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© The Rockefeller University Press, 0021-9525/1998//1535 $5.00
The Journal of Cell Biology, Volume 143, Number 6, , 1998 1535-1545

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

Folma Buss^*, John Kendrick-Jones, Corinne Lionne, Alex E. Knight, Graham P. Côté, and J. Paul Luzio^*

^* Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, Cambridge, CB2 2QR, United Kingdom; MRC Laboratory of Molecular Biology, Cambridge CB2 2QH, United Kingdom; and Department of Biochemistry, Queens University, Kingston, Ontario K7L 3N6, Canada

Myosin VI is an unconventional myosin that may play a role in vesicular membrane traffic through actin rich regions of the cytoplasm in eukaryotic cells. In this study we have cloned and sequenced a cDNA encoding a chicken intestinal brush border myosin VI. Polyclonal antisera were raised to bacterially expressed fragments of this myosin VI. The affinity purified antibodies were highly specific for myosin VI by immunoblotting and immunoprecipitation and were used to study the localization of the protein by immunofluorescence and immunoelectron microscopy. It was found that in NRK and A431 cells, myosin VI was associated with both the Golgi complex and the leading, ruffling edge of the cell as well as being present in a cytosolic pool. In A431 cells in which cell surface ruffling was stimulated by EGF, myosin VI was phosphorylated and recruited into the newly formed ruffles along with ezrin and myosin V. In vitro experiments suggested that a p21-activated kinase (PAK) might be the kinase responsible for phosphorylation in the motor domain. These results strongly support a role for myosin VI in membrane traffic on secretory and endocytic pathways.

Key Words: myosin • actin • cytoskeleton • membranes • Golgi complex

Abbreviations used in this paper: aa, amino acid; ab, antibody; GST, glutathione-S-transferase; His, Histidine; MIHCK, myosin I heavy chain kinases; MPR, cation-independent mannose 6-phosphate receptor; PAK, p21-activated protein kinase; PGT, globular tail ab.

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