Recombinant Lilium longiflorum Calcium and calcium/calmodulin-dependent serine/threonine-protein kinase (CCAMK)

Overview of CCAMK

Calcium/calmodulin-dependent protein kinase (CCaMK) is a serine/threonine-protein kinase that is unique to plants . Lilium longiflorum CCaMK (LlCCaMK) shares high sequence homology with CaMKII found in mammals, particularly in the kinase and CaM-binding domains . The full-length cDNA of DoCCaMK is 2071 bp long . The Lilium longiflorum CCaMK (Patil et al., 1995) has been characterized .

Structure and Properties

Lilium longiflorum CCaMK is a protein that consists of 520 amino acids . As deduced by Compute pI/MW, DoCCaMK has 514 amino acids, its isoelectric point is 5.92, and its molecular weight is 57.51 kDa . CCaMK contains several key domains :

• An N-terminal Ser-Thr kinase domain

• An autoinhibitory domain

• A CaM-binding domain

• A C-terminal neural visinin-like domain with three EF hands

Expression and Localization

CCaMK expression is significantly upregulated after symbiosis with Sebacina sp . The DoCCaMK-GFP protein localized in the nucleus and cell membrane .

Regulation of CCaMK

CCaMK is regulated by calcium in both negative and positive manners, creating a molecular switch sensitive to calcium concentrations associated with basal states and oscillations .

• Autophosphorylation: Calcium binding to the EF-hand domains promotes autophosphorylation, which negatively regulates CCaMK by stabilizing the inactive state of the protein .

• Calmodulin Binding: Calcium-dependent CaM binding overrides the effects of autophosphorylation and activates the protein . The differential calcium binding affinities of the EF-hand domains compared with those of CaM suggest that CCaMK is maintained in the inactive state at basal calcium concentrations and is activated via CaM binding during calcium oscillations .

Function

CCaMK is involved in several pathways and plays different roles in them . CIP73 transcripts are preferentially expressed in roots, and very low expression is detected in leaves, stems, and nodules . The expression in roots is significantly decreased after inoculation of Mesorhizobium loti . RNA interference knockdown of CIP73 expression by hairy root transformation in Lotus japonicus led to decreased nodule formation, suggesting that CIP73 performed an essential role in nodulation .

Interactions

CCaMK interacts with proteins and molecules, as detected by methods such as yeast two-hybrid, co-IP, and pull-down assays . The amino-terminal 80 amino acid residues (80–160) of CCaMK are required for interacting with CIP73 in yeast cells . Protein pull-down assay and bimolecular fluorescence complementation assay in Nicotiana benthamiana show that the full-length CCaMK could interact with CIP73 in vitro and in planta . CCaMK phosphorylates the amino terminus of CIP73 in a Ca2+/calmodulin-dependent manner in vitro .