Abstract
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.
| Original language | English |
|---|---|
| Pages (from-to) | 45-51 |
| Number of pages | 7 |
| Journal | Biochemical and Biophysical Research Communications |
| Volume | 410 |
| Issue number | 1 |
| DOIs | |
| Publication status | Published - Jun 24 2011 |
| Externally published | Yes |
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Keywords
- Ca-signaling
- CaMKK
- Phosphorylation
- Syndapin I
ASJC Scopus subject areas
- Biochemistry
- Biophysics
- Cell Biology
- Molecular Biology
Cite this
Identification of a novel CaMKK substrate. / Fujimoto, Tomohito; Hatano, Naoya; Nozaki, Naohito; Yurimoto, Saki; Kobayashi, Ryoji; Tokumitsu, Hiroshi.
In: Biochemical and Biophysical Research Communications, Vol. 410, No. 1, 24.06.2011, p. 45-51.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Identification of a novel CaMKK substrate
AU - Fujimoto, Tomohito
AU - Hatano, Naoya
AU - Nozaki, Naohito
AU - Yurimoto, Saki
AU - Kobayashi, Ryoji
AU - Tokumitsu, Hiroshi
PY - 2011/6/24
Y1 - 2011/6/24
N2 - 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.
AB - 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.
KW - Ca-signaling
KW - CaMKK
KW - Phosphorylation
KW - Syndapin I
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U2 - 10.1016/j.bbrc.2011.05.102
DO - 10.1016/j.bbrc.2011.05.102
M3 - Article
VL - 410
SP - 45
EP - 51
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
SN - 0006-291X
IS - 1
ER -