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Mutagenesis of Thr-286 in monomeric Ca2+/calmodulin-dependent protein kinase II eliminates
M N Waxham1, J Aronowski, S A Westgate
1Department of Neurology, University of Texas Medical School, Houston 77225.
Abstract:
We have examined the role of Thr-286 autophosphorylation in the autoregulation of Ca2+/calmodulin-dependent protein kinase II. Using site-directed mutagenesis, we have substituted alanine or serine for Thr-286, or isoleucine for Arg-283, in the 50-kDa subunit of the kinase and expressed each protein in bacteria. Activation and autophosphorylation of all four enzymes were stringently dependent on Ca2+/calmodulin, indicating that neither Arg-283 nor Thr-286 is an absolute requirement for the pseudosubstrate inhibition of the enzyme. Autophosphorylation of the Ile-283 or Ala-286 enzyme generated little, if any, Ca2+/calmodulin-independent kinase activity, unlike the parent (Thr-286) or Ser-286 enzyme. The enzymes expressed in bacteria are predominantly monomeric, indicating that the generation of Ca2+/calmodulin-independent activity does not require the cooperative interactions of subunits normally present in the brain holoenzyme.
Insights
Autophosphorylation of Thr-286 is crucial for the sustained activity of Ca2+/calmodulin-dependent protein kinase II. Mutations impairing this autophosphorylation prevent the enzyme
Area of Science:
- Biochemistry
- Molecular Biology
- Neuroscience
Background:
- Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a key enzyme in neuronal plasticity.
- Autoregulation of CaMKII activity is critical for synaptic function.
- The role of Thr-286 autophosphorylation in CaMKII autoregulation requires further elucidation.
Purpose of the Study:
- To investigate the specific role of Thr-286 autophosphorylation in the autoregulation of CaMKII.
- To determine if Arg-283 and Thr-286 are essential for pseudosubstrate inhibition.
- To assess the impact of mutations at Arg-283 and Thr-286 on CaMKII activity.
Main Methods:
- Site-directed mutagenesis was used to create mutant CaMKII enzymes.
- Mutations included substituting alanine or serine for Thr-286, and isoleucine for Arg-283.
- Mutant proteins were expressed in bacteria and their activation, autophosphorylation, and kinase activity were analyzed.
Main Results:
- All four mutant enzymes required Ca2+/calmodulin for activation and autophosphorylation.
- Neither Arg-283 nor Thr-286 was essential for pseudosubstrate inhibition.
- Mutations at Ile-283 and Ala-286 significantly reduced Ca2+/calmodulin-independent kinase activity compared to wild-type and Ser-286 enzymes.
- Monomeric bacterial enzyme expression indicated that subunit cooperation is not required for Ca2+/calmodulin-independent activity.
Conclusions:
- Thr-286 autophosphorylation is essential for generating sustained Ca2+/calmodulin-independent CaMKII activity.
- The findings highlight the importance of Thr-286 in CaMKII's functional autoregulation.
- CaMKII autoregulation leading to sustained activity does not necessitate the cooperative interactions of multiple subunits.