Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) phosphorylates and activates specific downstream protein kinases including CaMKI, CaMKIV and 5′-AMP-activated protein kinase. In order to examine the variety of CaMKK-mediated signaling pathways, we searched for novel CaMKK substrate(s) using N6-(1-methylbutyl)-ATP and genetically engineered CaMKKα mutant, CaMKKα (Phe230Gly), that was capable of utilizing this ATP analogue as a phosphate donor. Incubation of rat brain extracts with recombinant CaMKKα (Phe230Gly), but not with wild-type kinase, in the presence of N6-(1-methylbutyl)-ATP and Ca2+/CaM, induced significant threonine phosphorylation of a 50kDa protein as well as CaMKI phosphorylation at Thr177. The 50kDa CaMKK substrate was partially purified by using serial column chromatography, and was identified as Syndapin I by LC-MS/MS analysis. We confirmed that recombinant Syndapin I was phosphorylated by CaMKKα and β isoforms at Thr355 in vitro. Phosphorylation of HA-Syndapin I at Thr355 in transfected HeLa cells was significantly induced by co-expression of constitutively active mutants of CaMKK isoforms. This is the first report that CaMKK is capable of phosphorylating a non-kinase substrate suggesting the possibility of CaMKK-mediated novel Ca2+-signaling pathways that are independent of downstream protein kinases.
|Number of pages||7|
|Journal||Biochemical and Biophysical Research Communications|
|Publication status||Published - Jun 24 2011|
- Syndapin I
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology