Alternative Splicing of Rac1 Generates Rac1b, a Self-activating GTPase

Fiegen, Dennis; Haeusler, Lars−Christian; Blumenstein, Lars; Herbrand, Ulrike; Dvorský, Radovan; Vetter, Ingrid R.; Ahmadian, Mohammad Réza

doi:10.1074/jbc.m310281200

Cited by 133 publications

(102 citation statements)

References 50 publications

(59 reference statements)

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“…Hence, the Rac1b insert sequence may alter the intrinsic biochemical properties, as well as interaction with regulators and effectors. Consistent with this possibility, Matos et al (2003) and Fiegen et al (2003) determined that Rac1b possessed impaired intrinsic GTPase activity, yet it retained GAP responsiveness in vitro and in vivo. Thus, unlike the G12V or Q61L mutants of Rac1, Rac1b can still be inactivated by GAP activity.…”

Section: Introductionmentioning

confidence: 73%

Rac1b, a tumor associated, constitutively active Rac1 splice variant, promotes cellular transformation

et al. 2004

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A novel splice variant of Rac1, designated Rac1b, is expressed in human breast and colon carcinoma cells. Rac1b contains an additional 19 amino-acid insert immediately behind the switch II domain, a region important for Rac1 interaction with regulators and effectors. Recent studies showed that Rac1b exhibited the biochemical properties of a constitutively activated GTPase, yet it showed impaired interaction with downstream effectors, suggesting that Rac1b may be defective in biological activity. Whether Rac1b is a biologically active protein was not addressed. Therefore, we evaluated the biochemical, signaling and growth-promoting properties of authentic Rac1b. Similar to previous observations, we found that Rac1b showed enhanced intrinsic guanine nucleotide exchange activity, impaired intrinsic GTPase activity, and failed to interact with RhoGDI. Surprisingly, we found that Rac1b, like the constitutively-activated and transforming Rac1(Q61L) mutant, promoted growth transformation of NIH3T3 cells. Rac1b-expressing cells also showed a loss of density-dependent and anchorage-dependent growth. Surprisingly, unlike activated Rac1(61L), Rac1b did not show enhanced activation of the nuclear factor jB (NF-jB) transcription factor or stimulate cyclin D1 expression, the signaling activities that best correlate with Rac1 transforming activity. However, Rac1b did promote activation of the AKT serine/threonine kinase. Therefore, we suggest that Rac1b selectively activates a subset of Rac1 downstream signaling pathways to facilitate cellular transformation.

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Section: Introductionmentioning

confidence: 73%

Rac1b, a tumor associated, constitutively active Rac1 splice variant, promotes cellular transformation

et al. 2004

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“…Rac1b, an alternative splice variant of Rac1, contains a 19-amino acid insert immediately behind the switch II region (residues 60 -76) of Rac1. This has been shown to be sufficient to disrupt effector interaction itself (19,30,57,58 Previous studies demonstrated that Ras causes activation of Rac and that Rac function is critical for Rasmediated growth transformation. In these studies, Rac function was blocked by use of a dominant negative Rac1(17N) mutant (11,12), which blocks RacGEF activation of Rac (59), or by NSC23766, a cellpermeable small molecule that can bind directly to Rac1 and prevent its activation by Rac-specific RhoGEFs (27).…”

Section: Discussionmentioning

confidence: 99%

Specificity and Mechanism of Action of EHT 1864, a Novel Small Molecule Inhibitor of Rac Family Small GTPases

