Norbin Stimulates the Catalytic Activity and Plasma Membrane Localization of the Guanine-Nucleotide Exchange Factor P-Rex1

ABSTRACTRhoA is overexpressed in human cancer and contributes to aberrant cell motility and metastatic progression; however, regulatory mechanisms controlling RhoA activity in cancer are poorly understood. Neuroepithelial transforming gene 1 (Net1) is a RhoA guanine nucleotide exchange factor that is overexpressed in human cancer. It encodes two isoforms, Net1 and Net1A, which cycle between the nucleus and plasma membrane. Net1 proteins must leave the nucleus to activate RhoA, but mechanisms controlling the extranuclear localization of Net1 isoforms have not been described. Here, we show that Rac1 activation causes relocalization of Net1 isoforms outside the nucleus and stimulates Net1A catalytic activity. These effects do not require Net1A catalytic activity, its pleckstrin homology domain, or its regulatory C terminus. We also show that Rac1 activation protects Net1A from proteasome-mediated degradation. Replating cells on collagen stimulates endogenous Rac1 to relocalize Net1A, and inhibition of proteasome activity extends the duration and magnitude of Net1A relocalization. Importantly, we demonstrate that Net1A, but not Net1, is required for cell spreading on collagen, myosin light chain phosphorylation, and focal adhesion maturation. These data identify the first physiological mechanism controlling the extranuclear localization of Net1 isoforms. They also demonstrate a previously unrecognized role for Net1A in regulating cell adhesion.

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The α-Subunit of the Mammalian Guanine Nucleotide-Exchange Factor eIF-2B Is Essential for Catalytic Activity in Vitro

Biochemical and Biophysical Research Communications ◽

10.1006/bbrc.1996.0495 ◽

1996 ◽

Vol 220 (3) ◽

pp. 843-847 ◽

Cited By ~ 12

Author(s):

Bridget L. Craddock ◽

Christopher G. Proud

Keyword(s):

Catalytic Activity ◽

Guanine Nucleotide Exchange Factor ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Α Subunit ◽

Exchange Factor ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factor

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Activation of Rab8 guanine nucleotide exchange factor Rabin8 by ERK1/2 in response to EGF signaling

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1412089112 ◽

2014 ◽

Vol 112 (1) ◽

pp. 148-153 ◽

Cited By ~ 31

Author(s):

Juanfei Wang ◽

Jinqi Ren ◽

Bin Wu ◽

Shanshan Feng ◽

Guoping Cai ◽

...

Keyword(s):

Plasma Membrane ◽

Membrane Trafficking ◽

Guanine Nucleotide Exchange Factor ◽

Small Gtpases ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Exchange Factor ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factor ◽

Egf Signaling

Exocytosis is tightly regulated in many cellular processes, from neurite expansion to tumor proliferation. Rab8, a member of the Rab family of small GTPases, plays an important role in membrane trafficking from the trans-Golgi network and recycling endosomes to the plasma membrane. Rabin8 is a guanine nucleotide exchange factor (GEF) and major activator of Rab8. Investigating how Rabin8 is activated in cells is thus pivotal to the understanding of the regulation of exocytosis. Here we show that phosphorylation serves as an important mechanism for Rabin8 activation. We identified Rabin8 as a direct phospho-substrate of ERK1/2 in response to EGF signaling. At the molecular level, ERK phosphorylation relieves the autoinhibition of Rabin8, thus promoting its GEF activity. We further demonstrate that blocking ERK1/2-mediated phosphorylation of Rabin8 inhibits transferrin recycling to the plasma membrane. Together, our results suggest that ERK1/2 activate Rabin8 to regulate vesicular trafficking to the plasma membrane in response to extracellular signaling.

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Ypt32 recruits the Sec4p guanine nucleotide exchange factor, Sec2p, to secretory vesicles; evidence for a Rab cascade in yeast

The Journal of Cell Biology ◽

10.1083/jcb.200201003 ◽

2002 ◽

Vol 157 (6) ◽

pp. 1005-1016 ◽

Cited By ~ 199

Author(s):

Darinel Ortiz ◽

Martina Medkova ◽

Christiane Walch-Solimena ◽

Peter Novick

Keyword(s):

Plasma Membrane ◽

Guanine Nucleotide Exchange Factor ◽

Secretory Vesicles ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Exchange Factor ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factor ◽

