Phosphorylation status of human RNA-binding protein 8A in cells and its inhibitory regulation by Magoh

Abstract

The RNA-binding protein 8A (RBM8A)-mago-nashi homolog, proliferation-associated (Magoh) complex is a component of the exon junction complex (EJC) required for mRNA metabolism involving nonsense-mediated mRNA decay (NMD). RBM8A is a phosphorylated protein that plays some roles in NMD. However, the detailed status and mechanism of the phosphorylation of RBM8A is not completely understood. Therefore, in this study, we analyzed in detail RBM8A phosphorylation in human cells. Accordingly, analysis of the phosphorylation status of RBM8A protein in whole-cell lysates by using Phos-tag gels revealed that the majority of endogenous RBM8A was phosphorylated throughout the cell-cycle progression. Nuclear and cytoplasmic RBM8A and RBM8A in the EJC were also found to be mostly phosphorylated. We also screened the phosphorylated serine by mutational analysis using Phos-tag gels to reveal modifications of serine residues 166 and 168. A single substitution at position 168 that concomitantly abolished the phosphorylation of serine 166 suggested the priority of kinase reaction between these sites. Furthermore, analysis of the role of the binding protein Magoh in RBM8A phosphorylation revealed its inhibitory effect in vitro and in vivo. Thus, we conclude that almost all synthesized RBM8A proteins are rapidly phosphorylated in cells and that phosphorylation occurs before the complex formation with Magoh.

Keywords: Magoh; Phos-tag gel; RBM8A(Y14); mRNA; phosphorylation.

Figure 1

Phos-tag gel analysis of the RNA-binding protein 8A (RBM8A) in cell lysates. (a) The cells were transfected with control (M) and anti-RBM8A siRNA (lanes 2 and 3), and the cell lysates were collected. The samples were concomitantly separated on normal sodium dodecyl sulfate (SDS)-polyacrylamide gel (PAGE) or Phos-tag gel and transferred onto a nylon membrane. The membrane was blotted using an anti-RBM8A antibody and a horseradish peroxidase (HRP)-conjugated secondary antibody. (b) The cell lysates were treated with lambda PPase phosphatase and then separated on a Phos-tag gel. The membrane was blotted using an anti-RBM8A antibody. The black arrow indicates dephosphorylated RBM8A. In both the panels, the white arrow indicates the position of the RBM8A polypeptide in the normal gel (approximately 20 kDa), and the black arrow indicates the RBM8A protein in the Phos-tag gel. The asterisk (*) indicates a nonspecific signal

Figure 2

Phosphorylation of RBM8A at various cell-cycle phases. The samples were extracted from the growing and released cells at the G1/S stage by blocking. The cells were analyzed at 0, 0.5, 2, 6, 8, and 12 h after release. Flow cytometry analysis at each time point is shown in supplementary Figure 1, and the cells passed through the G1/S, S, and G2/M phases. The cell lysates were separated on normal SDS-PAGE and Phos-tag gels. After transfer onto a nylon membrane, the membrane was probed using an anti-RBM8A antibody. The positions of 20-, 25-, and 35-kDa bands are shown

Figure 3

Phosphorylation of RBM8A in the cytoplasmic and nucleoplasmic fractions. The cells were fractionated and each fraction was separated on normal and Phos-tag gels. After transfer onto a membrane, western blotting was performed. Lamin A/C and Caspase-3 were used as markers of nucleoplasmic (Nuc) and cytoplasmic (Cyto) fractions. The knockdown lysate was also analyzed to detect specific bands

Figure 4

Phosphorylation of RBM8A in the EJC. RBM8A or Casc3 was immunopurified and analyzed in normal and Phos-tag gels. Input is the whole-cell lysate, and NRS indicates the control normal rabbit serum used in immunoprecipitation (IP). Immunopurified proteins were detected by western blotting

Figure 5

Determination of phosphorylated serine residues by Phos-tag gel analysis. Myc-tagged RBM8A expression vectors were transfected into HeLa cells, and cell lysates were separated on a Phos-tag gel. Serine residues 42, 46, and 56 are located on the N-terminal (N) side, and both the serine residues 166 and 168 are located at the C-terminal side (C) of the RNA-binding motif of RBM8A. Single or multiple serine-to-alanine substitutions were introduced. C-SA contained both the 166-SA and 168-SA mutations, and N-SA contained the 42, 46, and 56 triple mutations. All-SA had all the mutations (substitutions at positions 42, 46, 56, 166, and 168)

Figure 6

Effect of Magoh on the phosphorylation of RBM8A by SRPK1. Recombinant RBM8A and Magoh were mixed to form a complex. After complex formation, SRPK1 was added to the reaction mixture and incubated. After separation in the Phos-tag gel, the polypeptides were transferred onto a nylon membrane. RBM8A was detected using a mouse anti-RBM8A antibody. (a) Representative images of western blotting. (b–e) The intensity of four bands detected in the Phos-tag gel was measured, and the intensities are shown in the graph. Bar shows average of three independent experiments and bar means standard deviation. The asterisk (*) indicates p < 0.05

Figure 7

The effect of Magoh overexpression on RBM8A phosphorylation. The cells were co-transfected with Flag-tagged Magoh and Myc-tagged RBM8A, and the cell lysates were separated on normal and Phos-tag gels. For western blotting, anti-Flag antibody was used for the detection of Magoh, and anti-Myc antibody was used for the detection of RBM8A. 166&168 SA mutant was used as the negative control of phosphorylation. (a) Representative images. (b) The intensity of band indicated with the white arrow was measured, and the ratio of band intensity from wild RBM8A with or without Magoh overexpression is shown in the graph. Bar shows the average of three independent experiments and bar shows the standard deviation. The asterisk (*) indicates p < 0.05