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Evidence that autophosphorylation at Thr-286/Thr-287 is required for full activation of calmodulin-dependent protein
A Ishida1, T Kitani, H Fujisawa
1Department of Biochemistry, Asahikawa Medical College, Japan.
Abstract:
Calmodulin-dependent protein kinase II (CaM-kinase II) undergoes a very rapid autophosphorylation at Thr-286/Thr-287 in the presence of Ca2+/calmodulin and ATP/Mg2+, and this has greatly hampered studies on the role of the autophosphorylation in the regulation of the enzyme activity, because it has been difficult to measure the activity of the non-autophosphorylated enzyme in the presence of Ca2+/calmodulin. In the present study, this difficulty was overcome by using adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) in place of ATP. When the enzyme was assayed with 2 microM ATP gamma S and 200 microM syntide-2 at 5 degrees C in the presence of Ca2+/calmodulin, the linear reaction rate of thiophosphorylation of syntide-2 was much slower than that of the enzyme which had previously undergone autothiophosphorylation. Under the limiting assay conditions, thiophosphorylation of the enzyme did not occur significantly during assay. Using this assay condition, activation by autothiophosphorylation was examined. When CaM-kinase II was autothiophosphorylated at 5 degrees C, the concomitant stimulation of both activities in the presence and absence of Ca2+/calmodulin was observed. The activity of the recombinant wild-type enzyme in the presence of Ca2+/calmodulin as well as in its absence was also markedly activated upon autothiophosphorylation, whereas those of the recombinant mutated enzyme, whose Thr-287 was replaced by Ala, was not activated at all. These results provide strong support for the contention that CaM-kinase II initially possesses a basal low level of the total activity and that the initial rapid autophosphorylation on Thr-286/Thr-287 results in full activation of the enzyme.
Insights
Calmodulin-dependent protein kinase II (CaM-kinase II) activity is regulated by autophosphorylation. Using ATPγS allowed researchers to study the enzyme
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Calmodulin-dependent protein kinase II (CaM-kinase II) autophosphorylation at Thr-286/Thr-287 is rapid and difficult to study.
- Previous methods hindered the measurement of non-autophosphorylated CaM-kinase II activity in the presence of Ca2+/calmodulin.
Purpose of the Study:
- To overcome the challenge of studying CaM-kinase II autophosphorylation.
- To investigate the role of Thr-286/Thr-287 autophosphorylation in CaM-kinase II activity regulation.
Main Methods:
- Utilized adenosine 5'-O-(3-thiotriphosphate) (ATPγS) instead of ATP for enzyme assays.
- Established assay conditions (2 μM ATPγS, 200 μM syntide-2, 5°C) to minimize enzyme thiophosphorylation during measurement.
- Examined the effect of autothiophosphorylation on CaM-kinase II activity in the presence and absence of Ca2+/calmodulin.
Main Results:
- Autothiophosphorylation of CaM-kinase II at 5°C significantly stimulated enzyme activity.
- Both wild-type and recombinant wild-type CaM-kinase II showed marked activation after autothiophosphorylation.
- A mutated enzyme with Thr-287 replaced by Ala did not exhibit activation, indicating the critical role of Thr-287.
Conclusions:
- CaM-kinase II possesses a low basal activity that is fully activated by rapid autophosphorylation at Thr-286/Thr-287.
- This study provides strong evidence for the regulatory role of autophosphorylation in CaM-kinase II activation.
- The developed assay method using ATPγS is effective for studying the kinetics of CaM-kinase II regulation.