NORMAL GENOMIC

• Weernink PA et al.
• Activation of type I phosphatidylinositol 4-phosphate 5-kinase isoforms bythe Rho GTPases, RhoA, Rac1, and Cdc42.
• J Biol Chem. 2004; 279: 7840-9
• Display abstract

• Type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K) catalyzes theformation of the phospholipid, phosphatidylinositol 4,5-bisphosphate(PIP(2)), which is implicated in many cellular processes. The Rho GTPases,RhoA and Rac1, have been shown previously to activate PIP5K and to bindPIP5K. Three type I PIP5K isoforms (Ialpha,Ibeta, and Igamma) have beenidentified; however, it is unclear whether these isoforms aredifferentially or even sequentially regulated by Rho GTPases. Here we showthat RhoA and Rac1, as well as Cdc42, but not the Ras-like GTPases, RalAand Rap1A, markedly stimulate PIP(2) synthesis by all three PIP5K isoformsexpressed in human embryonic kidney 293 cells, both in vitro and in vivo.RhoA-stimulated PIP(2) synthesis by the PIP5K isoforms was mediated by theRhoA effector, Rho-kinase. Stimulation of PIP5K isoforms by Rac1 and Cdc42was apparently independent of and additive with RhoA- and Rho-kinase, asshown by studies with C3 transferase and Rho-kinase mutants. RhoA, and toa lesser extent Rac1, but not Cdc42, interacted in anucleotide-independent form with all three PIP5K isoforms. Binding ofPIP5K isoforms to GTP-bound, but not GDP-bound, RhoA could be displaced byRho-kinase, suggesting a direct and constitutive PIP5K-Rho GTPase binding,which, however, does not trigger PIP5K activation. In summary, ourfindings indicate that synthesis of PIP(2) by the three PIP5K isoforms iscontrolled by RhoA, acting via Rho-kinase, as well as Rac1 and Cdc42,implicating that regulation of PIP(2) synthesis has a central position insignaling by these three Rho GTPases.

• Ward Y et al.
• The GTP binding proteins Gem and Rad are negative regulators of theRho-Rho kinase pathway.
• J Cell Biol. 2002; 157: 291-302
• Display abstract

• The cytoskeletal changes that alter cellular morphogenesis and motilitydepend upon a complex interplay among molecules that regulate actin,myosin, and other cytoskeletal components. The Rho family of GTP bindingproteins are important upstream mediators of cytoskeletal organization.Gem and Rad are members of another family of small GTP binding proteins(the Rad, Gem, and Kir family) for which biochemical functions have beenmostly unknown. Here we show that Gem and Rad interface with the Rhopathway through association with the Rho effectors, Rho kinase (ROK) alphaand beta. Gem binds ROKbeta independently of RhoA in the ROKbetacoiled-coil region adjacent to the Rho binding domain. Expression of Geminhibited ROKbeta-mediated phosphorylation of myosin light chain andmyosin phosphatase, but not LIM kinase, suggesting that Gem acts bymodifying the substrate specificity of ROKbeta. Gem or Rad expression ledto cell flattening and neurite extension in N1E-115 neuroblastoma cells.In interference assays, Gem opposed ROKbeta- and Rad opposedROKalpha-mediated cell rounding and neurite retraction. Gem did not opposecell rounding initiated by ROKbeta containing a deletion of the Gembinding region, demonstrating that Gem binding to ROKbeta is required forthe effects observed. In epithelial or fibroblastic cells, Gem or Radexpression resulted in stress fiber and focal adhesion disassembly. Inaddition, Gem reverted the anchorage-independent growth and invasivenessof Dbl-transformed fibroblasts. These results identify physiological rolesfor Gem and Rad in cytoskeletal regulation mediated by ROK.

• Bilodeau D, Lamy S, Desrosiers RR, Gingras D, Beliveau R
• Regulation of Rho protein binding to membranes by rhoGDI: inhibition ofreleasing activity by physiological ionic conditions.
• Biochem Cell Biol. 1999; 77: 59-69
• Display abstract

• The Rho GDP dissociation inhibitor (GDI) is an ubiquitously expressedregulatory protein involved in the cycling of Rho proteins betweenmembrane-bound and soluble forms. Here, we characterized the Rhosolubilization activity of a glutathione S-transferase (GST) - GDI fusionprotein in a cell-free system derived from rat kidney. Addition of GST-GDIto kidney brush border membranes resulted in the specific release of Cdc42and RhoA from the membranes, while RhoB and Ras were not extracted. Therelease of Cdc42 and RhoA by GST-GDI was dose dependent and saturable withabout 50% of both RhoA and Cdc42 extracted. The unextracted Rho proteinswere tightly bound to membranes and could not be solubilized by repeatedGST-GDI treatment. These results demonstrated that kidney brush bordermembranes contained two populations of RhoA and Cdc42. Furthermore, theGST-GDI solubilizing activity on membrane-bound Cdc42 and RhoA wasabolished at physiological conditions of salt and temperature in alltissues examined. When using bead-immobilized GST-GDI, KCl did not reducedthe binding of Rho proteins. However, washing brush border membranes withKCl prior treatment by GST-GDI inhibited the extraction of Rho proteins.Taken together, these results suggest that the binding of GDI tomembrane-bound Cdc42 and RhoA occurs easily under physiological ionicstrength conditions, but a complementary factor is required to extractthese proteins from membranes. These observations suggest that theshuttling activity of GDI upon Rho proteins could be normallydownregulated under physiological conditions.

