Proteins that associate with the GTP‐bound forms of the Ras superfamily of proteins are potential effector targets for these molecular switches. A 195 kDa protein was purified from cell lysates by affinity chromatography on immobilized cdc42Hs‐GTP and a corresponding cDNA was isolated. Sequence analysis revealed localized identities to calponin, the WW domain, unconventional myosins and to the rasGAP‐related domain (GRD) contained in IRA, NF‐1, SAR1 and rasGAP. p195 was found to be identical to IQGAP1, a protein previously reported to bind ras. Purified recombinant p195/IQGAP1 bound to and inhibited the GTPase activity of cdc42Hs and rac whereas no interaction with ras was detected. The C‐terminal half of IQGAP1 containing the GRD bound to cdc42 and rac in a GRD‐dependent fashion, but a smaller fragment containing only the GRD did not. Cdc42 was also co‐immunoprecipitated from cell lysates with antibody specific to p195/IQGAP1. Calmodulin also co‐immunoprecipitated with p195/IQGAP1 and was found to associate with fragments containing the IQ domain. Expression of a cDNA fragment encoding the GRD inhibited the CDC24/CDC42 pathway in yeast, but no effect on ras was observed. In mammalian cells, both endogenous and ectopically expressed p195/IQGAP1 were localized to lamellipodia and ruffling cell membranes, where co‐localization with actin was apparent. These results suggest that IQGAP1 is an effector target for cdc42Hs and may mediate the effects of this GTPase on cell morphology.
Most colorectal cancers (CRC) are initiated by mutations of APC, leading to increased b-catenin-mediated signaling. However, continued requirement of Wnt/b-catenin signaling for tumor progression in the context of acquired KRAS and other mutations is less well-established. To attenuate Wnt/b-catenin signaling in tumors, we have developed potent and specific small-molecule tankyrase inhibitors, G007-LK and G244-LM, that reduce Wnt/b-catenin signaling by preventing poly(ADP-ribosyl)ation-dependent AXIN degradation, thereby promoting b-catenin destabilization. We show that novel tankyrase inhibitors completely block ligand-driven Wnt/b-catenin signaling in cell culture and display approximately 50% inhibition of APC mutation-driven signaling in most CRC cell lines. It was previously unknown whether the level of AXIN protein stabilization by tankyrase inhibition is sufficient to impact tumor growth in the absence of normal APC activity. Compound G007-LK displays favorable pharmacokinetic properties and inhibits in vivo tumor growth in a subset of APC-mutant CRC xenograft models. In the xenograft model most sensitive to tankyrase inhibitor, COLO-320DM, G007-LK inhibits cell-cycle progression, reduces colony formation, and induces differentiation, suggesting that b-catenin-dependent maintenance of an undifferentiated state may be blocked by tankyrase inhibition. The full potential of the antitumor activity of G007-LK may be limited by intestinal toxicity associated with inhibition of Wnt/b-catenin signaling and cell proliferation in intestinal crypts. These results establish proof-of-concept antitumor efficacy for tankyrase inhibitors in APC-mutant CRC models and uncover potential diagnostic and safety concerns to be overcome as tankyrase inhibitors are advanced into the clinic. Cancer Res; 73(10); 3132-44. Ó2013 AACR.
Taken together, our data strongly implicate PAK4 in oncogenic transformation and suggest that PAK4 activity is required for Ras-driven, anchorage-independent growth.
Canonical Wnt signaling is controlled intracellularly by the level of β-catenin protein, which is dependent on Axin scaffolding of a complex that phosphorylates β-catenin to target it for ubiquitylation and proteasomal degradation. This function of Axin is counteracted through relocalization of Axin protein to the Wnt receptor complex to allow for ligand-activated Wnt signaling. AXIN1 and AXIN2 protein levels are regulated by tankyrase-mediated poly(ADP-ribosyl)ation (PARsylation), which destabilizes Axin and promotes signaling. Mechanistically, how tankyrase limits Axin protein accumulation, and how tankyrase levels and activity are regulated for this function, are currently under investigation. By RNAi screening, we identified the RNF146 RING-type ubiquitin E3 ligase as a positive regulator of Wnt signaling that operates with tankyrase to maintain low steady-state levels of Axin proteins. RNF146 also destabilizes tankyrases TNKS1 and TNKS2 proteins and, in a reciprocal relationship, tankyrase activity reduces RNF146 protein levels. We show that RNF146, tankyrase, and Axin form a protein complex, and that RNF146 mediates ubiquitylation of all three proteins to target them for proteasomal degradation. RNF146 is a cytoplasmic protein that also prevents tankyrase protein aggregation at a centrosomal location. Tankyrase auto-PARsylation and PARsylation of Axin is known to lead to proteasome-mediated degradation of these proteins, and we demonstrate that, through ubiquitylation, RNF146 mediates this process to regulate Wnt signaling.
Precise spatial and temporal regulation of Rho GTPases is required in controlling F-actin-based changes in cell morphology. The molecular mechanisms through which microtubules (MTs) modulate the activity of RhoGTPases and regulate the actin cytoskeleton are unclear. Here we show that p21-activated-kinase 4 (PAK4) mediates morphological changes through its association with the Rho-family guanine nucleotide exchange factor (GEF), GEF-H1. We show that this association is dependent upon a novel GEF-H1 interaction domain (GID) within PAK4. Further, we show that PAK4-mediated phosphorylation of Ser810 acts as a switch to block GEF-H1-dependent stress fiber formation while promoting the formation of lamellipodia in NIH-3T3 cells. We found that the endogenous PAK4-GEF-H1 complex associates with MTs and that PAK4 phosphorylation of MT-bound GEF-H1 releases it into the cytoplasm of NIH-3T3 cells, which coincides with the dissolution of stress fibers. Our observations propose a novel role for PAK4 in GEF-H1-dependent crosstalk between MTs and the actin cytoskeleton.
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