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Intermolecular autophosphorylation regulates myosin IIIa activity and localization in parallel actin bundles
Omar A Quintero1, Judy E Moore, William C Unrath
1Department of Cellular and Molecular Physiology, Penn State College of Medicine, Hershey, Pennsylvania 17033, USA.
Abstract:
Myosin IIIa (Myo3A) transports cargo to the distal end of actin protrusions and contains a kinase domain that is thought to autoregulate its activity. Because Myo3A tends to cluster at the tips of actin protrusions, we investigated whether intermolecular phosphorylation could regulate Myo3A biochemical activity, cellular localization, and cellular function. Inactivation of Myo3A 2IQ kinase domain with the point mutation K50R did not alter maximal ATPase activity, whereas phosphorylation of Myo3A 2IQ resulted in reduced maximal ATPase activity and actin affinity. The rate and degree of Myo3A 2IQ autophosphorylation was unchanged by the presence of actin but was found to be dependent upon Myo3A 2IQ concentration within the range of 0.1 to 1.2 microm, indicating intermolecular autophosphorylation. In cultured cells, we observed that the filopodial tip localization of Myo3A lacking the kinase domain decreased when co-expressed with kinase-active, full-length Myo3A. The cellular consequence of reduced Myo3A tip localization was decreased filopodial density along the cell periphery, identifying a novel cellular function for Myo3A in mediating the formation and stability of actin-based protrusions. Our results suggest that Myo3A motor activity is regulated through a mechanism involving concentration-dependent autophosphorylation. We suggest that this regulatory mechanism plays an essential role in mediating the transport and actin bundle formation/stability functions of Myo3A.
Insights
Myosin IIIa (Myo3A) activity and localization are regulated by concentration-dependent intermolecular autophosphorylation. This phosphorylation impacts Myo3A
Area of Science:
- Cell Biology
- Molecular Motor Function
- Protein Kinase Regulation
Background:
- Myosin IIIa (Myo3A) is a motor protein involved in cargo transport to actin protrusions.
- Its kinase domain is hypothesized to autoregulate Myo3A activity.
- Myo3A's tendency to cluster at protrusion tips suggests a role for intermolecular interactions.
Purpose of the Study:
- To investigate if intermolecular phosphorylation regulates Myo3A biochemical activity, cellular localization, and function.
- To elucidate the mechanism of Myo3A regulation at actin protrusion tips.
Main Methods:
- Site-directed mutagenesis (K50R) to inactivate the kinase domain.
- Biochemical assays measuring ATPase activity and actin binding affinity.
- Cell culture experiments co-expressing different Myo3A constructs.
- Microscopy to assess Myo3A localization and filopodial density.
Main Results:
- Phosphorylation of Myo3A 2IQ reduced maximal ATPase activity and actin affinity.
- Autophosphorylation rate was dependent on Myo3A 2IQ concentration, indicating intermolecular autophosphorylation.
- Reduced Myo3A tip localization in cells correlated with decreased filopodial density.
Conclusions:
- Myosin IIIa motor activity is regulated by concentration-dependent autophosphorylation.
- This mechanism is crucial for Myo3A's role in actin protrusion formation and stability.
- Intermolecular phosphorylation is a key regulatory mechanism for Myo3A function.
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