• Ahmed S et al.
• Cryptic Rac-binding and p21(Cdc42Hs/Rac)-activated kinase phosphorylationsites of NADPH oxidase component p67(phox).
• J Biol Chem. 1998; 273: 15693-701
• Display abstract

• Rac1 is a member of the Rho family of small molecular mass GTPases thatact as molecular switches to control actin-based cell morphology as wellas cell growth and differentiation. Rac1 and Rac2 are specificallyrequired for superoxide formation by components of the NADPH oxidase. Inbinding assays, Rac1 interacts directly with p67(phox), but not with theother oxidase components: cytochrome b, p40(phox), or p47(phox) (Prigmore,E., Ahmed, S., Best, A., Kozma, R. , Manser, E., Segal, A. W., and Lim, L.(1995) J. Biol. Chem. 270, 10717-10722). Here, the Rac1/2 interaction withp67(phox) has been characterized further. Rac1 and Rac2 can bind top67(phox) amino acid residues 170-199, and the N terminus (amino acids1-192) of p67(phox) can be used as a specific inhibitor of Rac signaling.Deletion of p67(phox) C-terminal sequences (amino acids 193-526), theC-terminal SH3 domain (amino acids 470-526), or the polyproline-rich motif(amino acids 226-236) stimulates Rac1 binding by approximately 8-fold.p21(Cdc42Hs/Rac)-activated kinase (PAK) phosphorylates p67(phox) aminoacid residues adjacent to the Rac1/2-binding site, and thisphosphorylation is stimulated by deletion of the C-terminal SH3 domain orthe polyproline-rich motif. These data suggest a role for crypticRac-binding and PAK phosphorylation sites of p67(phox) in control of theNADPH oxidase.

• Ridley AJ
• The GTP-binding protein Rho.
• Int J Biochem Cell Biol. 1997; 29: 1225-9
• Display abstract

• RhoA, RhoB and RhoC are three closely related proteins, and are members of the Ras super-family of small GTP-binding proteins. They bind and hydrolyse GTP, and are active in the GTP-bound form. Their activity in cells is regulated by exchange factors, GTPase activating proteins and guanine nucleotide dissociation inhibitors. Several potential downstream target proteins for Rho proteins have been identified, including protein kinases and adaptor-type proteins. Rho proteins regulate actin cytoskeletal organization; for example in fibroblasts RhoA induces the formation of actin stress fibres. Rho proteins are also involved in regulating secretion, pinocytosis and clathrin coat-mediated endocytosis, transcriptional activation and stimulation of DNA synthesis. In addition, there is evidence that Rho proteins can play a role in cell transformation, and thus Rho proteins or components of their signalling pathways may be potential targets for the development of anti-cancer therapies.

• Chou MM, Blenis J
• The 70 kDa S6 kinase complexes with and is activated by the Rho family Gproteins Cdc42 and Rac1.
• Cell. 1996; 85: 573-83
• Display abstract

• The 70 kDa ribosomol S6 kinase (pp70S6k) plays an important role in theprogression of cells through G1 phase of the cell cycle. However, littleis known of the signaling molecules that mediate its activation. Wedemonstrate that Rho family G proteins regulate pp70S6k activity in vivo.Activated alleles of Cdc42 and Rac1, but not RhoA, stimulate pp70S6kactivity in multiple cell types. Activation requires an intact effectordomain and isoprenylation of Cdc42 and Rac1. Coexpression of Dbl, anexchange factor for Cdc42, also activates pp70S6k. Growth factor-inducedactivation of pp70S6k is abrogated by dominant negative alleles of Cdc42and Rac1. In addition, Cdc42 and Rac1 form GTP-dependent complex with thecatalytically inactive form of pp70S6k in vitro and in vivo, suggesting amechanism by which these G proteins activate pp70S6k.

• Robertson D, Paterson HF, Adamson P, Hall A, Monaghan P
• Ultrastructural localization of ras-related proteins using epitope-taggedplasmids.
• J Histochem Cytochem. 1995; 43: 471-80
• Display abstract

• To determine the ultrastructural distribution of H-ras, the rho proteinsrho-A, rho-B, rho-C, and the rac1 protein (members of the ras GTP-bindingprotein family), we used cDNA expression plasmids in which a shortsequence coding for the epitope recognized by the anti c-myc monoclonalantibody 9E10 has been inserted at the N-terminus. Each of the expressedproteins has this epitope as a tag, allowing its localization by light andelectron microscopy by the same antibody. After nuclear microinjection ofthese plasmids into MDCK or Rat 2 cells, expression of the protein (6-18hr later) was confirmed by immunofluorescence labeling with 9E10 imaged byconfocal microscopy. For ultrastructural localization of these taggedproteins, a method was devised to process microinjected cells in situ intolow-temperature resin. The proteins were localized on the sections using9E10 detected with colloidal gold conjugates. Ha-ras protein was localizedalmost exclusively on the cell membranes. Rho-A and rho-C werepredominantly associated with the submembraneous actin network, and rho-Bwas found in association with multivesicular bodies. Rac1 protein inducesthe formation of large pinocytotic vesicles and was detected on thecytoplasmic face of these vacuoles. These experiments demonstrate thesuccessful use of this approach for detection of de novo synthesizedproteins from microinjected plasmids by both light and electron microscopyon a small (< 50 cells) sample size.