Related Experiment Video
Updated: Jan 15, 2026
Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells
Published on: November 26, 2019
Characterization of Autonomous and Ca2+/Calmodulin-Dependent Activities of CaMKK Isoforms In Vitro and in Mouse
Satomi Ohtsuka1, Yerun Chen1, Masaki Magari1
1Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University, Okayama 700-8530, Japan.
Abstract:
Ca2+/CaM-dependent protein kinase kinase (CaMKK) phosphorylates and activates downstream kinases, including CaMKI, CaMKIV, PKB, and AMPK, regulating various cellular functions such as neuronal morphogenesis, metabolic control, and pathophysiological pathways, such as cancer progression. CaMKKα/1 is tightly regulated by an autoinhibitory mechanism. CaMKKβ/2 activity is highly Ca2+/CaM-independent (autonomous activity) in vitro and Ca2+/CaM-dependent in cultured cells. Whether these two activity states of CaMKKβ/2 exist in vivo and the detailed regulatory mechanisms for the transition of both activity states remain unclear due to the difficulty in distinguishing the two activity states. In this study, we detected Ca2+-dependent and autonomous CaMKK activity in HeLa cells and successfully separated both activity states of CaMKKβ/2 in mouse brain and testis extracts using a recently developed CaMKK inhibitor (TIM-063)-coupled sepharose, which binds to the catalytic domain in the active state but not in the autoinhibited state. Furthermore, lambda protein phosphatase treatment converted the Ca2+/CaM-dependent form to the autonomous form of CaMKKβ/2, which was not affected by Ala mutation of Ser128, Ser132, and Ser136. The two activity forms of CaMKKβ/2 had equivalent Ca2+/CaM-binding ability. The findings demonstrate the presence of autonomous and Ca2+/CaM-dependent forms of CaMKKβ/2 independently in mouse tissues and cultured cells. The transition of these states of CaMKKβ/2 may be dynamically regulated by the phosphorylation/dephosphorylation of serine residues in the N-terminal regulatory domain.
Insights
Calcium/calmodulin-dependent protein kinase kinase beta (CaMKKβ/2) exists in both autonomous and Ca2+/calmodulin-dependent forms in vivo. Phosphorylation/dephosphorylation of N-terminal serine residues regulates CaMKKβ/2 activity states.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Signaling
Background:
- Calcium/calmodulin-dependent protein kinase kinase (CaMKK) regulates crucial cellular functions by phosphorylating downstream kinases.
- CaMKKα is autoinhibited, while CaMKKβ exhibits Ca2+/calmodulin-independent (autonomous) activity in vitro but Ca2+/calmodulin-dependent activity in cells.
- The in vivo existence and regulation of CaMKKβ's distinct activity states remain poorly understood.
Purpose of the Study:
- To investigate the in vivo presence and regulation of autonomous and Ca2+/calmodulin-dependent CaMKKβ/2 activity states.
- To differentiate and characterize the two activity states of CaMKKβ/2 in biological samples.
Main Methods:
- Utilized a novel CaMKK inhibitor (TIM-063)-coupled sepharose to separate active (bound) and autoinhibited (unbound) CaMKKβ/2 states.
- Employed lambda protein phosphatase treatment to assess the role of phosphorylation in state transitions.
- Analyzed Ca2+/calmodulin-binding abilities of both CaMKKβ/2 forms.
Main Results:
- Detected both Ca2+-dependent and autonomous CaMKK activity in HeLa cells.
- Successfully isolated autonomous and Ca2+/calmodulin-dependent CaMKKβ/2 from mouse brain and testis extracts.
- Lambda protein phosphatase treatment converted the Ca2+/calmodulin-dependent form to the autonomous form, independent of specific serine mutations.
- Both CaMKKβ/2 forms exhibited similar Ca2+/calmodulin-binding capacities.
Conclusions:
- Demonstrated the independent existence of autonomous and Ca2+/calmodulin-dependent CaMKKβ/2 forms in mouse tissues and cultured cells.
- Suggests that dynamic phosphorylation/dephosphorylation of N-terminal serine residues regulates the transition between CaMKKβ/2 activity states.
Related Concept Videos
01:16Calmodulin-dependent Signaling
The Ca2+-CaM complex does not have enzymatic activity by itself. Instead, the complex binds downstream target proteins, including membrane proteins or enzymes,...
01:25cAMP-dependent Protein Kinase Pathways