Activation of p115-RhoGEF Requires Direct Association of Gα13 and the Dbl Homology Domain

Chen, James Z.; Guo, Liang; Hadas, Jana; Gutowski, Stephen; Sprang, Stephen R.; Sternweis, Paul C.

doi:10.1074/jbc.m111.333716

Cited by 38 publications

(37 citation statements)

References 35 publications

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“…GNA13 is a guanine nucleotide binding protein or G protein that is thought to play a role in cell migration through regulation of the exchange activity of RGS-containing RhoGEFs. [30][31][32] We confirmed these observations by knockdown of either CDK2, CyclinA2 (CCNA2) or GNA13 using multiple shRNAs followed by cell proliferation assays in OCIP23 cells in the presence of different concentrations of MK-1775 (Fig. 7C, D, E).…”

Section: Pooled Rna Interference Screen Identifies Genetic Suppressorsupporting

confidence: 67%

Cytokinetic effects of Wee1 disruption in pancreatic cancer

et al. 2016

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The Wee1 kinase, which is activated in response to DNA damage, regulates exit from G2 through inhibitory phosphorylation of Cdk1/Cdc2, and is an attractive drug target. However, recent work has highlighted effects of Cdk2 phosphorylation by Wee1 on movement through S-phase, suggesting the potential to sensitize to S-phase specific agents by Wee1 inhibitors. In this paper we applied multiparametric flow cytometry to patient-derived pancreatic cancer xenograft tumor cells to study the cell cycle perturbations of Wee1 disruption via the small molecule inhibitor MK-1775, and genetic knockdown. We find that in vitro treatment with MK-1775, and to a lesser degree, Wee1 RNA transcript knockdown, results in the striking appearance of S-phase cells prematurely entering into mitosis. This effect was not seen in vivo in any of the models tested. Here, although we noted an increase of S-phase cells expressing the damage response marker gH2AX, treatment with MK-1775 did not significantly sensitize cells to the cytidine analog gemcitabine. Treatment with MK-1775 did result in a transient but large increase in cells expressing the mitotic marker phosphorylated H3S10 that reached a peak 4 hours after treatment. This suggests a role for Wee1 regulating the progression of genomically unstable cancer cells through G2 in the absence of extrinsically-applied DNA damage. A single dose of 8Gy ionizing radiation resulted in the time-dependent accumulation of Cyclin A2 positive/phosphorylated H3S10 negative cells at the 4N position, which was abrogated by treatment with MK-1775. Consistent with these findings, a genomescale pooled RNA interference screen revealed that toxic doses of MK-1775 are suppressed by CDK2 or Cyclin A2 knockdown. These findings support G2 exit as the more significant effect of Wee1 inhibition in pancreatic cancers.

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Section: Pooled Rna Interference Screen Identifies Genetic Suppressorsupporting

confidence: 67%

Cytokinetic effects of Wee1 disruption in pancreatic cancer

et al. 2016

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“…Together, these observations reveal that thrombin modulates cell migration via its PARs that are coupled to various G proteins in different cell types. Some studies have reported that G␣ 12/13 associates with RhoGEFs, such as p115 RhoGEF, involving their RH and DH domains, and enhances their GEF activity (63). It was also reported that the enhancement of the RhoGEF activity by G␣ 12/13 was greater if the RhoGEF is tyrosine-phosphorylated as compared with its nonphosphorylated form (64).…”

Section: Discussionmentioning

confidence: 99%

Novel Role for p21-activated Kinase 2 in Thrombin-induced Monocyte Migration

Gadepalli

Kotla

Heckle

et al. 2013

Journal of Biological Chemistry

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“…1) and full-length RhoA were inserted into pGEX-KG-TEV either with or without a C-terminal His 6 tag as described previously (10). Mutations were inserted by PCR sewing.…”

Section: Methodsmentioning

confidence: 99%

“…Whereas the RH domain of p115RhoGEF was first characterized as a GAP for the ␣ subunits, it is also necessary for the RhoGEF to mediate direct regulation of RhoA by G protein-coupled receptors that activate G 12 and G 13 (8,9). Recent studies indicate that the main function of the RH domain in activation is to bind and orient the GTPase so that the ␣-helical domain of G␣ 13 can interact effectively, albeit with low affinity, to the back side of the DH domain (10). In a mechanism not yet defined, this interaction stimulates the turnover rate of p115RhoGEF for activation of RhoA.…”

mentioning

confidence: 99%