Interaction of plant chimeric calcium/calmodulin-dependent protein kinase with a homolog of eukaryotic elongation factor-1α

Abstract

A chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) was previously cloned and characterized in this laboratory. 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-1α1) has high similarity with the eukaryotic elongation factor-1α (EF-1α). CCaMK phosphorylated LlEF-1αl in a Ca2+/calmodulin-dependent manner. The phosphorylation site for CCaMK (Thr-257) was identified by site-directed mutagenesis. Interestingly, Thr- 257 is located in the putative tRNA-binding region of LlEF-1α1. An isoform of Ca2+-dependent protein kinase (CDPK) phosphorylated multiple sites of LIEF-1α1 in a Ca2+-dependent but calmodulin-independent manner. Unlike CDPK, CCaMK phosphorylated only one site, and this site is different from CDPK phosphorylation sites. This suggests that the phosphorylation of EF- 1α1 by these two kinases may have different functional significance. Although the phosphorylation of LIEF-1α1 by CCaMK is Ca2+/calmodulin- dependent, in vitro binding assays revealed that CCaMK binds to LIEF-1α1 in a Ca2+-independent manner. This was further substantiated by coimmunoprecipitation of CCaMK and EF-1α using the protein extract from lily anthers. Dissociation of CCaMK from EF-1α by Ca2+ and phosphorylation of EF-1α by CCaMK in a Ca2+/calmodulin-dependent manner suggests that these interactions may play a role in regulating the biological functions of EF- 1α.