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Activated heterotrimeric G protein ?i subunits inhibit Rap-dependent cell adhesion and promote cell migration.

ABSTRACT: Our recent work uncovered novel roles for activated G?i signaling in the regulation of neutrophil polarity and adhesion. G?iGTP-mediated enhancement of neutrophil polarization was dependent on inhibition of cAMP/PKA signaling, whereas reversal of G??-stimulated adhesion was cAMP/PKA independent. To uncover the mechanism for G?i regulation of adhesion, we analyzed the effects of constitutively active G?i1(Q204L) expression on adhesion driven by constitutively active Rap1a(G12V) or its downstream effector Radil in neutrophil-like HL-60 cells, or in HT-1080 fibrosarcoma cells. In HT-1080 cells, Rap1a(G12V) or Radil cause an increase in cell spreading and adhesion to fibronectin, which are both reversed by G?i1(Q204L) but not WT G?i1 In contrast, G?i1(Q204L) did not reverse Rap1-GTP-interacting adaptor molecule (RIAM)-dependent increases in cell adhesion. This indicates that adhesion regulation by G?i-GTP occurs downstream of Rap1a and Radil, but is upstream of components such as integrins and talin that are regulated by both Radil and RIAM. HL-60 neutrophil-like cells expressing Rap1a(G12V) or Radil have an elongated phenotype because of enhanced uropod adhesion as they attempt to migrate on fibronectin. This elongated phenotype driven by Rap1a(G12V) or Radil is reversed by G?i1(Q204L), but not by WT G?i1 expression, suggesting that G?i-GTP also regulates adhesion in immune cells at the level of, or downstream of, Radil. These data identify a novel role of G?i-GTP in regulation of cell adhesion and migration. Cell migration involves cycles of adhesion and de-adhesion, and we propose that the dynamic spatiotemporal balance between G??-promoted adhesion and G?i-GTP reversal of adhesion is important for this process.

SUBMITTER: To JY

PROVIDER: S-EPMC5798289 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Activated heterotrimeric G protein αi subunits inhibit Rap-dependent cell adhesion and promote cell migration.

To Jesi Y JY Smrcka Alan V AV

The Journal of biological chemistry 20171219 5

Our recent work uncovered novel roles for activated Gαi signaling in the regulation of neutrophil polarity and adhesion. GαiGTP-mediated enhancement of neutrophil polarization was dependent on inhibition of cAMP/PKA signaling, whereas reversal of Gβγ-stimulated adhesion was cAMP/PKA independent. To uncover the mechanism for Gαi regulation of adhesion, we analyzed the effects of constitutively active Gαi1(Q204L) expression on adhesion driven by constitu ...[more]

PMID: 29259127

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Project description:IntroductionBreast cancer is one of the most common types of cancer in women. One of the genes that were found mutated in breast cancer is casein kinase 1 epsilon (CK1epsilon). Because CK1epsilon is a crucial regulator of the Wnt signaling cascades, we determined how these CK1epsilon mutations interfere with the Wnt pathway and affect the behavior of epithelial breast cancer cell lines.MethodsWe performed in silico modeling of various mutations and analyzed the kinase activity of the CK1epsilon mutants both in vitro and in vivo. Furthermore, we used reporter and small GTPase assays to identify how mutation of CK1epsilon affects different branches of the Wnt signaling pathway. Based on these results, we employed cell adhesion and cell migration assays in MCF7 cells to demonstrate a crucial role for CK1epsilon in these processes.ResultsIn silico modeling and in vivo data showed that autophosphorylation at Thr 44, a site adjacent to the breast cancer point mutations in the N-terminal lobe of human CK1epsilon, is involved in positive regulation of the CK1epsilon activity. Our data further demonstrate that, in mammalian cells, mutated forms of CK1epsilon failed to affect the intracellular localization and phosphorylation of Dvl2; we were able to demonstrate that CK1epsilon mutants were unable to enhance Dvl-induced TCF/LEF-mediated transcription, that CK1epsilon mutants acted as loss-of-function in the Wnt/beta-catenin pathway, and that CK1epsilon mutants activated the noncanonical Wnt/Rac-1 and NFAT pathways, similar to pharmacological inhibitors of CK1. In line with these findings, inhibition of CK1 promoted cell migration as well as decreased cell adhesion and E-cadherin expression in the breast cancer-derived cell line MCF7.ConclusionsIn summary, these data suggest that the mutations of CK1epsilon found in breast cancer can suppress Wnt/beta-catenin as well as promote the Wnt/Rac-1/JNK and Wnt/NFAT pathways, thus contributing to breast cancer development via effects on cell adhesion and migration. In terms of molecular mechanism, our data indicate that the breast cancer point mutations in the N-terminal lobe of CK1epsilon, which are correlated with decreased phosphorylation activities of mutated forms of CK1epsilon both in vitro and in vivo, interfere with positive autophosphorylation at Thr 44.

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Activated heterotrimeric G protein ?i subunits inhibit Rap-dependent cell adhesion and promote cell migration.

Publications

Activated heterotrimeric G protein α<sub>i</sub> subunits inhibit Rap-dependent cell adhesion and promote cell migration.

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