in solution will be characterized. Phosphorylation of substrate proteins will be characterized, and the effect of phosphorylation on these proteins will be determined. Initial work will focus on serine acetyltransferase whose activity is regulated on cultured cells or protoplasts for characterization of the role of CDPKg in vivo. Autophosphorylation of CDPKg and phosphorylation of substrate proteins in cells or protoplasts in response to changes in by phosphorylation. 2. Use a model system based intracellular free calcium concentration induced artificially or in response to stimuli will be measured by immunoprecipitation of proteins metabolically labeled with 32P. The hypothesis that activation of CDPKg and phosphorylation of substrate proteins identified by interaction cloning are involved in cellular responses to stress or pathogen attack will be tested. Initial work will focus on serine acetyltransferase. Nontechnical How environmental changes are detected in order to evoke specific responses in plants is one of the large questions of plant biology for which there is presently little information. Calcium ions appear to have an important role in transmitting the signal from sensors to downstream effects of protein synthesis and alterations. This project will analyze how a calcium-dependent protein kinase attaches phosphate molecules to specific proteins in response to biotic and abiotic stress. This modification of specific proteins is proposed to be a critical step in the stress reponse. This research is important because it will address a fundamental aspect of how plant perceive stress situation and transmit the signal to initiate a response.