Journal of Biological Chemistry
Volume 285, Issue 46, November 2010, Pages 35770-35782
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Cell Biology
Intermolecular Autophosphorylation Regulates Myosin IIIa Activity and Localization in Parallel Actin Bundles*

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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 μm, 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.

Actin
Cytoskeleton
Enzyme Mechanisms
Molecular Motors
Myosin
Actin Protrusions
Autophosphorylation
Filopodia
Inner Ear Hair Cells
Stereocilia

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*

This work was supported, in whole or in part, by National Institutes of Health Grants EY018141 and HL093531 (to C. M. Y.) and NIH DIR Grant DC000002-22 (to B. K.).

The on-line version of this article (available at http://www.jbc.org) contains supplemental “Experimental Procedures” and Figs. S1–S3.

1

Both authors contributed equally to this work.