Among 18 family members in the myosin superfamily, myosin IX is unique by possessing a GTPase activating protein (GAP) for Rho. It is also attention-grabbing since it is a single-headed processive motor, as well as a minus-end directed motor. Although many biochemical properties have been revealed, its physiological function is largely unknown. As an initial step to address this question, I attempted to find the binding partner of myosin IXb using the yeast two-hybrid screen. Through the screen using the tail domain of myosin IXb as bait I found BIG1, a guanine nucleotide exchange factor (GEF) for ADP-ribosylation factor (Arfl), as a potential binding partner for myosin IXb. The interaction between myosin IXb and BIG1 was demonstrated by co-immunoprecipitation of endogenous myosin IXb and BIG1 with anti-BIG1 antibodies in normal rat kidney (NRK) cells. Using the isolated proteins, it was demonstrated that myosin IXb and BIG1 directly bind to each other. Various truncation mutants of the myosin IXb tail domain were produced and it was revealed that the binding region of myosin IXb to BIG1 is the zinc finger/GAP domain. Interestingly, the GAP activity of myosin IXb was significantly inhibited by addition of BIG1 with IC50 of 0.06 μM. The RhoA binding to myosin IXb was inhibited by the addition of BIG1 with a concentration similar to that which inhibit the GAP activity. Likewise, RhoA inhibited the BIG1 binding of myosin IXb. These results suggest that BIG1 and RhoA compete with each other for the binding to myosin IXb, thus resulting in the inhibition of the GAP activity by BIG1. The present study identified BIG1, the ArfGEF, as a new binding partner for myosin IXb, which inhibited the GAP activity of myosin IXb.
Together, the results imply that the RhoGAP activity of myosin IXb is down-regulated by BIG1 at the Golgi, where myosin IXb could be involved in the regulation of actin cytoskeleton through the Rho-signaling pathway.
| Identifer | oai:union.ndltd.org:umassmed.edu/oai:escholarship.umassmed.edu:gsbs_diss-1294 |
| Date | 01 May 2005 |
| Creators | Saeki, Nobutaka |
| Publisher | eScholarship@UMassChan |
| Source Sets | University of Massachusetts Medical School |
| Detected Language | English |
| Type | text |
| Source | Morningside Graduate School of Biomedical Sciences Dissertations and Theses |
| Rights | Copyright is held by the author, with all rights reserved. |
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