L-plastin Ser5 phosphorylation is modulated by the PI3K/SGK pathway and promotes breast cancer cell invasiveness

in Cell Communication and Signaling (2021), 19(22), 1-22

Background: Metastasis is the predominant cause for cancer morbidity and mortality accounting for approxima‑ tively 90% of cancer deaths. The actin‑bundling protein L‑plastin has been proposed as a ... [more ▼]

Background: Metastasis is the predominant cause for cancer morbidity and mortality accounting for approxima‑ tively 90% of cancer deaths. The actin‑bundling protein L‑plastin has been proposed as a metastatic marker and phos‑ phorylation on its residue Ser5 is known to increase its actin‑bundling activity. We recently showed that activation of the ERK/MAPK signalling pathway leads to L‑plastin Ser5 phosphorylation and that the downstream kinases RSK1 and RSK2 are able to directly phosphorylate Ser5. Here we investigate the involvement of the PI3K pathway in L‑plastin Ser5 phosphorylation and the functional effect of this phosphorylation event in breast cancer cells. Methods: To unravel the signal transduction network upstream of L‑plastin Ser5 phosphorylation, we performed computational modelling based on immunoblot analysis data, followed by experimental validation through inhi‑ bition/overexpression studies and in vitro kinase assays. To assess the functional impact of L‑plastin expression/ Ser5 phosphorylation in breast cancer cells, we either silenced L‑plastin in cell lines initially expressing endogenous L‑plastin or neoexpressed L‑plastin wild type and phosphovariants in cell lines devoid of endogenous L‑plastin. The established cell lines were used for cell biology experiments and confocal microscopy analysis. Results: Our modelling approach revealed that, in addition to the ERK/MAPK pathway and depending on the cellular context, the PI3K pathway contributes to L‑plastin Ser5 phosphorylation through its downstream kinase SGK3. The results of the transwell invasion/migration assays showed that shRNA‑mediated knockdown of L‑plastin in BT‑20 or HCC38 cells significantly reduced cell invasion, whereas stable expression of the phosphomimetic L‑plastin Ser5Glu variant led to increased migration and invasion of BT‑549 and MDA‑MB‑231 cells. Finally, confocal image analysis combined with zymography experiments and gelatin degradation assays provided evidence that L‑plastin Ser5 phosphorylation promotes L‑plastin recruitment to invadopodia, MMP‑9 activity and concomitant extracellular matrix degradation. Conclusion: Altogether, our results demonstrate that L‑plastin Ser5 phosphorylation increases breast cancer cell invasiveness. Being a downstream molecule of both ERK/MAPK and PI3K/SGK pathways, L‑plastin is proposed here as a potential target for therapeutic approaches that are aimed at blocking dysregulated signalling outcome of both pathways and, thus, at impairing cancer cell invasion and metastasis formation. [less ▲]

Constitutive activation of oncogenic PDGFRalpha-mutant proteins occurring in GIST patients induces receptor mislocalisation and alters PDGFRalpha signalling characteristics.

in Cell Communication and Signaling (2015), 13

BACKGROUND: Gastrointestinal stromal tumours (GIST) are mainly characterised by the presence of activating mutations in either of the two receptor tyrosine kinases c-KIT or platelet-derived growth factor ... [more ▼]

BACKGROUND: Gastrointestinal stromal tumours (GIST) are mainly characterised by the presence of activating mutations in either of the two receptor tyrosine kinases c-KIT or platelet-derived growth factor receptor-alpha (PDGFRalpha). Most mechanistic studies dealing with GIST mutations have focused on c-KIT and far less is known about the signalling characteristics of the mutated PDGFRalpha proteins. Here, we study the signalling capacities and corresponding transcriptional responses of the different PDGFRalpha proteins under comparable genomic conditions. RESULTS: We demonstrate that the constitutive signalling via the oncogenic PDGFRalpha mutants favours a mislocalisation of the receptors and that this modifies the signalling characteristics of the mutated receptors. We show that signalling via the oncogenic PDGFRalpha mutants is not solely characterised by a constitutive activation of the conventional PDGFRalpha signalling pathways. In contrast to wild-type PDGFRalpha signal transduction, the activation of STAT factors (STAT1, STAT3 and STAT5) is an integral part of signalling mediated via mutated PDGF-receptors. Furthermore, this unconventional STAT activation by mutated PDGFRalpha is already initiated in the endoplasmic reticulum whereas the conventional signalling pathways rather require cell surface expression of the receptor. Finally, we demonstrate that the activation of STAT factors also translates into a biologic response as highlighted by the induction of STAT target genes. CONCLUSION: We show that the overall oncogenic response is the result of different signatures emanating from different cellular compartments. Furthermore, STAT mediated responses are an integral part of mutated PDGFRalpha signalling. [less ▲]

Light microscopy applications in systems biology: opportunities and challenges.

