Characterization of molecular mechanisms of CaMKKα/1 oligomerization.

Abstract

Calcium/calmodulin-dependent protein kinase kinase (CaMKK) is an activating kinase for calcium/calmodulin-dependent protein kinase type 1 (CaMKI), calcium/calmodulin-dependent protein kinase type IV (CaMKIV), RAC-alpha serine/threonine-protein kinase (PKB), and AMP-activated protein kinase (AMPK) that has been reported to form an active oligomer in cells. Glutathione S-transferase (GST) pulldown assay from the extracts of COS-7 cells expressing GST- and His₆-CaMKKα/1 mutants showed that the C-terminal region containing the autoinhibitory and calmodulin (CaM)-binding sequence (residues 438-463) is required for CaMKKα/1 homo-oligomerization. This was confirmed by the fact that the GST-CaMKKα/1 C-terminal domain (residues 435-505) directly interacted with EGFP-CaMKKα/1 residues 435-505 as well as with wild-type CaMKKα/1. Notably, once oligomerized in cells, CaMKKα/1 is neither exchangeable between the oligomeric complexes nor dissociated by Ca²⁺/CaM binding. These results support stable oligomerization of CaMKK in the cells by intermolecular self-association of its C-terminal region containing a regulatory domain.