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

Cell Commun Signal. 2021 Feb 22;19(1):22. doi: 10.1186/s12964-021-00710-5.

Abstract

Background: Metastasis is the predominant cause for cancer morbidity and mortality accounting for approximatively 90% of cancer deaths. The actin-bundling protein L-plastin has been proposed as a metastatic marker and phosphorylation 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 inhibition/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. Video abstract.

Keywords: Actin-bundling; ERK/MAPK pathway; Extracellular matrix degradation; Invadopodia; Invasion; L-plastin; Metastasis; PI3K pathway; RSK; SGK.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Breast Neoplasms / metabolism*
  • Breast Neoplasms / pathology
  • Cell Line
  • Cell Movement
  • Female
  • Humans
  • Membrane Glycoproteins / genetics
  • Membrane Glycoproteins / metabolism*
  • Microfilament Proteins / genetics
  • Microfilament Proteins / metabolism*
  • Models, Biological
  • Neoplasm Invasiveness
  • Phosphatidylinositol 3-Kinases / metabolism*
  • Phosphorylation
  • Protein Kinases / metabolism*
  • Serine / metabolism

Substances

  • Membrane Glycoproteins
  • Microfilament Proteins
  • plastin
  • Serine
  • Protein Kinases