Intermolecular autophosphorylation regulates myosin IIIa activity and localization in parallel actin bundles.
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.
Quintero
Omar A
OA
Department of Cellular and Molecular Physiology, Penn State College of Medicine, Hershey, Pennsylvania 17033, USA.
Moore
Judy E
JE
Unrath
William C
WC
Manor
Uri
U
Salles
Felipe T
FT
Grati
M'hamed
M
Kachar
Bechara
B
Yengo
Christopher M
CM
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002522:Chlorocebus aethiops; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D018069:Hair Cells, Vestibular / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence; D009154:Mutation; D018995:Myosin Heavy Chains / Q000235:genetics / Q000378:metabolism*; D025102:Myosin Type III / Q000235:genetics / Q000378:metabolism*; D009925:Organ of Corti / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011554:Pseudopodia / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D000199:Actins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C491724:MYO3A protein, human; D025102:Myosin Type III; D018995:Myosin Heavy Chains
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