Analysis of RhoA-binding proteins reveals an interaction domain conserved in heterotrimeric G protein beta subunits and the yeast response regulator protein Skn7.
To identify potential RhoA effector proteins, we conducted a two-hybrid screen for cDNAs encoding proteins that interact with a Gal4-RhoA.V14 fusion protein. In addition to the RhoA effector ROCK-I we identified cDNAs encoding Kinectin, mDia2 (a p140 mDia-related protein), and the guanine nucleotide exchange factor, mNET1. ROCK-I, Kinectin, and mDia2 can bind the wild type forms of both RhoA and Cdc42 in a GTP-dependent manner in vitro. Comparison of the ROCK-I and Kinectin sequences revealed a short region of sequence homology that is both required for interaction in the two-hybrid assay and sufficient for weak interaction in vitro. Sequences related to the ROCK-I/Kinectin sequence homology are present in heterotrimeric G protein beta subunits and in the Saccharomyces cerevisiae Skn7 protein. We show that beta2 and Skn7 can interact with mammalian RhoA and Cdc42 and yeast Rho1, both in vivo and in vitro. Functional assays in yeast suggest that the Skn7 ROCK-I/Kinectin homology region is required for its function in vivo.
Alberts
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Transcription Laboratory, Imperial Cancer Research Fund Laboratories, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom. alberts@cc.ucsf.edu
Bouquin
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N
Johnston
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Treisman
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D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry; D017124:Conserved Sequence; D018076:DNA, Complementary; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D005656:Fungal Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D051379:Mice; D008969:Molecular Sequence Data; D015513:Oncogene Proteins / Q000737:chemistry; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry; D011956:Receptors, Cell Surface / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein
D002352:Carrier Proteins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D047908:Intracellular Signaling Peptides and Proteins; C088547:KTN1 protein, human; C511012:Ktn1 protein, mouse; D046911:Macromolecular Substances; D008565:Membrane Proteins; C490988:Net1 protein, mouse; D015513:Oncogene Proteins; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; C083962:SKN7 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D019204:GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein