Novozhylov D. Search for Ca2+-dependent protein kinases related to plant microtubules and elucidation of their role in tubulin phosphorylation

The initial search was performed based on the homology of A. thaliana calcium-dependent protein (CDPK) and calmodulin-dependent kinases (CRK, CDPK-related kinases) and animal protein kinases involved in the regulation of the cytoskeleton according to the literature data. The search was performed by N-J (Neighbor-Joining) clustering of catalytic domain sequences as the most evolutionarily conservative structures. The closest homologues for the CaMK2 protein kinase - CPK7, CPK14, CPK32 and CPK21; for protein kinases RSK - CPK17, CPK34, CRK2; for protein kinases DAPK - CPK20, CPK27; for protein kinase CHK2 - CPK16, CPK18, CPK28, CRK4 and CRK6. Based on the created profiles of phosphorylation sites, consensus sequences were found in the sequences of various isotypes of A. thaliana tubulins, which made it possible to determine protein kinases capable of phosphorylating plant tubulin - CaMK1A, CaMK2A, CaMKK2 from H. sapiens and CaMK2A from R. norvegicus. Their closest homologues among A. thaliana protein kinases were determined: CPK20, CPK21, and GRIK2. The possibility of using calmodulin-targeted CaMK2 protein kinase inhibitors to suppress the activity of plant Ca2+-dependent protein kinases was analyzed. The presence of a potential binding site for inhibitors KN-93 and KN-62 in the structure of the Ca2+-binding domain of CPK1 from A. thaliana, similar to that in the structure of calmodulin 1 from H. sapiens, was determined, and the similarity of the amino acid environment of these sites for potential ligands was proved. Molecular docking has shown the presence of energetically favorable binding sites for KN-93 and KN-62 in the structures of human calmodulin and calcium-dependent protein kinase 1 of arabidopsis, which makes it possible to propose KN-93 and KN-62 as inhibitors of plant protein kinases of the CDPK subfamily.