Doctoral Dissertations

Suk-Heung Oh

Date of Award

5-1992

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Daniel M. Roberts

Committee Members

Jayant G. Joshi, Elizabeth E. Howell, Gary Stacey

Abstract

A recombinant DNA-derived calmodulin (VU-1, lys 115) was used as a substrate to purify a specific calmodulin-lysine N-methyltransferase to homogeneity from sheep brain. The enzyme is a monomer with an apparent molecular weight of 38,000. The enzyme is highly specific for lysine 115 of calmodulin since the site-directed mutants of calmodulin with substitutions at this position (VU-3, lys to arg, 115; VU-4, lys to ile, 115) were unable to be methylated. These calmodulins were also strong competitive inhibitors of methylation. Substrates of different protein methyltransferases also were not methylated by the enzyme. The enzyme requires μ calcium for activity, suggesting that the calcium bound form of calmodulin is preferred as a substrate.

A radiometric assay based on the purified calmodulin­ lysine N-methyltransferase has been developed to analyze the degree of methylation of calmodulins from developing plant tissues. By this approach, the presence of undermethylated calmodulin from plant tissues was detected. Further, there are developmental differences in calmodulin methylation in plants. Calmodulin methylation levels were lower in apical root segments and in the young lateral roots compared with the mature, differentiated root tissues. The endogenous methylation of calmodulin from carrot culture cells in the early exponential stage of growth is substantially lower than that from non-proliferating cells in the stationary stage. The level of endogenous calmodulin methylation rapidly decreases upon inoculation of these cells into fresh media. Calmodulin protein levels were similar in cells from exponential and stationary stages. These findings suggest that calmodulin methyltransferase activity is regulated depending upon the growth or developmental stage, and/or that other factors, such as the availability of co-substrate, s­ adenosyl methionine and calcium ion fluxes, are involved in controlling calmodulin methylation during plant cell growth and development. The data also suggest that proliferating cells have lower endogenous methylation of lysine 115 of calmodulin.

We utilized carrot embryogenesis to study the calmodulin system in undifferentiated tissues vs. differentiated tissues. Calmodulin, calmodulin methylation, and calmodulin-binding proteins were analyzed during the formation and germination of embryos. During the germination of carrot embryos, the level of calmodulin was doubled and calmodulin binding to a 54,000 Mr protein increased substantially. The changes in calmodulin methylation were not as drastic as those seen in the proliferating cells. The 54,000 Mr calmodulin-binding protein was purified by using chromatography on calmodulin-Sepharose. These data suggest that changes in the calmodulin regulatory pathway occur during the germination of carrot embryos. These changes may reflect an altered role of calcium signal transduction during early growth and development of germinating embryos.

In order to address questions of the biological significance of calmodulin methylation in plants, transgenic tobacco plants expressing VU-1, VU-3 or VU-4 calmodulin have been generated. The VU-calmodulins were stably expressed in transgenic tobacco plants. The expression levels in transgenic tobacco tissues were maximally twice that of endogenous calmodulin. As expected, the transgenic VU-1 calmodulin is methylated at position 115, similar to the endogenous tobacco calmodulin. However, VU-3 calmodulin is incapable of methylation. These differences in posttranslational modification result in altered plant NAO kinase activation, with VU-3 calmodulin activating the enzyme to a level three-fold higher than transgenic VU-1 and endogenous tobacco calmodulins. As previously shown by Besl (1991), severe defects in growth and development (i.e., dwarfism and low seed yield) were observed in tobacco plants that express VU-3 calmodulin. The same abnormalities in growth and development were observed with 20% of the F0 VU-4 transgenic plants.

Overall, the data show unmethylated calmodulins exist in vivo, and that the methylation process is controlled developmentally. Combined with the transgenic plant and NAO kinase activator data, the results suggest that post translational methylation of calmodulin could be a mechanism for regulating calmodulin activities in vivo.

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