The role of PCNA as a scaffold protein in cellular signaling is functionally conserved between yeast and humans

Abstract

Proliferating cell nuclear antigen (PCNA), a member of the highly conserved DNA sliding clamp family, is an essential protein for cellular processes including DNA replication and repair. A large number of proteins from higher eukaryotes contain one of two PCNA-interacting motifs: PCNA-interacting protein box (PIP box) and AlkB homologue 2 PCNA-interacting motif (APIM). APIM has been shown to be especially important during cellular stress. PIP box is known to be functionally conserved in yeast, and here, we show that this is also the case for APIM. Several of the 84 APIM-containing yeast proteins are associated with cellular signaling as hub proteins, which are able to interact with a large number of other proteins. Cellular signaling is highly conserved throughout evolution, and we recently suggested a novel role for PCNA as a scaffold protein in cellular signaling in human cells. A cell-penetrating peptide containing the APIM sequence increases the sensitivity toward the chemotherapeutic agent cisplatin in both yeast and human cells, and both yeast and human cells become hypersensitive when the Hog1/p38 MAPK pathway is blocked. These results suggest that the interactions between APIM-containing signaling proteins and PCNA during the DNA damage response is evolutionary conserved between yeast and mammals and that PCNA has a role in cellular signaling also in yeast.

Keywords: DNA damage response; MAPK; PCNA; conservation; hypersensitivity; phosphatidylinositol; signaling.

Figure 1

Several human and yeast PI kinases have a conserved APIM sequence. Pathways generating PIs in human (A) and yeast (B) by PI kinases. Black arrows refer to activity that can be performed by APIM‐containing PI kinases, while green arrows and letters refer to activity performed only by non‐APIM‐containing PI kinases. The activity of PI phosphatases is not shown. (C) Conservation of APIM in yeast and human PI kinases. The letters (a–g) in the column for catalytic activity in (C) refer to the arrows in (A).

Figure 2

The APIM‐peptide is imported into yeast and affects cellular growth. (A) Confocal image of live S. cerevisiae 2–5 minutes after addition of FAM‐labeled APIM‐peptide. (B and C) WT S. cerevisiae treated with different concentrations of APIM‐peptide alone (B) and in combination with 125 μm cisplatin (C). (D) Fus3 ^−/− and (E) Hog1 ^−/− S. cerevisiae treated with 125 μm cisplatin in combination with APIM‐peptide. Optical densities were measured every hour for 24 h (15 h are shown in the plot). Concentrations and symbol explanations are indicated in the panel below. Data shown as mean from n technical replicates: controls (no treatment; n = 9), APIM‐peptide (n = 3), cisplatin (n = 7), and combinatorial treatment (n = 2). Two independent biological replicates were analyzed for the WT and Hog1 ^−/− strains giving identical results. (F‐I) Cell growth (MTT assay) of Du145 cells (F and G) and JJN‐3 cells (H and I) treated with APIM‐peptide (6 μm), p38 inhibitor SB 203580 (10 μm) (F and H), cisplatin (0.6 μm for Du145; 0.4 μm for JJN‐3), and combinations of these three agents (G and I). Symbols are explained in the panel below. Data shown as mean from at least three parallel wells from one representative experiment of three independent experiments.