Abstract
Profilin-1 (PFN1) plays an important role in the control of actin dynamics, and could represent an important therapeutic target in several diseases. We previously identified PFN1 as a huntingtin aggregation inhibitor, and others have implicated it as a tumor-suppressor. Rho-associated kinase (ROCK) directly phosphorylates PFN1 at Ser-137 to prevent its binding to polyproline sequences. This negatively regulates its anti-aggregation activity. However, the phosphatase that dephosphorylates PFN1 at Ser-137, and thus activates it, is unknown. Using a phospho-specific antibody against Ser-137 of PFN1, we characterized PFN1 dephosphorylation in cultured cells based on immunocytochemistry and a quantitative plate reader-based assay. Both okadaic acid and endothall increased pS137-PFN1 levels at concentrations more consistent with their known IC50s for protein phosphatase 1 (PP1) than protein phosphatase 2A (PP2A). Knockdown of the catalytic subunit of PP1 (PP1Cα), but not PP2A (PP2ACα), increased pS137-PFN1 levels. PP1Cα binds PFN1 in cultured cells, and this interaction was increased by a phosphomimetic mutation of PFN1 at Ser-137 (S137D). Together, these data define PP1 as the principal phosphatase for Ser-137 of PFN1, and provide mechanistic insights into PFN1 regulation by phosphorylation.
Highlights
Profilins are small actin-binding proteins that are essential for all eukaryotic cells
We previously determined by Western blot (WB) that pS137-PFN1 levels in cultured cells are regulated by RhoA/Rho-associated kinase (ROCK) signaling
The RhoA activator lysophosphatidic acid (LPA) mildly increased pS137-PFN1 levels in cultured cells, which was blocked by the ROCK inhibitor Y-27632 [6]
Summary
Profilins are small actin-binding proteins that are essential for all eukaryotic cells They play a role in many cellular processes including cell motility, cytokinesis, gene transcription, endocytosis and neuronal plasticity [1,2,3,4]. These activities depend on their interactions with three major cellular ligands: globular actin (Gactin), polyproline-containing proteins, and phosphatidylinositols (e.g. phosphatidylinositol 4,5-bisphosphate, PIP2). Two testis-specific profilins, PFN3 and PFN4, are recently described, and differ substantially from PFN1 and PFN2a in their primary sequences [5]. PFN3 binds polyproline-ligands poorly, and PFN4 completely lacks this activity [5]
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