Regulation of CaMKII by phospho-Thr253 or phospho-Thr286 sensitive targeting alters cellular function.
Calcium/calmodulin-stimulated protein kinase II (CaMKII) is an important mediator of synaptic function that is regulated by multi-site phosphorylation and targeting through interactions with proteins. A new phosphorylation site at Thr253 has been identified in vivo, that does not alter CaMKII activity, but does alter CaMKII function through interactions with binding proteins. To identify these proteins, as well as to examine the specific effects following Thr253 or Thr286 phosphorylation on these interactions, we developed an in vitro overlay binding assay. We demonstrated that the interaction between CaMKII and its binding proteins was altered by the phosphorylation state of both the CaMKII and the partner, and identified a CaMKII-specific sequence that was responsible for the interaction between CaMKII and two interacting proteins. By comparing CaMKII binding profiles in tissue and cell extracts, we demonstrated that the CaMKII binding profiles varied with cell type, and also showed that overexpression of a CaMKII Thr253 phospho-mimic mutant in human neuroblastoma and breast cancer cells dramatically altered the morphology and growth rates when compared to overexpression of non-phosphorylated CaMKII. This data highlights the importance of the microenvironment in regulating CaMKII function, and describes a potentially new mechanism by which the functions of CaMKII can be regulated.
Skelding
Kathryn A
KA
Hunter Medical Research Institute, The University of Newcastle, Callaghan, New South Wales, Australia.
Suzuki
Tatsuo
T
Gordon
Sarah
S
Xue
Jing
J
Verrills
Nicole M
NM
Dickson
Phillip W
PW
Rostas
John A P
JA
D000818:Animals; D001665:Binding Sites; D001681:Biological Assay; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000378:metabolism*; D002457:Cell Extracts; D002460:Cell Line; D006801:Humans; D008297:Male; D010766:Phosphorylation; D010769:Phosphothreonine / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D013329:Structure-Activity Relationship; D013347:Subcellular Fractions / Q000201:enzymology
D002457:Cell Extracts; D010769:Phosphothreonine; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2