Date of Award
Doctor of Philosophy (PhD)
Biochemistry & Molecular Biophysics
E. Michael Ostap
Myo1c is a member of the myosin superfamily that links the dynamic actin cytoskeleton to the membrane, 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. We found that the tail domain binds tightly and specifically to PIP2 in a non-cooperative manner. It binds with slightly higher affinity to Ins(1,4,5)P3 as well as other inositol phosphates which may act as inhibitors to membrane binding in the cell. By sequence and secondary structure analysis, we identified this phosphatidylinositol binding site in the tail to be a putative pleckstrin homology (PH) domain. Point mutations of residues known to be essential for phosphatidylinositol binding in previously characterized PH domains inhibit myo1c binding to PIP2 in vitro and eradicate correct localization and membrane binding in vivo. The extended sequence of this binding site is conserved within many other myosin-Is across species, suggesting they also contain a putative PH domain. We also characterized a previously identified membrane binding site within the IQ motifs in the regulatory domain. This region is not phosphatidylinositol specific, but binds anionic phospholipids in a Ca2+ dependent manner; nevertheless, this site is not essential for in vivo membrane binding. As a result, we have determined that myo1c contains two lipid binding sites, a polybasic region that binds to high levels of PS in a Ca2+ dependent manner and a putative PH domain that binds tightly and specifically to phosphatidylinositols.
Hokanson, David, "MYO1C Binds to PiP2 With High Affinity Through a Putative Ph Domain" (2007). Publicly Accessible Penn Dissertations. 1.