Abstract
Doublecortin-like protein kinase (DCLK), a Ser/Thr protein kinase predominantly expressed in brain and eyes, is believed to play crucial roles in neuronal functions. However, the regulatory mechanisms for DCLK activation and its physiological targets are still unknown. In the present study, we found that a deletion mutant consisting of the catalytic domain of zebrafish DCLK, zDCLK(377-677), exhibited the highest activity among various mutants. Since fully active zDCLK(377-677) showed essentially the same substrate specificity as wild-type zDCLK, we used it to search for physiological substrates of zDCLK. When a zebrafish brain extract was resolved by isoelectric focusing and then phosphorylated by zDCLK(377-677), a highly basic protein with a molecular mass of ∼90 kDa was detected. This protein was identified as synapsin II by mass spectrometric analysis. Synapsin II was found to interact with the catalytic domain of zDCLK and was phosphorylated at Ser-9 and Ser-58. When synaptosomes were isolated from zebrafish brain, both synapsin II and zDCLK were found to coexist in this preparation. Furthermore, synapsin II in the synaptosomes was efficiently phosphorylated by zDCLK. These results suggest that zDCLK mediates its neuronal functions through phosphorylation of physiological substrates such as synapsin II.
| Original language | English |
|---|---|
| Pages (from-to) | 711-722 |
| Number of pages | 12 |
| Journal | Journal of Biochemistry |
| Volume | 147 |
| Issue number | 5 |
| DOIs | |
| Publication status | Published - Jan 1 2010 |
| Externally published | Yes |
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Keywords
- autoinhibitory domain
- catalytic domain
- doublecortin-like protein kinase
- synapsin IIsynaptosomes
ASJC Scopus subject areas
- Biochemistry
- Molecular Biology
- Medicine(all)
Cite this
Identification of an endogenous substrate of zebrafish doublecortin-like protein kinase using a highly active truncation mutant. / Shimomura, Sachiko; Nagamine, Tadashi; Hatano, Naoya; Sueyoshi, Noriyuki; Kameshita, Isamu.
In: Journal of Biochemistry, Vol. 147, No. 5, 01.01.2010, p. 711-722.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Identification of an endogenous substrate of zebrafish doublecortin-like protein kinase using a highly active truncation mutant
AU - Shimomura, Sachiko
AU - Nagamine, Tadashi
AU - Hatano, Naoya
AU - Sueyoshi, Noriyuki
AU - Kameshita, Isamu
PY - 2010/1/1
Y1 - 2010/1/1
N2 - Doublecortin-like protein kinase (DCLK), a Ser/Thr protein kinase predominantly expressed in brain and eyes, is believed to play crucial roles in neuronal functions. However, the regulatory mechanisms for DCLK activation and its physiological targets are still unknown. In the present study, we found that a deletion mutant consisting of the catalytic domain of zebrafish DCLK, zDCLK(377-677), exhibited the highest activity among various mutants. Since fully active zDCLK(377-677) showed essentially the same substrate specificity as wild-type zDCLK, we used it to search for physiological substrates of zDCLK. When a zebrafish brain extract was resolved by isoelectric focusing and then phosphorylated by zDCLK(377-677), a highly basic protein with a molecular mass of ∼90 kDa was detected. This protein was identified as synapsin II by mass spectrometric analysis. Synapsin II was found to interact with the catalytic domain of zDCLK and was phosphorylated at Ser-9 and Ser-58. When synaptosomes were isolated from zebrafish brain, both synapsin II and zDCLK were found to coexist in this preparation. Furthermore, synapsin II in the synaptosomes was efficiently phosphorylated by zDCLK. These results suggest that zDCLK mediates its neuronal functions through phosphorylation of physiological substrates such as synapsin II.
AB - Doublecortin-like protein kinase (DCLK), a Ser/Thr protein kinase predominantly expressed in brain and eyes, is believed to play crucial roles in neuronal functions. However, the regulatory mechanisms for DCLK activation and its physiological targets are still unknown. In the present study, we found that a deletion mutant consisting of the catalytic domain of zebrafish DCLK, zDCLK(377-677), exhibited the highest activity among various mutants. Since fully active zDCLK(377-677) showed essentially the same substrate specificity as wild-type zDCLK, we used it to search for physiological substrates of zDCLK. When a zebrafish brain extract was resolved by isoelectric focusing and then phosphorylated by zDCLK(377-677), a highly basic protein with a molecular mass of ∼90 kDa was detected. This protein was identified as synapsin II by mass spectrometric analysis. Synapsin II was found to interact with the catalytic domain of zDCLK and was phosphorylated at Ser-9 and Ser-58. When synaptosomes were isolated from zebrafish brain, both synapsin II and zDCLK were found to coexist in this preparation. Furthermore, synapsin II in the synaptosomes was efficiently phosphorylated by zDCLK. These results suggest that zDCLK mediates its neuronal functions through phosphorylation of physiological substrates such as synapsin II.
KW - autoinhibitory domain
KW - catalytic domain
KW - doublecortin-like protein kinase
KW - synapsin IIsynaptosomes
UR - http://www.scopus.com/inward/record.url?scp=77953708856&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=77953708856&partnerID=8YFLogxK
U2 - 10.1093/jb/mvq005
DO - 10.1093/jb/mvq005
M3 - Article
C2 - 20097902
AN - SCOPUS:77953708856
VL - 147
SP - 711
EP - 722
JO - Journal of Biochemistry
JF - Journal of Biochemistry
SN - 0021-924X
IS - 5
ER -