Saeki, N. , Tokuo, H. & Ikebe, M. BIG1 is a binding partner of myosin IXb and regulates its Rho-GTPase activating protein activity. J. Biol. Chem. 280, 10128-10134

Department of Physiology, University of Massachusetts Medical School, 55 Lake Ave., Worcester, Massachusetts 01655, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 04/2005; 280(11):10128-34. DOI: 10.1074/jbc.M413415200
Source: PubMed


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.

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    • "Despite this, we did not detect any perturbation in the localization to the Golgi of, or stability of GM130, GRASP65, giantin, or p115. BIG1 interacts with myosin IXb [51]. Little is known of the function of myosin IXb but it has been localized throughout the cytoplasm and on the Golgi apparatus. "
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    ABSTRACT: Transport of molecules from one subcellular compartment to another involves the recruitment of cytosolic coat protein complexes to a donor membrane to concentrate cargo, deform the membrane and ultimately to form an independent carrier. Small-GTP-binding proteins of the Arf family are central to many membrane trafficking events. Arfs are activated by guanine nucleotide exchange factors (GEFs) which results in their recruitment to membranes and subsequent engagement with Arf-effectors, many of which are coat proteins. Among the human BFA-sensitive large Arf-GEFs, the function of the two closely related BIG1 and BIG2 is still not clear, and recent studies have raised the question of functional redundancy between the two proteins. Here we have used small-interfering RNA on human cells and a combination of fixed and live-cell imaging to investigate the differential functions of BIG1 and BIG2 in endomembrane organization and function. Importantly, in this direct comparative study, we show discrete functions for BIG1 and BIG2. Our results show that depletion of BIG2 but not of BIG1 induces a tubulation of the recycling endosomal compartment, consistent with a specific role for BIG2 here. In contrast, suppression of BIG1 induces the formation of Golgi mini-stacks still polarized and functional in terms of cargo export. A key finding from our work is that suppression of BIG1 expression results in a fragmentation of the Golgi apparatus. Our data indicate that the human BFA-sensitive large Arf-GEFs have non-redundant functions in cell organization and membrane trafficking. BIG1 is required to maintain the normal morphology of the Golgi; BIG2 is important for endosomal compartment integrity and cannot replace the function of BIG1 in Golgi organization.
    Full-text · Article · Mar 2010 · PLoS ONE
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    ABSTRACT: Class IX myosins are found in animals from invertebrates to vertebrates. Invertebrates contain a single myosin IX gene, whereas vertebrates contain two myosin IX genes, MYO9A and MYO9B. Mammalian Myo9b, the only class IX myosin studied so far, has unique motor properties. It is the first myosin for which ATP hydrolysis is the rate-limiting step in the chemical cycle and although it is a single-headed myosin, it can take multiple steps along F-actin before dissociating. Class IX myosins are motorized signaling molecules that contain in their tail domain a Rho GTPase-activating protein (GAP) activity. Mammalian members of myosin class IX negatively regulate the monomeric GTP-binding proteins RhoA-C. In cells, Myo9b accumulates in regions of active actin polymerization such as in extending lamellipodia. In these regions Myo9b might locally down regulate contractility and adhesion that are controlled by Rho activity and thereby contribute to sustained lamellipodial extension and cell polarity.
    No preview · Chapter · Jan 1970
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    ABSTRACT: BIG1 and BIG2 are large (∼200 kDa) guanine nucleotide‐exchange proteins for ADP‐ribosylation factors, or ARFs, that were isolated based on sensitivity of their guanine nucleotide‐exchange activity to inhibition by brefeldin A. The intracellular distributions of BIG1 and BIG2 differ from those of other ARF guanine nucleotide‐exchange proteins. In addition to its presence in Golgi membranes, BIG2 is seen in peripheral vesicular structures that most likely represent recycling endosomes, and BIG1 is found in nuclei of serum‐starved HepG2 cells. Several binding partners for BIG1 and BIG2 that were identified via yeast two‐hybrid screens include FKBP13 and myosin IXb for BIG1 and, for BIG2, the regulatory RIα subunit of protein kinase A, Exo70, and the GABA receptor β subunit. Autosomal recessive periventricular heterotopia with microcephaly, a disorder of human embryonic development due to defective vesicular trafficking, has been attributed to mutations in BIG2. Methods for purification of BIG1 and BIG2 from HepG2 cells are presented here, along with a summary of information regarding their structure and function.
    No preview · Article · Feb 2002 · Methods in Enzymology
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