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 N-6-(1-methylbutyl)-ATP and genetically engineered CaMKK alpha mutant, CaMKK alpha (Phe(230)Gly), that was capable of utilizing this ATP analogue as a phosphate donor. Incubation of rat brain extracts with recombinant CaMKK alpha (Phe(230)Gly), but not with wild-type kinase, in the presence of N-6-(1-methylbutyl)-ATP and Ca2+/CaM, induced significant threonine phosphorylation of a 50 kDa protein as well as CaMKI phosphorylation at Thr(177). The 50 kDa 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 CaMKK alpha and beta isoforms at Thr(355) in vitro. Phosphorylation of HA-Syndapin I at Thr(355) 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. (C) 2011 Elsevier Inc. All rights reserved.