Protein kinase a-dependent phosphorylation of serine 119 in the proto-oncogenic serine/arginine-rich splicing factor 1 modulates its activity as a splicing enhancer protein

Abstract

Serine/arginine-rich splicing factor 1 (SRSF1), previously designated SF2/ASF, belongs to a family of SR proteins that regulate constitutive and alternative splicing. SRSF1 expression is increased in tumors from several tissues and elicits changes in key target genes involved in tumor genesis. Several protein kinases phosphorylate SRSF1, which regulates its localization and function. It is previously reported that protein kinase A (PKA) phosphorylates SRSF1, but the importance of this modification is not well characterized. Here, we show that PKA phosphorylates SRSF1 on serine 119 in vitro. Phosphorylation of SRSF1 on this site enhanced the RNA binding capacity of SRSF1 in vivo and reduced the protein's capacity to activate splicing of the Minx transcript in vitro. We also confirm an interaction between SRSF1 and PKA Cα1 and demonstrate that this interaction is not dependent on serine 119 phosphorylation but requires active PKA Cα1. We conclude that PKA phosphorylation of SRSF1 at serine 119 regulates SFRS1-dependent RNA binding and processing but not its interaction with PKA.

Keywords: PKA; SRSF1; phosphorylation; pre-mRNA splicing regulation.

Figure 1.

Domains and putative PKA phosphorylation sites in SRSF1. Schematic representation of SRSF1 showing the sequence and location of the 4 putative PKA phosphorylation sites predicted by the NetPHosK server. The deleted sequence in SRSF1 ΔRS (aa 202-248) is expanded.

Figure 2.

PKA phosphorylates SRSF1 at serine 119 in vitro. Purified SRSF1 (lanes 1 and 2), SRSF1 S119A (lanes 3 and 4), SRSF1 ΔRS (lanes 5 and 6), and SRSF1 ΔRS S119A (lanes 7 and 8) were incubated with active or heat-inactivated PKA Cα1 and γ-[³²P]-ATP in a reaction buffer. The samples were analyzed by SDS-PAGE followed by Coomassie staining (lower panel) and autoradiography (upper panel).

Figure 3.

SRSF1 and SRSF1 S119A are localized in the nucleus. Immunofluorescence of PF-fixated U2OS cells transfected with SRSF1 wild-type (upper panel) or SRSF1 S119A (lower panel) and stained with anti-SRSF1 (left column, blue). DNA was visualized by Hoechst staining (middle column, blue). Photograph overlays are shown to the right (Merge).

Figure 4.

SRSF1 interacts with catalytically active PKA Cα1. (A) 293T cells were co-transfected with either PKA Cα1 and SRSF1 (lanes 1-3) or PKA Cα1 and SRSF1 S119A (lanes 4-9). The cell lysates were adjusted to equal protein concentration for each experiment and precleared with magnetic beads before input samples were collected (lanes 1, 4, and 7). The lysates were subjected to IPs using anti-SRSF1 (lanes 3 and 6), anti-PKA Cα (lane 9), mouse IgG (lanes 2 and 5), or rabbit IgG (lane 8) and magnetic beads. All samples were analyzed by SDS-PAGE and immunoblotting using anti-SRSF1 (upper panel) and anti-PKA C (lower panel). (B) 293T cells were co-transfected with either SRSF1 and PKA Cα1 (lanes 1-3) or SRSF1 and PKA Cα1 K73M (lanes 4-9). The cell lysates were adjusted to equal protein concentration for each experiment and precleared with magnetic beads before input samples were collected (lanes 1, 4, and 7). The lysates were subjected to IPs using anti-PKA Cα (lanes 3 and 6), anti-SRSF1 (lane 9), mouse IgG (lane 8), or rabbit IgG (lanes 2 and 5) and magnetic beads. All samples were analyzed by SDS-PAGE and immunoblotting using anti-SRSF1 (upper panel) and anti-PKA C (lower panel).

Figure 5.

Mutation of serine 119 decreases the ability of SRSF1 to interact with RNA. (A) 293T cells were transfected with SRSF1 (lanes 1 and 2) or SRSF1 S119A (lanes 3 and 4). Twenty hours posttransfection, the cells were UV cross-linked and lysed. The cell lysates were adjusted to equal protein concentration and treated with T1 RNase before IPs with magnetic beads conjugated with either mouse IgG (lanes 1 and 3) or anti-SRSF1 (lanes 2 and 4). Immunoprecipitated samples were dephosphorylated, labeled with γ-[³²P]-ATP by PNK kinase, and run on a denaturating polyacrylamide gel before analysis by autoradiography. The immunoblot (lower panel) shows the amount of SRSF1 in the cell lysate. The arrows indicate accumulation of specific RNA species. (B) Lanes in unsaturated images were manually detected in Adobe Photoshop using identical frames. The obtained intensities were adjusted for background and analyzed in GraphPad Prism by a paired t test (n = 3).

Figure 6.

Mutation of serine 119 enhances the ability of SRSF1 to activate splicing of the Minx transcript in vitro. (A) In vitro splicing of Minx pre-mRNA in HeLa-NE (lane 1) or S100 extracts (lanes 2-5). The splicing-deficient S100 extracts were supplemented with either no addition (lane 2), SR mix (lane 3), SRSF1 (lane 4), or SRSF1 S119A (lane 5). The identities of the RNAs are shown schematically at the right side of the panel. (B) Unsaturated images were quantified in Scion Image, adjusted for background, and analyzed by a paired t test in GraphPad Prism (n = 9).