The Journaling of Olsen 368

Pancreatic acini were also pre-incubated with the conventional PKC isoform inhibitor Gö6976 (2 µM) and stimulated with CCK or PMA . The response to both agonists was abolished in the presence of Gö6976 (Fig. 3A). These results further support the participation of PKCα in RhoA translocation induced by CCK. To test for further inhibition related to a different conventional PKC isoform, pancreatic acini expressing DN-PKCα or β-Gal, were pre-incubated with or without GF X . Pancreatic acini were stimulated with CCK and RhoA translocation was analyzed. The results showed that there is no further inhibition in the response to CCK; all the treatment abolished RhoA translocation as the same extent (Fig. 3B), which indicates that there is no other conventional PKC isoform involved.
Measurement of Rac activity in cell lysates 10 min after FGF2 stimulation demonstrated a 1.5-fold increase in cells expressing WT RhoG (Fig. 1 c), whereas A37 RhoG significantly blunted Rac1 activation. Conversely, the constitutively active V12 RhoG mutant induced high Rac1 activity that did not change after FGF2 treatment. Second, Rabbit anti Rho-GDI (Phosphospecific) Polyclonal Antibody we examined the effect of knocking down RhoG expression, in this case measuring Rac1 activity in live cells using an intramolecular fluorescence resonance energy transfer probe (Raichu-Rac1). This probe measures the local balance of GEF and GAP activities but is insensitive to regulation by RhoGDI (Itoh et al., 2002).

Immunoblots were probed with antibodies raised against Cdc42 and the myc epitope. 3HA-CDC42 RDI1–3myc cells carrying either GAL1-GST-SKM1 on a plasmid or the empty plasmid were induced with galactose for 120 min. CLA4 overexpression decreases the cytoplasmic pool of Rho1 and Cdc42. Cells expressing the indicated 3HA-tagged Rho GTPase and carrying either GAL1-myc-CLA4 on a plasmid or the empty plasmid, respectively, were induced for 120 min. Cells were lysed, and equal amounts of protein extract were separated by centrifugation at 100,000 × g. Rho proteins were detected by immunoblotting using antibodies against the HA epitope.
Oocytes were stored in 1x modified Barth’s solution (88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.82 mM MgSO4, 0.33 mM NaNO3, 0.41 mM CaCl2, 10 mM HEPES, pH 7.4) with 100 µg/mL gentamicin sulfate, 6 µg/mL tetracycline and 25 µg/mL ampicillin at 16°C. Prior to manual defolliculation with forceps, oocytes were treated with 8 mg/mL type I collagenase in 1x modified Barth’s solution for 1 hr at 16°C on an orbital shaker. The overexpression of WT-RhoGDI1 or S34D-RhoGDI1 inhibits CCK-induced amylase secretion whereas S96D-RhoGDI1 does not. RhoGDI1 interacts with inactive RhoA and Rac1 and upon CCK stimulation both complexes are dissociated. Results were expressed as means ± SEM of 3 to 6 separate experiments. The statistical analysis was performed by ANOVA following by the Student-Newman-Keuls test.
A body of evidence shows that Rho GTPases can regulate COX-2 protein expression [16–18] and that COX-2 is overexpressed in many types of cancer. Strikingly, western blot analysis revealed that siRhoGDI cells displayed significantly higher levels of COX-2 when compared to parental and siLuc control cells (Fig. 3A). The upregulation of COX-2 protein was selective to loss of RhoGDI as silencing D4-GDI did not increase COX-2 protein expression (Fig. 3A). To confirm a direct inverse correlation with RhoGDI and COX-2, we rescued RhoGDI expression in siRhoGDI cells by transfecting a plasmid encoding RhoGDI mutant cDNA. In this construct the siRhoGDI targeted region was mutated to prevent knockdown by the pre-existing siRhoGDI in the cells. When RhoGDI expression was restored, the level of COX-2 was reduced to basal levels as observed in parental cells (Fig. 3B).

Comparison of IT-Cdc42 to a Cdc42 activity reporter (mRFP-wGBD; Benink and Bement 2005) revealed that IT-Cdc42 localized throughout the active Cdc42 zone, but extended slightly beyond it towards the wound center (Fig1B,C; see also below). We also tested the behavior of IT-Rac and found that it concentrated around wounds in the same region as IT-Cdc42 as expected from previous experiments (SuppFig2; Abreu-Blanco, Verboon, & Parkhurst, 2014; Benink & Bement, 2001). These results indicate that the IT and Cy3-tagged GTPase variants can interact with diverse regulators required to achieve their normal enrichment at wounds. Increased cardiac expression of Rho GDIα inhibited the activity of Rho family members.
To date there are three human RhoGDIs that have been identified, including RhoGDI (RhoGDI-1or RhoGDI-α), D4-GDI (RhoGDI-2 or RhoGDI-β), and RhoGDI-3. Together they are responsible for the regulation of the entire Rho GTPase family consisting of at least 22 members. RhoGDI is the founding member and binds to all Rho GTPases that have been examined (e.g., Rac1, Cdc42, and RhoA). Increasing evidence shows that RhoGDI protein expressions are aberrantly regulated in cancer cells when compared to normal counterparts. The altered expression of RhoGDI protein is expected to directly impact Rho GTPases activity and downstream signaling cascades. However, conflicting results have been reported for RhoGDI expression in breast cancer cells .

Accumulating evidence shows that RhoGDI and D4-GDI are aberrantly expressed in certain types of human cancers . For example while ovarian cancers have been shown to display high levels of both RhoGDI and D4-GDI compared to normal tissue; RhoGDI has been shown to be under expressed in both primary non-small cell lung cancer and malignant gliomas . Similarly, D4-GDI expression is reported to be significantly lower in primary bladder carcinoma when compared to normal tissue [26–28] and has also been identified as a suppressor of metastasis.
Recently, the heterotrimeric G protein Gα13 has been shown to participate in the activation of RhoA induced by CCK in isolated pancreatic acini . Regulates the GDP/GTP exchange reaction of the Rho proteins by inhibiting the dissociation of GDP from them, and the subsequent binding of GTP to them. Retains Rho proteins such as CDC42, RAC1 and RHOA in an inactive cytosolic pool, regulating their stability and protecting them from degradation. Actively involved in the recycling and distribution of activated Rho GTPases in the cell, mediates extraction from membranes of both inactive and activated molecules due its exceptionally high affinity for prenylated forms. Through the modulation of Rho proteins, may play a role in cell motility regulation.
In contrast, membrane extraction of Rho4 by Rdi1 results in the degradation of this Rho protein. We found that this proteolytic pathway includes the proteasome, vacuolar proteases, and the GSK-3β homologue Ygk3. Recent evidence from the laboratory of Bokoch has identified pak 21-kinase, an effector protein for Cdc42, as the RhoGDI kinase, which phosphorylates RhoGDI and facilitates the dissociation of Rac1 from Rac1/GDI complex. Therefore, it is likely that GSIS involves potential cross-talk between multiple Rho GTPases (e.g., Cdc42 and Rac1) as we originally proposed previously .

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