Shutes

Onesto

Picard

et al. 2007

Journal of Biological Chemistry

286

263

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There is now considerable experimental evidence that aberrant activation of Rho family small GTPases promotes the uncontrolled proliferation, invasion, and metastatic properties of human cancer cells. Therefore, there is considerable interest in the development of small molecule inhibitors of Rho GTPase function. However, to date, most efforts have focused on inhibitors that indirectly block Rho GTPase function, by targeting either enzymes involved in post-translational processing or downstream protein kinase effectors. We recently determined that the EHT 1864 small molecule can inhibit Rac function in vivo. In this study, we evaluated the biological and biochemical specificities and biochemical mechanism of action of EHT 1864. We determined that EHT 1864 specifically inhibited Rac1-dependent platelet-derived growth factor-induced lamellipodia formation. Furthermore, our biochemical analyses with recombinant Rac proteins found that EHT 1864 possesses high affinity binding to Rac1, as well as the related Rac1b, Rac2, and Rac3 isoforms, and this association promoted the loss of bound nucleotide, inhibiting both guanine nucleotide association and Tiam1 Rac guanine nucleotide exchange factor-stimulated exchange factor activity in vitro. EHT 1864 therefore places Rac in an inert and inactive state, preventing its engagement with downstream effectors. Finally, we evaluated the ability of EHT 1864 to block Rac-dependent growth transformation, and we determined that EHT 1864 potently blocked transformation caused by constitutively activated Rac1, as well as Rac-dependent transformation caused by Tiam1 or Ras. Taken together, our results suggest that EHT 1864 selectively inhibits Rac downstream signaling and transformation by a novel mechanism involving guanine nucleotide displacement.Rho family proteins (20 human members) comprise a major branch of the Ras superfamily of small GTPases (1, 2). Like Ras, Rho GTPases function as GDP/GTP-regulated binary on-off switches. In resting, quiescent cells, Rho GTPases exist predominately in their inactive, GDP-bound state. Upon growth factor stimulation, Rho-specific guanine nucleotide exchange factors (RhoGEFs) 4 are activated, and they stimulate the intrinsic guanine nucleotide exchange activity to promote formation of the active GTP-bound protein. Rho-GTP binds preferentially to downstream effector proteins. Rho-specific GTPaseactivating proteins then stimulate the intrinsic GTP hydrolysis activity of Rho GTPases, returning the protein to its inactive state and terminating effector interaction.RhoA, Rac1, and Cdc42 are the best studied and understood members of the Rho GTPase family (1, 2). Rho GTPases are regulators of a diverse variety of cellular processes that include regulation of cell proliferation, actin cytoskeleton reorganization, and gene expression (3). Like Ras and other members of the Ras superfamily (4, 5), the aberrant activity of Rho GTPases has been implicated in cancer and other human diseases (6 -10). In particular, the Rac subfamily has also been linked to c...

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“…The resulting Rac1b protein fails to interact with Rho-GDI in cells and displays both an increased intrinsic nucleotide exchange activity and a decreased rate of GTP hydrolysis in vitro. Accordingly, Rac1b is isolated predominantly in the active GTP-bound state from cell lines (21)(22)(23)(24). Unexpectedly, several classic Rac signaling pathways including lamellipodia formation or the activation of PAK1 or c-Jun-NH 2 -kinase activities were not activated by Rac1b.…”

Section: Introductionmentioning

confidence: 98%

Increased Rac1b Expression Sustains Colorectal Tumor Cell Survival

Matos

Jordan

2008

Molecular Cancer Research

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The small GTPase Rac1 can stimulate various signaling pathways that contribute to cell transformation. In particular, the activation of the NFKB transcription factor initiates an antiapoptotic response and promotes cell cycle progression through increased cyclin D1 expression. As a potential oncogenic mechanism to up-regulate this pathway, the overexpression of the Rac1b splicing variant was reported in some colorectal tumors. Rac1b exists predominantly in the active GTP-bound state and selectively promotes the pathway leading to NFKB activation. Here, we studied the role of endogenous Rac1b in colorectal cancer cells. We found that depletion of Rac1b by small interfering RNAs inhibited endogenous NFKB activation and reduced cell viability to 50% within 48 hours. This reduction was due to increased apoptosis, although a reduced G 1 -S progression rate was also observed. These data show, for the first time, that colorectal cells expressing alternative spliced Rac1b also depend on Rac1b signaling to sustain their survival.

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Alternative Splicing of Rac1 Generates Rac1b, a Self-activating GTPase

Cited by 133 publications

References 50 publications

Rac1b, a tumor associated, constitutively active Rac1 splice variant, promotes cellular transformation

Rac1b, a tumor associated, constitutively active Rac1 splice variant, promotes cellular transformation

Specificity and Mechanism of Action of EHT 1864, a Novel Small Molecule Inhibitor of Rac Family Small GTPases

Increased Rac1b Expression Sustains Colorectal Tumor Cell Survival

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