in Cell Communication and Signaling (2013), 11(1), 1-19

Biological systems present multiple scales of complexity, ranging from molecules to entire populations. Light microscopy is one of the least invasive techniques used to access information from various ... [more ▼]

Biological systems present multiple scales of complexity, ranging from molecules to entire populations. Light microscopy is one of the least invasive techniques used to access information from various biological scales in living cells. The combination of molecular biology and imaging provides a bottom-up tool for direct insight into how molecular processes work on a cellular scale. However, imaging can also be used as a top-down approach to study the behavior of a system without detailed prior knowledge about its underlying molecular mechanisms. In this review, we highlight the recent developments on microscopy-based systems analyses and discuss the complementary opportunities and different challenges with high-content screening and high-throughput imaging. Furthermore, we provide a comprehensive overview of the available platforms that can be used for image analysis, which enable community-driven efforts in the development of image-based systems biology. [less ▲]

Interferon-γ-induced activation of Signal Transducer and Activator of Transcription 1 (STAT1) up-regulates the tumor suppressing microRNA-29 family in melanoma cells

in Cell Communication and Signaling (2012), 10

Background: The type-II-cytokine IFN-γ is a pivotal player in innate immune responses but also assumes functions in controlling tumor cell growth by orchestrating cellular responses against neoplastic ... [more ▼]

Background: The type-II-cytokine IFN-γ is a pivotal player in innate immune responses but also assumes functions in controlling tumor cell growth by orchestrating cellular responses against neoplastic cells. The role of IFN-γ in melanoma is not fully understood: it is a well-known growth inhibitor of melanoma cells in vitro. On the other hand, IFN-γ may also facilitate melanoma progression. While interferon-regulated genes encoding proteins have been intensively studied since decades, the contribution of miRNAs to effects mediated by interferons is an emerging area of research.We recently described a distinct and dynamic regulation of a whole panel of microRNAs (miRNAs) after IFN-γ-stimulation. The aim of this study was to analyze the transcriptional regulation of miR-29 family members in detail, identify potential interesting target genes and thus further elucidate a potential signaling pathway IFN-γ → Jak→ P-STAT1 → miR-29 → miR-29 target genes and its implication for melanoma growth. Results: Here we show that IFN-γ induces STAT1-dependently a profound up-regulation of the miR-29 primary cluster pri-29a∼b-1 in melanoma cell lines. Furthermore, expression levels of pri-29a∼b-1 and mature miR-29a and miR-29b were elevated while the pri-29b-2∼c cluster was almost undetectable. We observed an inverse correlation between miR-29a/b expression and the proliferation rate of various melanoma cell lines. This finding could be corroborated in cells transfected with either miR-29 mimics or inhibitors. The IFN-γ-induced G1-arrest of melanoma cells involves down-regulation of CDK6, which we proved to be a direct target of miR-29 in these cells. Compared to nevi and normal skin, and metastatic melanoma samples, miR-29a and miR-29b levels were found strikingly elevated in certain patient samples derived from primary melanoma. Conclusions: Our findings reveal that the miR-29a/b1 cluster is to be included in the group of IFN- and STAT-regulated genes. The up-regulated miR-29 family members may act as effectors of cytokine signalling in melanoma and other cancer cells as well as in the immune system. © 2012 Schmitt et al.; licensee BioMed Central Ltd. [less ▲]

Signal transduction, receptors, mediators and genes: Younger than ever - The 13th meeting of the Signal Transduction Society focused on aging and immunology

in Cell Communication and Signaling (2010), 8

The 13th meeting of the Signal Transduction Society was held in Weimar, from October 28 to 30, 2009. Special focus of the 2009 conference was "Aging and Senescence", which was co-organized by the SFB 728 ... [more ▼]

The 13th meeting of the Signal Transduction Society was held in Weimar, from October 28 to 30, 2009. Special focus of the 2009 conference was "Aging and Senescence", which was co-organized by the SFB 728 "Environmentally-Induced Aging Processes" of the University of Düsseldorf and the study group 'Signal Transduction' of the German Society for Cell Biology (DGZ). In addition, several other areas of signal transduction research were covered and supported by different consortia associated with the Signal Transduction Society including the long-term associated study groups of the German Society for Immunology and the Society for Biochemistry and Molecular Biology, and for instance the SFB/Transregio 52 "Transcriptional Programming of Individual T Cell Subsets" located in Würzburg, Mainz and Berlin. The different research areas that were introduced by outstanding keynote speakers attracted more than 250 scientists, showing the timeliness and relevance of the interdisciplinary concept and exchange of knowledge during the three days of the scientific program. This report gives an overview of the presentations of the conference. © 2010 Entschladen et al; licensee BioMed Central Ltd. Entschladen, F.; Institute of Immunology, Witten/Herdecke UniversityGermany; email: entschladen@uni-wh.de [less ▲]