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. 2022 Feb 28;18(3):e10820. doi: 10.15252/msb.202110820

Figure 5. Identification of human S/T kinases modulating the AAR2–PRP8 protein interaction.

Figure 5

  1. Schematic of phospho‐dependent interaction of the spliceosomal AAR2 and PRP8. AAR2, a spliceosomal assembly protein, and PRP8 from a stable complex, which dissociates upon phosphorylation of AAR2. PPR8 is then free to bind the spliceosomal interaction partner protein BRR2 (Galej et al, 2013; Weber et al, 2013). This regulatory interaction switch is thought to be conserved between yeast and human. A protein kinase that can modulate the dissociation of AAR2 from PRP8 is unknown.
  2. Superposition of the yeast Aar2 (orange, PDB entry 3SBT; (Weber et al, 2011)) 3D structure and a homology model of human AAR2 derived from HHpred (marine), based on the yeast Aar2p structure (Santos et al, 2015). The inset on the right shows a close‐up view of the C‐terminal domain, where serines 253 (yeast) and 284 (human) are displayed as sticks. Residues 154–170 and 318–356 of yeast Aar2 as well as residues 1–18, 170–201, and 359–384 of human AAR2 are not shown for clarity.
  3. Y2H interaction experiment with human AAR2 (FL) as bait and PRP8 (aa1755‐2335) as prey. Growth on selective media is abolished when AAR2 carries a S284E phospho‐mimicry amino acid substitution. Mutation of second candidate phospho‐site, AAR2(T356), does not influence the AAR2–PRP8 protein interaction.
  4. Interaction of AAR2 and PRP8 in human cells. HA‐tagged PRP8 (aa1755‐2335) was co‐expressed in HEK293 cells with either wild‐type AAR2, AAR2(S284A) or AAR2(S284E) mutant versions. Immunoprecipitation of PRP8 led to a coprecipitation of wild‐type AAR2 and phospho‐null AAR2(S284A). In contrast, the phospho‐mimicry AAR2(S284E) version did not bind PRP8.
  5. Results of the phospho‐Y2H screen to identify kinases modulating the interaction of AAR2 and PRP8. Growth on selective media (SD5), of strains carrying AAR2 bait and PRP8 prey plasmids is reduced through co‐expression of either SGK2 or CK2α1(CSNK2A1). Growth reduction is visible in a 1:10 dilution series on selective agar (SD5) in comparison to the control media (SD3). Ctrl: nonrelated kinases.
  6. Interaction of AAR2 and PRP8 in human cells in the presence of SGK and CK2α1 kinases. Experiment as in (D). Co‐expression of wild‐type SGK2 and CK2α1 reduces the amount of AAR2 co‐immunoprecipitated with RPR8.