Densin-180 forms a ternary complex with the (alpha)-subunit of Ca2+/calmodulin-dependent protein kinase II and (alpha)-actinin.
Densin-180 is a transmembrane protein that is tightly associated with the postsynaptic density in CNS neurons and is postulated to function as a synaptic adhesion molecule. Here we report the identification of the alpha-subunit of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and alpha-actinin-4 as potential binding partners for the densin-180 intracellular segment. We demonstrate by yeast two-hybrid and biochemical assays that the intracellular portion of densin-180, the alpha-subunit of CaMKII (CaMKIIalpha), and alpha-actinin interact with each other at distinct binding sites and can form a ternary complex stabilized by multiple interactions. Densin-180 binds specifically to the association domain of CaMKIIalpha and does not bind with high affinity to holoenzymes of CaMKII that contain beta-subunit. The PDZ (PSD-95, DIg, Z0-1) domain of densin contributes to its binding to alpha-actinin. A distinct domain of alpha-actinin interacts with the kinase domains of both alpha- and beta-subunits of CaMKII. Autophosphorylation of CaMKII increases its affinity for densin-180 from an EC(50) of >1 micrometer to an EC(50) of <75-150 nM. In contrast, phosphorylation of densin-180 by CaMKII at serine-1397 only slightly decreases its affinity for CaMKII. The specific interaction of densin-180 with holoenzymes of CaMKII containing only alpha-subunit and the increased affinity of CaMKII for densin-180 after autophosphorylation suggest that densin-180 may be involved in localization of activated CaMKII synthesized in dendrites.
Walikonis
R S
RS
Division of Biology, California Institute of Technology, Pasadena, California 91125, USA.
Oguni
A
A
Khorosheva
E M
EM
Jeng
C J
CJ
Asuncion
F J
FJ
Kennedy
M B
MB
D000185:Actinin / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D046911:Macromolecular Substances; D008840:Microfilament Proteins; D010766:Phosphorylation; D011233:Precipitin Tests; D016548:Prosencephalon / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000502:physiology; D021122:Protein Subunits; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020539:Sequence Analysis, Protein; D012795:Sialoglycoproteins / Q000235:genetics / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013574:Synaptosomes / Q000737:chemistry / Q000378:metabolism; D020798:Two-Hybrid System Techniques
C111453:ACTN4 protein, human; C491751:Actn4 protein, rat; C504461:LRRC7 protein, human; C504463:Lrrc7 protein, rat; D046911:Macromolecular Substances; D008840:Microfilament Proteins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D012795:Sialoglycoproteins; D000185:Actinin; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases