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Acanthamoeba myosin I heavy chain kinase is activated by phosphatidylserine-enhanced phosphorylation
H Brzeska1, T J Lynch, E D Korn
1Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
Abstract:
The actin-activated Mg2(+)-ATPase activities of myosins I from Acanthamoeba castellanii are fully expressed only when a single amino acid on their heavy chain is phosphorylated by myosin I heavy chain kinase. Here we show that kinase isolated by a procedure designed to minimize its phosphorylation during purification can incorporate up to 7.5 mol of phosphate/mol of enzyme when incubated with ATP, possibly by autophosphorylation. The rate of phosphorylation is enhanced about 20-fold by phosphatidylserine but is unaffected by calcium ions. Phosphorylation increases the rate at which the kinase phosphorylates the regulatory site of myosin I by about 50-fold. These results suggest that (auto?)phosphorylation may regulate the activity of myosin I heavy chain kinase in vivo. The stimulation of kinase phosphorylation by phosphatidylserine (other phospholipids have not yet been tested) is of particular interest because myosin I has been shown to be tightly associated with membranes, especially the plasma membrane.
Insights
Myosin I heavy chain kinase activity is regulated by phosphorylation. Phosphatidylserine significantly enhances this kinase
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Motors
Background:
- Myosins I are essential molecular motors involved in cellular processes.
- Their activity is regulated by phosphorylation of the heavy chain.
- Myosin I heavy chain kinase (MIHCK) is responsible for this critical phosphorylation.
Purpose of the Study:
- To investigate the regulation of Acanthamoeba myosin I heavy chain kinase (MIHCK).
- To determine the effect of autophosphorylation and phospholipids on MIHCK activity.
- To understand the in vivo regulation of MIHCK.
Main Methods:
- Purification of MIHCK with minimized endogenous phosphorylation.
- In vitro kinase assays to measure phosphorylation rates.
- Assays to assess the effect of ATP, phosphatidylserine, and calcium ions on kinase activity.
Main Results:
- Purified MIHCK can undergo autophosphorylation, incorporating up to 7.5 mol of phosphate/mol of enzyme.
- Phosphatidylserine significantly enhances MIHCK phosphorylation rate by approximately 20-fold.
- Phosphorylation of MIHCK by ATP increases its ability to phosphorylate myosin I by about 50-fold.
- Calcium ions do not affect MIHCK phosphorylation.
Conclusions:
- Autophosphorylation may play a role in regulating MIHCK activity.
- Phosphatidylserine stimulation suggests a link between membrane association and kinase regulation.
- These findings provide insights into the in vivo regulation of myosin I activity through its kinase.