Chimeric Calcium/Calmodulin-Dependent Protein Kinase Mediated Signaling in Plants

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This renewal project is focussed on this laboratory's long standing interest in understanding the calcium/calmodulin-mediated signaling in plants. Changes in intracellular calcium concentration can affect a number of physiological processes through the action of calmodulin (CaM), a ubiquitous calcium-binding protein. Calcium/CaM-regulated protein phosphorylation is believed to play a pivotal role in amplifying and diversifying the action of calcium-mediated signals. Although calcium/CaM-dependent protein phosphorylation is implicated in regulating a number of cellular processes in plants, not much is known about calcium/CaM-dependent protein kinases and their role in calcium signaling. This laboratory has cloned a chimeric calcium/CaM-dependent protein kinase (CCaMK) gene from developing anthers (Proc. Natl. Acad. Sci. 92: 4897-4901, 1995, communicated by Professor Edwin Krebs, nobel laureate). CCaMK is characterized by the presence of a kinase domain, an autoinhibitory domain, a CaM-binding domain and a neural visinin-like calcium-binding domain in a single polypeptide. One of the unique features of CCaMK is the presence of a visinin-like calcium-binding domain, an additional calcium sensing mechanism not previously known to exist in kinases (J. Biol. Chem., 271: 8126-8132, 1996; J. Biochem 121: 984-990, 1997). The effect of autophosphorylation of CCaMK and its interaction with CaM was studied by using 35S-labeled CaM and dansylated CaM as probes. The affinity of CCaMK for CaM (CaM trapping) increases over eight-fold upon autophosphorylation. These results suggest the existence of a new state of CCaMK when it has a higher affinity for CaM. CCaMK is expressed in a stage-specific manner in pollen mother cells and tapetal cells during anther development (Planta 209: 161-171, 1999). We hypothesize that CCaMK phosphorylates protein(s) in these target cells in a calcium/CaM-dependent manner and the phosphorylation triggers stage-specific changes during microsporogenesis and microspore maturation. To study the functional significance, transgenic plants carrying the antisense construct of CCaMK were produced. Results indicate that the transgenic plants are male sterile, suggesting an important role for CCaMK in anther development. Based on this work, Washington State University Research Foundation has obtained a U.S. patent entitled 'Compositions and Methods for Production of Male Sterile Plants' and a global patent application is pending. To investigate the biological functions of CCaMK, the yeast two-hybrid system was used to isolate genes encoding proteins that interact with CCaMK. One of the cDNA clones obtained from the screening (LIEF-1 alpha 1) has high similarity with the eukaryotic elongation factor-1 alpha (EF-1 alpha). CCaMK phosphorylates LlEF-1 alpha 1 in a calcium/CaM-dependent manner. A CDPK isoform cloned and characterized in this laboratory (CRPK2) phosphorylates multiple sites of LIEF-1 alpha 1 in a calcium-dependent but CaM-independent manner. Unlike CDPK, CCaMK phosphorylates only one site (Thr-257), and this site is different from CDPK phosphorylation sites. Interestingly, Thr-257 is located in the putative tRNA-binding region of LIEF-1 alpha 1 (J. Biol. Chem., 274: 12001-12008, 1999). We propose to continue this investigation by emphasizing the following: the functional significance of CCaMK in anther development by characterizing antisense plants (T1, T2, T3 and T4 generations) and by obtaining T-DNA insertion mutants; protein-protein interaction between CCaMK and its target proteins in pollen mother cells and tapetal cells; and the structure/function relationships of CCaMK, especially the role of the visinin-like calcium-binding domain in autophosphorylation and substrate phosphorylation. A better understanding of the regulation and function of CCaMK will increase our knowledge of calcium/CaM-mediated signaling in plants while at the same time promoting teaching, training and learning at all levels.