Abstract
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.
Highlights
Members of the Rho family of GTPases regulate diverse cellular processes ranging from cytoskeletal organization to gene expression and cell transformation
Plasmids D2 and D3 carried the same partial Kinectin cDNA [20]; interestingly, Kinectin was previously identified as a putative RhoA effector, the cDNA fragment isolated in our screen does not overlap that isolated in the previous screen (15; see “Discussion”)
We identified cDNAs encoding two previously characterized effectors, ROCK-I and Kinectin, together with cDNAs for mNET1, the mouse homolog of NET1 [40], a putative guanine nucleotide exchange factor (GEF), and a novel protein, mDia2, which is related to p140 mDia [17]
Summary
Plasmids—Plasmids were constructed by standard techniques; details are available on request. CDNA clone D1 encodes IWNSDPREFT(ROCK-I codons 300 –1030)-KKKVNSRDL. CDNA clone D4 encodes IWNSDPRNLPSSP-(ROCK-I codons 456 –1028)-VNLERSMNRRY. CDNA clone D9 encodes IWNSDPREFT-(ROCK-I codons 349 –1025)-GELERSMNRRY. GAD-Kinectin-(1053–1327) (cDNA clones D2 and D3) encodes IWNSDPRDLP-(Kinectin codons 1053–1327). GAD-Kinectin-(1053–1327)-⌬HR encodes IEFPMGRDLP(Kinectin codons 1053–1327) with codons 1191–1215 replaced by GS. GAD-mDia2-(47–257) (cDNA clone D7) encodes mDia IWNSDPREF(mDia codons 47– 800)-VNSREIYES. GST-ROCK-(831–1010)-⌬HR encodes ROCK(831–1010)-ES, with codons 950 –966 deleted. GST-SKN7⌬HR encodes (Skn codons 1– 623) with codons 237–260 replaced by G. In Vitro Binding Assay—Equimolar amounts of each GST-fusion protein (ϳ100 –300 ng) were bound to glutathione-Sepharose beads and incubated with 10 ng of GTP␥S- or GDP-loaded RhoA.WT-9E10 or Cdc.WT-9E10 at 4 °C for 2 h, with agitation, in RB containing 0.5 mg/ml bovine serum albumin. Following fractionation by SDS-PAGE, GTPases were detected by immunoblotting with the 9E10 antibody
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