Phosphorylation of Schizosaccharomyces pombe Dss1 mediates direct binding to the ubiquitin-ligase Dma1 in vitro

Abstract

Intrinsically disordered proteins (IDPs) are often multifunctional and frequently posttranslationally modified. Deleted in split hand/split foot 1 (Dss1-Sem1 in budding yeast) is a highly multifunctional IDP associated with a range of protein complexes. However, it remains unknown if the different functions relate to different modified states. In this work, we show that Schizosaccharomyces pombe Dss1 is a substrate for casein kinase 2 in vitro, and we identify three phosphorylated threonines in its linker region separating two known disordered ubiquitin-binding motifs. Phosphorylations of the threonines had no effect on ubiquitin-binding but caused a slight destabilization of the C-terminal α-helix and mediated a direct interaction with the forkhead-associated (FHA) domain of the RING-FHA E3-ubiquitin ligase defective in mitosis 1 (Dma1). The phosphorylation sites are not conserved and are absent in human Dss1. Sequence analyses revealed that the Txx(E/D) motif, which is important for phosphorylation and Dma1 binding, is not linked to certain branches of the evolutionary tree. Instead, we find that the motif appears randomly, supporting the mechanism of ex nihilo evolution of novel motifs. In support of this, other threonine-based motifs, although frequent, are nonconserved in the linker, pointing to additional functions connected to this region. We suggest that Dss1 acts as an adaptor protein that docks to Dma1 via the phosphorylated FHA-binding motifs, while the C-terminal α-helix is free to bind mitotic septins, thereby stabilizing the complex. The presence of Txx(D/E) motifs in the disordered regions of certain septin subunits may be of further relevance to the formation and stabilization of these complexes.

Keywords: FHA; IDP; NMR; Sem1; forkhead-associated domain; phosphorylation; septin; ubiquitin.

FIGURE 1

Sequence alignment of different eukaryotic Dss1 sequences. Dss1 has three conserved regions responsible for the majority of its interactions, two disordered ubiquitin‐binding sites (DisUBM 1 + 2) and a third region that populates a transiently folded α‐helix (Helix). The two DisUBMs are separated by a poorly conserved linker region. Top panel shows sequence alignments of different eukaryotic Dss1 proteins and a corresponding sequence logo representing the conservation. Conserved residues are marked in orange with the color darkness reflecting the degree of conservation. Bottom panel shows which residues of Dss1 are involved in binding to a set of different binding partners as determined experimentally (Ellisdon et al., ; Liu et al., ; Tomko & Hochstrasser, ; Yang et al., 2002). Dark green is known, and light green suggested interaction sites with Brca1.

FIGURE 2

Dss1 is phosphorylated by CK2 at three sites. (a) Schematic of Dss1 and sequence alignment of the three threonine phosphorylation sites in comparison with the CK2 consensus motif. (b) GFP immunoprecipitates (IPs) from dss1Δ cells labeled with 32P‐orthophosphate and overexpressing GFP, GFP‐Dss1, and GFP‐Dss1 AAA (T31A, T35A, and T39A) were resolved by SDS‐PAGE and analyzed by autoradiography. As a control for expression and purity, GFP and GFP‐Dss1 (WT and AAA variant) were purified from larger (unlabeled) cultures, analyzed by SDS‐PAGE, and stained with Coomassie Brilliant Blue (CBB). (c) Maldi‐TOF spectra showing phosphorylation of Dss1 after incubation with CK2 for 4, 24, and 48 h. Peaks correspond to additions of one (8188 Da), two (8286 Da), or three phosphorylations (8348 Da). Right: Phosphorylation was also monitored using SDS‐PAGE; arrow colors correspond to the color coding of the mass spectrum.

FIGURE 3

Dss1 is phosphorylated on T35, T39, and T31, without affecting mono‐ubiquitin binding or fitness. (a) Identification of phosphorylated residues using NMR spectroscopy. Assignments of Dss1 before and after phosphorylation by CK2 confirmed phosphorylation on T31, T35, and T39. CSPs plotted against the sequence residues upon phosphorylation by CK2. Lower panel shows a zoom of the baseline to identify potential small perturbations outside the linker region. The positions of the two DisUBMs and the helix are indicated in orange boxes and the phosphorylation sites are in magenta dotted lines. (b) Solid media growth assays at 29°C and 35°C of cells deleted for Dss1 (dss1Δ) transformed to overexpress GFP‐tagged Dss1 wild‐type (WT), Dss1 AAA (T31A, T35A, and T39A) or Dss1 DDD (T31D, T35D, and T39D). Each row represents a dilution series of the indicated strains applied to the agar in 5 μL droplets. The dss1Δ strain transformed with vector alone served as a control. (c) CSP of residues E19 (circles), A22 (squares), and Q58 (triangles) of Dss1 (green) and 3p‐Dss1 (blue) plotted as a function of ubiquitin concentration. Dotted lines represent fits to a one‐site binding site model; however, saturation was not reached. (d) Ubiquitin binding to nonphosphorylated (green) and phosphorylated (blue) ¹⁵N‐Dss1 was monitored using NMR spectroscopy. Mono‐ubiquitin was titrated into Dss1 in steps from 1:2 ratio (light colors) to 1:10 ratio (dark colors), and the total CSPs were mapped onto the sequence. (*) residues that were not assigned, (•) overlapping peaks and (▲) disappearing peaks. (e) SCS analysis of C′ shifts before (green) and after (blue) phosphorylation of Dss1. The differences in SCSs are shown in cyan. SCS, secondary chemical shift.

FIGURE 4

3p‐Dss1 binds to Dma1‐FHA through interactions with the phosphorylated linker. (a) Far‐UV CD spectrum of Dma1‐FHA. The distinct shape of the spectrum is indicative of aromatic exciton couplings, common to β‐sandwich structured proteins. Inserted is the AlphaFold2 structure of the FHA domain of S. pombe Dma1 (Jumper et al., ; Varadi et al., 2022). (b) ¹H,¹⁵N‐HSQC spectra of 3p‐Dss1 in the absence (blue) and presence (red) of Dma1‐FHA added in a 1:1 molar ratio. The marked peaks represent residues residing within the α‐helix. Insert at the bottom left shows the indole NHs. (c) Upper panel: Sequence mapping of the peak intensity ratios from the ¹H,¹⁵N‐HSQC spectra. Lower panel: CSP analysis of 3p‐Dss1 upon interaction with Dma1‐FHA. The majority of peaks within the linker region disappear or are perturbed upon addition of Dma1‐FHA. The positions of the two DisUBMs and the helix are indicated in orange boxes and the phosphorylation sites are in magenta dotted lines.

FIGURE 5

Linker sequence properties of Dss1 species and potential models for complex stabilization. (a) Abundance and properties of the Dss1 linker focusing on threonine‐based motif. The distribution of the number of Txx(D/E) SLiMs in the linkers is shown to the right. (b) Potential models showing different possible modes of complex stabilization using disordered regions and FHA domain interactions. Left: stabilization of the Dma1/Septin complex involving Dss1 as adaptor as seen in Schizosaccharomyces pombe (2, 3, and 4 indicate Spn2, Spn3, and Spn4). Right: stabilization of the RNF8/Septin complex in humans involving disordered loops in septin 6 (2, 6, and 7 indicate Sptn2, Sptn6, and Sptn 7).