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MBC in Press, published online ahead of print March 7, 2007
Mol. Biol. Cell 10.1091/mbc.E06-08-0741

A more recent version of this article appeared on May 1, 2007
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Submitted on August 24, 2006
Revised on January 29, 2007
Accepted on February 26, 2007

A Ser/Thr Kinase Required for Membrane-associated Assembly of the MSP Motility Apparatus in the Amoeboid Sperm of Ascaris

Kexi Yi,* Shawnna M. Buttery,*{dagger} Murray Stewart,{ddagger} and Thomas M. Roberts*

*Department of Biological Science, Florida State University, Tallahassee, FL 32306; {ddagger}Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 2QH, United Kingdom

Monitoring Editor: Yu-li Wang

Leading edge protrusion in the amoeboid sperm of Ascaris suum is driven by the localized assembly of the MSP cytoskeleton in the same way that actin assembly powers protrusion in other types of crawling cell. Reconstitution of this process in vitro led to the identification of two accessory proteins required for MSP polymerization: an integral membrane phosphoprotein, MPOP, and a cytosolic component, MFP2. Here, we identify and characterize a 34 kDa cytolic protein, MSP polymerization activating kinase, (MPAK) that links the activities of MPOP and MFP2. Depletion/add-back assays of sperm extracts showed that MPAK, which is a member of the casein kinase 1 family of Ser/Thr protein kinases, is required for motility. MPOP and MPAK comigrated by native gel electrophoresis, coimmunoprecipitated, and colocalized by immunofluorescence indicating that MPOP binds to and recruits MPAK to the membrane surface. MPAK, in turn, phosphorylated MFP2 on threonine residues resulting in incorporation of MFP2 into the cytoskeleton. Beads coated with MPAK assembled a surrounding cloud of MSP filaments when incubated in MPAK-depleted sperm extract, but only when supplemented with detergent-solubilized MPOP. Our results suggest that interactions involving MPOP, MPAK, and MFP2 focus MSP polymerization to the plasma membrane at the leading edge of the cell thereby generating protrusion and minimizing nonproductive filament formation elsewhere.

{dagger}Present address: Dana Farber Cancer Institute, Boston, MA 02115.

Address correspondence to: Thomas M. Roberts (roberts{at}bio.fsu.edu)






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