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Vol. 17, Issue 11, 4856-4865, November 2006

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Myo1c Binds Phosphoinositides through a Putative Pleckstrin Homology Domain

David E. Hokanson^*, Joseph M. Laakso^*, Tianming Lin^*, David Sept, and E. Michael Ostap^*

*The Pennsylvania Muscle Institute and Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6085; and Department of Biomedical Engineering and Center for Computational Biology, Washington University, St. Louis, MO 63130

Submitted May 23, 2006; Accepted August 31, 2006
Monitoring Editor: Carole Parent

Myo1c is a member of the myosin superfamily that binds phosphatidylinositol-4,5-bisphosphate (PIP₂), links the actin cytoskeleton to cellular membranes and plays roles in mechano-signal transduction and membrane trafficking. We located and characterized two distinct membrane binding sites within the regulatory and tail domains of this myosin. By sequence, secondary structure, and ab initio computational analyses, we identified a phosphoinositide binding site in the tail to be a putative pleckstrin homology (PH) domain. Point mutations of residues known to be essential for polyphosphoinositide binding in previously characterized PH domains inhibit myo1c binding to PIP₂ in vitro, disrupt in vivo membrane binding, and disrupt cellular localization. The extended sequence of this binding site is conserved within other myosin-I isoforms, suggesting they contain this putative PH domain. We also characterized a previously identified membrane binding site within the IQ motifs in the regulatory domain. This region is not phosphoinositide specific, but it binds anionic phospholipids in a calcium-dependent manner. However, this site is not essential for in vivo membrane binding.

This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E06-05-0449) on September 13, 2006.

Address correspondence to: E. Michael Ostap (ostap{at}mail.med.upenn.edu)

Abbreviations used: GFP, green fluorescence protein; GFP-myo1c-tail^IQ1-3, GFP-tagged myo1c (residues 690-1028); GFP-myo1c-tail^IQ2–3, GFP-tagged myo1c (residues 721-1028); GFP-myo1c-tail^IQ3, GFP-tagged myo1c (residues 744-1028); GFP-myo1c-tail^IQ0, GFP-tagged myo1c (residues 768-1028); GFP-K892A and GFP-R903A, point mutations in GFP-myo1c-tail^IQ1-3; GFP-myo1c-K892A and GFP-myo1c-K903A, point mutations in GFP-myo1c; Ins(1,2,6)P₃, D-myo-inositol-1,2,6-trisphosphate; Ins(1,3,4)P₃, D-myo-inositol-1,3,4-trisphosphate; Ins(1,4,5)P₃, D-myo-inositol-1,4,5-trisphosphate; Ins(1,3,4,5)P₄, D-myo-inositol-1,3,4,5-tetrakisphosphate; Ins(1,2,5,6)P₄, D-myo-inositol-1,2,5,6-tetrakisphosphate; Ins(1,3,4,6)P₄, D-myo-inositol-1,3,4,6-tetrakisphosphate; Ins(1,2,3,5,6)P₅, D-myo-inositol-1,2,3,5,6-pentakisphosphate; Ins(3)P₁, D-myo-inositol-3-monophosphate; InsP₆, D-myo-inositol hexakisphosphate; LUV, large unilamellar vesicle; myo1c-tail^IQ1-3, myo1c (residues 690-1028); myo1c-motor^IQ1-3, myo1c (residues 1–767); PH, pleckstrin homology; PIP₂, phosphatidylinositol-4,5-bisphosphate; PC, phosphatidylcholine; PS, phosphatidylserine.

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