Abstract
Myosin IXb, a member of the myosin superfamily, is a molecular motor that possesses a GTPase activating protein (GAP) for Rho. Through the yeast two-hybrid screening using the tail domain of myosin IXb as bait we found BIG1, a guanine nucleotide exchange factor for ADP-ribosylation factor (Arf1), 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 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 the addition of BIG1 with IC(50) of 0.06 microm. The RhoA binding to myosin IXb was inhibited by the addition of BIG1 with the concentration similar to the inhibition of 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 Arf guanine nucleotide exchange factor, as a new binding partner for myosin IXb, which inhibited the GAP activity of myosin IXb. The findings raise a concept that the myosin transports the signaling molecule as a cargo that functions as a regulator for the myosin molecule.
Highlights
GTPase activating protein (GAP) and about 60 for guanine nucleotide exchange factor (GEF) [2]
To confirm the interaction of BIG1 with bait, an additional yeast two-hybrid assay was performed in which the DNA binding domain and the gene activation domain were switched for BIG1 and the myosin IXb tail domain
For myosin IX, it was previously shown that the tail domain contains a RhoGAP domain
Summary
GAPs and about 60 for GEFs [2]. Even though the numbers may be lowered because they are likely to contain some redundant counts [3], the numbers exceed far more than the number of Rho GTPases (i.e. 20, Ref. 2), which leads us to think that Rho GTPases must be tightly regulated in a spatial and temporal manner. The RhoA binding to myosin IXb was inhibited by the addition of BIG1 with the concentration similar to the inhibition of the GAP activity.
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