Phosphorylation on Ser5 increases the F-actin-binding activity of L-plastin and promotes its targeting to sites of actin assembly in cells

Author:

Janji Bassam1,Giganti Adeline1,De Corte Veerle2,Catillon Marie1,Bruyneel Erik3,Lentz Delphine1,Plastino Julie4,Gettemans Jan2,Friederich Evelyne1

Affiliation:

1. Laboratory for Molecular Biology, Genomics and Modelling, Public Research Centre for Health (CRP-Santé), 84 Val Fleuri, 1526 Luxembourg

2. Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University, Albert Baertsoenkaai 3, 9000 Ghent, Belgium and Flanders Interuniversity, Institute for Biotechnology (V.I.B.), 9052 Ghent, Belgium

3. Laboratory of Experimental Cancerology, Department of Radiotherapy and Nuclear Medicine, Ghent University Hospital (1P7), De Pintelaan 185, 9000 Ghent, Belgium

4. Laboratoire Physicochimie “Curie”, UMR168 CNRS/Institut Curie, 11, rue Pierre et Marie Curie, 75231 Paris CEDEX 05, France

Abstract

L-plastin, a malignant transformation-associated protein, is a member of a large family of actin filament cross-linkers. Here, we analysed how phosphorylation of L-plastin on Ser5 of the headpiece domain regulates its intracellular distribution and its interaction with F-actin in transfected cells and in in vitro assays. Phosphorylated wild-type L-plastin localised to the actin cytoskeleton in transfected Vero cells. Ser5Ala substitution reduced the capacity of L-plastin to localise with peripheral actin-rich membrane protrusions. Conversely, a Ser5Glu variant mimicking a constitutively phosphorylated state, accumulated in actin-rich regions and promoted the formation of F-actin microspikes in two cell lines. Similar to phosphorylated wild-type L-plastin, this variant remained associated with cellular F-actin in detergent-treated cells, whereas the Ser5Ala variant was almost completely extracted. When compared with non-phosphorylated protein, phosphorylated L-plastin and the Ser5Glu variant bound F-actin more efficiently in an in vitro assay. Importantly, expression of L-plastin elicited collagen invasion in HEK293T cells, in a manner dependent on Ser5 phosphorylation. Based on our findings, we propose that conversely to other calponin homology (CH)-domain family members, phosphorylation of L-plastin switches the protein from a low-activity to a high-activity state. Phosphorylated L-plastin might act as an integrator of signals controlling the assembly of the actin cytoskeleton and cell motility in a 3D-space.

Publisher

The Company of Biologists

Subject

Cell Biology

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