BIG1 is a binding partner of myosin IXb and regulates its Rho-GTPase activating protein activity
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.
Chapter: Class Ix Myosins[Show abstract] [Hide abstract]
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.01/1970: pages 391-401;
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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.Methods in Enzymology 02/2002; 345:397-404. DOI:10.1016/S0076-6879(05)04017-6 · 2.19 Impact Factor
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ABSTRACT: Small G proteins, which are essential regulators of multiple cellular functions, are activated by guanine nucleotide exchange factors (GEFs) that stimulate the exchange of the tightly bound GDP nucleotide by GTP. The catalytic domain responsible for nucleotide exchange is in general associated with non-catalytic domains that define the spatio-temporal conditions of activation. In the case of small G proteins of the Arf subfamily, which are major regulators of membrane trafficking, GEFs form a heterogeneous family whose only common characteristic is the well-characterized Sec7 catalytic domain. In contrast, the function of non-catalytic domains and how they regulate/cooperate with the catalytic domain is essentially unknown. Based on Sec7-containing sequences from fully-annotated eukaryotic genomes, including our annotation of these sequences from Paramecium, we have investigated the domain architecture of large ArfGEFs of the BIG and GBF subfamilies, which are involved in Golgi traffic. Multiple sequence alignments combined with the analysis of predicted secondary structures, non-structured regions and splicing patterns, identifies five novel non-catalytic structural domains which are common to both subfamilies, revealing that they share a conserved modular organization. We also report a novel ArfGEF subfamily with a domain organization so far unique to alveolates, which we name TBS (TBC-Sec7). Our analysis unifies the BIG and GBF subfamilies into a higher order subfamily, which, together with their being the only subfamilies common to all eukaryotes, suggests that they descend from a common ancestor from which species-specific ArfGEFs have subsequently evolved. Our identification of a conserved modular architecture provides a background for future functional investigation of non-catalytic domains.BMC Genomics 02/2005; 6:20. DOI:10.1186/1471-2164-6-20 · 4.04 Impact Factor