Regulation of Myosin IIIa Motor/Kinase Activity

1 online resource (99 pages) : PDF

2009

University of North Carolina at Charlotte

Myosin IIIa, a motor protein that localizes to actin-rich regions of photoreceptors and to stereocilia of inner ear hair cells, is associated with the hearing process in vertebrates and with phototransduction for invertebrates. It contains a large Pak-like kinase domain at its N-terminus that has previously been shown to autophosphorylate residue(s) on the motor domain. We have characterized the autophosphorylation process and its influence on the motor's ATPase activity and actin affinity using a phosphorylated version of our previously developed construct human MIIIa 2IQ, contrasting its activity with the kinase-dead MIIIa 2IQ K50R construct, which has a single substitution mutation in the kinase's catalytic region. We have determined that autophosphorylation is an intermolecular process with rates ranging from ~0.05 min-1 to ~0.30 min-1 (0.01 - 1.2 µM MIIIa). Although proposed target autophosphorylation sites are near the motor's actin binding region, the autophosphorylation rate is not affected by the presence of actin filaments. We demonstrated that the MIIIa 2IQ ATPase rate in the presence of 20 µM actin decreases over time of pre-incubation with ATP (~40% in 1 h). The ATPase of fully phosphorylated MIIIa 2IQ has lower kcat and higher KATPase than unphosphorylated MIIIa 2IQ or the K50R construct, while its steady-state actin affinity in the actin co-sedimentation assay (KActin) is decreased. We propose that phosphorylation of the myosin IIIa motor by its activated kinase domain reduces the molecule's actin affinity. This concentration-dependent autophosphorylation acts as a downregulatory mechanism that serves to maintain myosin IIIa/cargo concentration at actin bundle tips within physiologically determined limits for maintenance of stereocilia length and flexibility. This model is further supported by immunolocalization results (Kachar lab, NIH) in which a GFP-tagged version of the kinase-dead construct was demonstrated to localize more effectively to stereocilia tips than GFP-tagged wild type.

doctoral dissertations

Biochemistry
Neurosciences
Cytology

Ph.D.

Kinase Domain
Myosin IIIa
Stereocilia

Biology

Yengo, Christopher

Schrum, Laura
Marriott, Ian
Jacobs, Donald
Gibas, Cynthia

Thesis (Ph.D.)--University of North Carolina at Charlotte, 2009.

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