The localization of myosin VI at the Golgi complex and leading edge of fibroblasts and its phosphorylation and recruitment into membrane ruffles of A431 cells after growth factor stimulation

Myosin VI is an unconventional myosin that may play a role in vesicular mem brane traffic through actin rich regions of the cytoplasm in eukaryotic cel ls. In this study we have cloned and sequenced a cDNA encoding a chicken in testinal brush border myosin VI. Polyclonal antisera were raised to bacteri ally expressed fragments of this myosin VI, The affinity purified antibodie s were highly specific for myosin VI by immunoblotting and immunoprecipitat ion and were used to study the localization of the protein by immunofluores cence and immunoelectron microscopy. It was found that in NRK and A431 cell s, myosin VI was associated with both the Golgi complex and the leading, ru ffling edge of the cell as well as being present in a cytosolic pool. In A4 31 cells in which cell surface ruffling was stimulated by EGF, myosin VI wa s phosphorylated and recruited into the newly formed ruffles along with ezr in and myosin V. In vitro experiments suggested that a p21-activated kinase (PAK) might be the kinase responsible for phosphorylation in the motor dom ain. These results strongly support a role for myosin VI in membrane traffi c on secretory and endocytic pathways.