Regulation of Myosin VI Function Through Phosphorylation

Lorraine Ida Kelley
University of California, San Diego

Myosins are motor proteins that utilize the energy of ATP hydrolysis to move cellular components along actin filaments. Of these motors, Myosin VI (Myo6) is required for cochlear development, as patients with mutations of the Myo6 gene suffer from deafness and balance disorders. Myo6 has an N-terminal motor domain, which binds actin and a C-terminal tail domain, which binds cargo. This tail binds both uncoated vesicles and clathrin-coated pits suggesting roles in endocytic trafficking. The purpose of my research is to elucidate how targeting to endocytic compartments is regulated. I hypothesized that the differential targeting may be due to signaling pathways that phosphorylate Myo6. The Myo6 tail domain has several potential Casein Kinase II, Protein Kinase A and Protein Kinase C sites. To investigate the role of phosphorylation in vivo, I performed site directed mutagenesis to alter conserved serine residues in a Green Fluorescent Protein (GFP)-tagged Myo6 tail domain construct. The altered constructs were then transfected into cultured cells that exhibit Myo6??s characteristic targeting to determine the effects of the mutations on targeting. In a parallel experiment, phosphorylation of Myo6 was analyzed in vitro by incubating the tail domain with potential kinases. Phosphorylation sites were determined using MALDI Mass Spectrometry. The results of both the in vitro and in vivo analysis will serve to underscore phosphorylation of Myo6 as a notable means of regulating endocytic functioning.