Polarized Transport ◽

Exchange Activity

SEC2 is an essential gene required for polarized growth of the yeast Saccharomyces cerevisiae. It encodes a protein of 759 amino acids that functions as a guanine nucleotide exchange factor for the small GTPase Sec4p, a regulator of Golgi to plasma membrane transport. Activation of Sec4p by Sec2p is needed for polarized transport of vesicles to exocytic sites. Temperature-sensitive (ts) mutations in sec2 and sec4 result in a tight block in secretion and the accumulation of secretory vesicles randomly distributed in the cell. The proper localization of Sec2p to secretory vesicles is essential for its function and is largely independent of Sec4p. Although the ts mutation sec2-78 does not affect nucleotide exchange activity, the protein is mislocalized. Here we present evidence that Ypt31/32p, members of Rab family of GTPases, regulate Sec2p function. First, YPT31/YPT32 suppress the sec2-78 mutation. Second, overexpression of Ypt31/32p restores localization of Sec2-78p. Third, Ypt32p and Sec2p interact biochemically, but Sec2p has no exchange activity on Ypt32p. We propose that Ypt32p and Sec4p act as part of a signaling cascade in which Ypt32p recruits Sec2p to secretory vesicles; once on the vesicle, Sec2p activates Sec4p, enabling the polarized transport of vesicles to the plasma membrane.

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Role of the guanine nucleotide exchange factor in Akt2-mediated plasma membrane translocation of GLUT4 in insulin-stimulated skeletal muscle

Cellular Signalling ◽

10.1016/j.cellsig.2014.07.002 ◽

2014 ◽

Vol 26 (11) ◽

pp. 2460-2469 ◽

Cited By ~ 17

Author(s):

Nobuyuki Takenaka ◽

Naoto Yasuda ◽

Yuma Nihata ◽

Tetsuya Hosooka ◽

Tetsuya Noguchi ◽

...

Keyword(s):

Skeletal Muscle ◽

Plasma Membrane ◽

Guanine Nucleotide Exchange Factor ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Membrane Translocation ◽

Exchange Factor ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factor

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GTPgammaS-induced actin polymerisation in vitro: ATP- and phosphoinositide-independent signalling via Rho-family proteins and a plasma membrane-associated guanine nucleotide exchange factor

Journal of Cell Science ◽

10.1242/jcs.111.11.1583 ◽

1998 ◽

Vol 111 (11) ◽

pp. 1583-1594

Author(s):

V.L. Katanaev ◽

M.P. Wymann

Keyword(s):

Plasma Membrane ◽

Guanine Nucleotide Exchange Factor ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Toxin B ◽

Exchange Factor ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factor ◽

Rho Family ◽

Rho Family Proteins

In a cell-free system from neutrophil cytosol GTP(γ)S can induce an increase in the number of free filament barbed ends and massive actin polymerisation and cross-linking. GTP(γ)S stimulation was susceptible to an excess of GDP, but not Bordetella pertussis toxin and could not be mimicked by aluminium fluoride, myristoylated GTPgammaS. Gialpha2 or Gbeta1gamma2 subunits of trimeric G proteins. In contrast, RhoGDI and Clostridium difficile toxin B (inactivating Rho family proteins) completely abrogated the effect of GTPgammaS. When recombinant, constitutively activated and GTPgammaS-loaded Rac1, RhoA, or Cdc42 proteins alone or in combination were probed at concentrations >100 times the endogenous, however, they were ineffective. Purified Cdc42/Rac-interactive binding (CRIB) domain of WASP or C3 transferase did not prevent actin polymerisation by GTPgammaS. The action of GTPgammaS was blocked by mM [Mg2+], unless a heat- and trypsin-sensitive component present in neutrophil plasma membrane was added. Liberation of barbed ends seems therefore to be mediated by a toxin B-sensitive cytosolic Rho-family protein, requiring a membrane-associated guanine nucleotide exchange factor (GEF) for its activation by GTPgammaS under physiologic conditions. The inefficiency of various protein kinase and phosphatase inhibitors (staurosporine, genistein, wortmannin, okadaic acid and vanadate) and removal of ATP by apyrase, suggests that phosphate transfer reactions are not required for the downstream propagation of the GTPgammaS signal. Moreover, exogenously added phosphoinositides failed to induce actin polymerisation and a PtdIns(4,5)P2-binding peptide did not interfere with the response to GTPgammaS. The speed and simplicity of the presented assay applicable to protein purification techniques will facilitate the further elucidation of the molecular partners involved in actin polymerisation.

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