Biochemical characterization of calmodulin isoforms and identification of calmodulin-binding proteins in Arabidopsis

A central question in plant biology is how plant cells discriminate among diverse stimuli to generate precise physiological responses when many stimuli and their responses are coupled by the same signal of changes in intracellular Ca$\sp{2+}$ concentration. The identification of four calmodulin (CaM) isoforms in Arabidopsis provides a good opportunity to address this fundamental question. The goal of this research was to address whether CaM isoforms confer specificity to Ca$\sp{2+}$-mediated signal transduction pathways. A combination of in vitro biochemical enzyme activation and molecular biological approaches were employed to tackle this question. First, CaM isoforms produced from E. coli expression systems were used to test their abilities to activate pea NAD kinase. There was a small, but significant difference among the CaM isoforms' ability to activate pea NAD kinase in vitro. Minor differences in K$\sb{0.5}$, but not V$\sb{\rm max}$, were observed between CaM isoforms and chimeric proteins tagged at their N-termini with a 12-residue c-myc epitope. Nonsense mutations of the C-terminal 7 residues of CaM-6 (CaM-6M), however, drastically impaired the ability of the protein to activate NAD kinase. The requirement for free Ca$\sp{2+}$ to activate NAD kinase by wt and mutant CaM-6 proteins was identical. In contrast, CaM-6M bound synthetic peptide substrates with lower apparent affinity than CaM-6. Because of the importance of NAD kinase as a model system for studying CaM structure and function relationships, a systematic series of experiments were performed to purify NAD kinase with the goal of obtaining direct amino acid sequence information. The method reported here yielded higher purity of the enzyme ($\sim$5000 fold) with enhanced enzyme stability than any previously published methods. Methods of detecting CaM-binding proteins (CaM-BPs) were optimized by conjugating several reporter molecules CaM. The feasibilities of conjugates as ligand probes for detecting CaM-BPs in protein fractions after separation in SDS-PAGE and in screening cDNA expression library were evaluated. Thirty-two clones encoding putative CaM-BPs were isolated from an Arabidopsis flower library. Eighteen clones were partially sequenced. Data base comparisons revealed that some of the sequences shared significant homology with enzymes involved in cell division, phospholipid signaling and other cellular process.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Plant Biology

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2011

Author dc:creator

• Liao, Birong

Contributors dc:contributor

• Zielinski, Raymond E.

• Biology, Molecular
• Biology, Cell
• Biology, Plant Physiology

Statement dc:rights

• Copyright 1996 Liao, Birong

Language dc:language

eng

Identifier

9780591199178
AAI9712352
(UMI)AAI9712352

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/23354