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Profilin2a-phosphorylation as a regulatory mechanism for actin dynamics.

FASEB journal : official publication of the Federation of American Societies for Experimental Biology (2020-01-08)
Lisa Marie Walter, Peter Franz, Robert Lindner, Georgios Tsiavaliaris, Niko Hensel, Peter Claus
ABSTRACT

Profilin is a major regulator of actin dynamics in multiple specific processes localized in different cellular compartments. This specificity is not only meditated by its binding to actin but also its interaction with phospholipids such as phosphatidylinositol (4,5)-bisphosphate (PIP2 ) at the membrane and a plethora of proteins containing poly-L-proline (PLP) stretches. These interactions are fine-tuned by posttranslational modifications such as phosphorylation. Several phospho-sites have already been identified for profilin1, the ubiquitously expressed isoform. However, little is known about the phosphorylation of profilin2a. Profilin2a is a neuronal isoform important for synapse function. Here, we identified several putative profilin2a phospho-sites in silico and tested recombinant phospho-mimetics with regard to their actin-, PLP-, and PIP2 -binding properties. Moreover, we assessed their impact on actin dynamics employing a pyrene-actin polymerization assay. Results indicate that distinct phospho-sites modulate specific profilin2a functions. We could identify a molecular switch site at serine residue 71 which completely abrogated actin binding-as well as other sites important for fine-tuning of different functions, for example, tyrosine 29 for PLP binding. Our findings suggest that differential profilin2a phosphorylation is a sensitive mechanism for regulating its neuronal functions. Moreover, the dysregulation of profilin2a phosphorylation may contribute to neurodegeneration.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Cyanogen bromide-activated-Sepharose® 4B, lyophilized powder
Sigma-Aldrich
Thermolysin from Geobacillus stearothermophilus, Type X, lyophilized powder, 30-350 units/mg protein (E1%/280)
Sigma-Aldrich
Anti-Profilin 2 (C-terminal) antibody produced in rabbit, IgG fraction of antiserum, buffered aqueous solution
Sigma-Aldrich
Atto 655-maleimide, BioReagent, suitable for fluorescence, ≥90% (coupling rate)

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