TY - JOUR
T1 - The active-site cysteine residue of Ca 2+ /calmodulin-dependent protein kinase I is protected from irreversible modification via generation of polysulfidation
AU - Takata, Tsuyoshi
AU - Tsukuda, Ayaka
AU - Tsuchiya, Yukihiro
AU - Akaike, Takaaki
AU - Watanabe, Yasuo
N1 - Funding Information:
This work was supported in part by Grant-in-Aid for Scientific Research on Innovative Areas “Oxygen Biology: a new criterion for integrated understanding of life” [No. 26111008 ] (T.A.) (Y.W.), Scientific Research C [No. 18K11083 ] (Y.W.), Early-Career Scientists [No. 18K14853 ] (T.T.), Young Scientists B [No. 15K18994 ] (Y.T.) from Japan Society for the Promotion of Science (JSPS) ; and Grants-in-Aid for Program for the Strategic Research Foundation at Private Universities [No. S1311012 ] (Y.W.) of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan ; and Grant-in-Aid from the Showa Pharmaceutical University for Young Scientists [H27-3], [H28-2] (T.T.), [H23-2 ] (Y.T.).
Publisher Copyright:
© 2019 Elsevier Inc.
PY - 2019/5/1
Y1 - 2019/5/1
N2 - Ca 2+ /calmodulin (CaM)-dependent protein kinase (CaMK) I is activated by the phosphorylation of a crucial activation loop Thr 177 by upstream kinases, CaMK kinase (CaMKK), and regulates axonal or dendritic extension and branching. Reactive sulfur species (RSS) modulate protein functions via polysulfidation of the reactive Cys residues. Here, we report that the activity of CaMKI was reversibly inhibited via its polysulfidation at Cys 179 by RSS. In vitro incubation of CaMKI with the exogenous RSS donor Na 2 S 3 resulted in a dose-dependent inhibition of the phosphorylation at Thr 177 by CaMKK and inactivation of the enzymatic activity. Dithiothreitol (DTT), a small molecule reducing reagent, rescued these inhibitions. Conversely, mutated CaMKI (C179V) was resistant to the Na 2 S 3 -induced inactivation. In transfected cells expressing CaMKI, ionomycin-induced CaMKI activity was decreased upon treatment with Na 2 S 4 , whereas cells expressing mutant CaMKI (C179V) proved resistant to this treatment. A biotin-polyethylene glycol-conjugated maleimide capture assay revealed that CaMKI was a target for polysulfidation in cells. Furthermore, the polysulfidation of CaMKI protected Cys 179 from its irreversible modification, known as protein succination. Thus, we propose that CaMKI was reversibly inhibited via polysulfidation of Cys 179 by RSS, thereby protecting it from irreversible modification.
AB - Ca 2+ /calmodulin (CaM)-dependent protein kinase (CaMK) I is activated by the phosphorylation of a crucial activation loop Thr 177 by upstream kinases, CaMK kinase (CaMKK), and regulates axonal or dendritic extension and branching. Reactive sulfur species (RSS) modulate protein functions via polysulfidation of the reactive Cys residues. Here, we report that the activity of CaMKI was reversibly inhibited via its polysulfidation at Cys 179 by RSS. In vitro incubation of CaMKI with the exogenous RSS donor Na 2 S 3 resulted in a dose-dependent inhibition of the phosphorylation at Thr 177 by CaMKK and inactivation of the enzymatic activity. Dithiothreitol (DTT), a small molecule reducing reagent, rescued these inhibitions. Conversely, mutated CaMKI (C179V) was resistant to the Na 2 S 3 -induced inactivation. In transfected cells expressing CaMKI, ionomycin-induced CaMKI activity was decreased upon treatment with Na 2 S 4 , whereas cells expressing mutant CaMKI (C179V) proved resistant to this treatment. A biotin-polyethylene glycol-conjugated maleimide capture assay revealed that CaMKI was a target for polysulfidation in cells. Furthermore, the polysulfidation of CaMKI protected Cys 179 from its irreversible modification, known as protein succination. Thus, we propose that CaMKI was reversibly inhibited via polysulfidation of Cys 179 by RSS, thereby protecting it from irreversible modification.
KW - Ca /calmodulin-dependent protein kinase (CaMK)
KW - Phosphorylation
KW - Redox regulation
KW - polysulfidation
KW - reactive sulfur species (RSS)
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U2 - 10.1016/j.niox.2019.02.008
DO - 10.1016/j.niox.2019.02.008
M3 - Article
C2 - 30844494
AN - SCOPUS:85062444112
VL - 86
SP - 68
EP - 75
JO - Nitric Oxide - Biology and Chemistry
JF - Nitric Oxide - Biology and Chemistry
SN - 1089-8603
ER -