Ser71 Phosphorylation Inhibits Actin-Binding of Profilin-1 and Its Apoptosis-Sensitizing Activity

Abstract

The essential actin-binding factor profilin-1 (Pfn1) is a non-classical tumor suppressor with the abilities toboth inhibit cellular proliferation and augment chemotherapy-induced apoptosis. Besides actin, Pfn1 interacts with proteins harboring the poly-L-proline (PLP) motifs. Our recent work demonstrated that both nuclear localization and PLP-binding are required for tumor growth inhibition by Pfn1, and this is at least partially due to Pfn1 association with the PLP-containing ENL protein in the Super Elongation Complex (SEC) and the transcriptional inhibition of pro-cancer genes. In this paper, by identifying a phosphorylation event of Pfn1 at Ser71 capable of inhibiting its actin-binding and nuclear export, we provide in vitro and in vivo evidence that chemotherapy-induced apoptotic sensitization by Pfn1 requires its cytoplasmic localization and actin-binding. With regard to tumor growth inhibition byPfn1, our data indicate a requirement for dynamic actin association and dissociation rendered by reversible Ser71phosphorylation and dephosphorylation. Furthermore, genetic and pharmacological experiments showed that Ser71 of Pfn1 can be phosphorylated by protein kinase A (PKA). Taken together, our data provide novel mechanistic insights into the multifaceted anticancer activities of Pfn1 and how they are spatially-defined in the cell and differentially regulated by ligand-binding.

Phospho-Ser784-VCP Drives Resistance of Pancreatic Ductal Adenocarcinoma to Genotoxic Chemotherapies and Predicts the Chemo-Sensitizing Effect of VCP Inhibitor

“…All cell lines including PANC-1, MIA PaCa-2, Capan-1, Pa01C, and HEK293T were mycoplasma-tested annually and cultured in high glucose DMEM supplemented with 10% fetal bovine serum and 50 mg/mL gentamicin. Lentiviruses were produced by transient transfection of HEK293T cells as previously described [25,[29][30][31].…”

“…Briefly, cells were grown in 96-well plates, treated, and detergent extracted for 8 min, fixed by 4% paraformaldehyde for 15 min, washed by PBS/0.1% Triton X-100, blocked with 5% normal goat serum for 60 min, and incubated with primary antibodies (1:1000; custom-made mouse anti-pSer 784 -VCP and rabbit anti-γH2AX, Cell Signaling #9718) overnight at 4 • C. Cells were washed with PBS/0.1% Triton X-100, incubated with Alexa 488 or 594-conjugated secondary antibodies (Invitrogen, Waltham, MA, USA) for 2 h at room temperature, washed with PBS/0.1% Triton X-100, and counterstained with DAPI. Fluorescence images were acquired on an inverted Olympus IX70 microscope equipped with CellSens software as previously described [25,30,31].…”

“…For colony formation assay, PDAC cells were plated in triplicates in 24-well plates at a 400cells-per-well density, allowed to adhere overnight, treated with SN38 or Etoposide for 16 hours, and grown in drug-free media for 14 days. End-point viable cells were quantified by Alamar blue and colonies were visualized by crystal violet staining as previously described [25,30,31].…”

Cofilin-1 and profilin-1 expression in lung microvascular endothelial cells exposed to titanium dioxide nanoparticles

“…The phosphorylation of Ser 71 inhibits actin-binding of profilin-1. 30 The mechanisms by which toxic nanoparticles induce pulmonary disease remain to be elucidated. In this study, we examined whether nanoparticles play a role in lung disease employing two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS) of endothelial cells, and examined protein expression in the lungs of a mouse model of asthma.…”

Suppressive Effects of Tyrosine (Y129) Phosphorylation of Profilin I on Breast Cancer Progression

“…Post-translational modifications (PTM), such as nitration and phosphorylation (S137, Y219), of profilin 1 alter its biological functions [16][17][18]. Profilin is also regulated by phosphorylation at sites T89, S71, Y129 [19][20][21]. It has been demonstrated that phosphorylation of profilin is counter acted by protein phosphatase-1, and the switch between the two states regulates cell proliferation and survival [22].…”