IDENTIFICATION OF DOMAINS ESSENTIAL FOR THE ASSEMBLY OF CALCIUM CALMODULIN-DEPENDENT PROTEIN-KINASE-II HOLOENZYMES/

Ca2+/calmodulin-dependent protein kinase II (CaM kinase II), as isolat ed from brain, is a multimeric complex composed predominantly of two s ubunits, alpha and beta, products of unique genes. Little is known abo ut how subunit composition influences holoenzyme structure or how the domain(s) of each subunit interact to form holoenzymes. We show here t hat holoenzymes composed of only alpha or only beta subunits exhibit d ifferent biophysical properties. The S values of alpha and beta are 17 .2 and 14.5 S while the Stokes's radii are 85 and 111 Angstrom respect ively, indicating their structures are different. C-terminal truncatio ns of the a subunit show that amino acids 382-478 are necessary for ho loenzyme formation and that amino acids 427-478 contribute to holoenzy me stability, Additionally, the C-terminal domains of both the alpha s ubunit, alpha 315-478, and beta subunit, beta 314-542, formed oligomer s indicating the sufficiency of the C-terminal domain for multimer for mation. Using the yeast two-hybrid system we show, in vivo, that full- length subunits, alpha 1-478 and beta 1-542, interact with themselves or each other interchangeably. Additionally, the C-terminal domains of the alpha subunit, alpha 315-478 and beta subunit, beta 314-542 assoc iated with themselves in a manner indistinguishable from their associa tion with full-length alpha or beta subunits. Further studies revealed that the C-terminal domains of the alpha and beta subunits contain in formation necessary for interaction with beta but not alpha, These dat a are summarized into a model describing the assembly of CaM kinase II holoenzymes.