Identification of PP1-Gadd34 substrates involved in the unfolded protein response using K-BIPS, a method for phosphatase substrate identification

Abstract

Phosphorylation is a key post-translational modification in cell signaling, which is regulated by the equilibrium activities of kinases and phosphatases. The biological significance of many phosphorylation events remains poorly characterized due to the scarcity of tools to discover phosphatases substrates. In prior work, we established kinase-catalyzed biotinylation where kinases accept the γ-modified ATP analog, ATP-biotin, to label phosphoproteins. Here, we developed a novel method to study substrates of phosphatases using kinase-catalyzed biotinylation termed K-BIPS (Kinase-catalyzed Biotinylation to Identify Phosphatase Substrates). In a proof-of-concept experiment, K-BIPS was initially used to explore the substrates of phosphatases inhibited by okadaic acid. Many known phosphatase substrates were observed, confirming K-BIPS as a valid phosphatase substrate identification tool. Then, as a further application, K-BIPS was used to discover the substrates of the PP1-Gadd34 phosphatase complex in the context of unfolded protein response (UPR). In addition to the known substrate eIF2α, K-BIPS revealed several novel substrates, suggesting a more prominent role for the PP1-Gadd34 complex in UPR than previously appreciated. Overall, the two studies establish K-BIPS as a powerful tool to discover the cellular substrates of phosphatases.

Figure 1

Protein phosphorylation and the K-BIPS method. (A) Kinases transfer a phosphoryl group from ATP to cellular proteins. Phosphatases reverse the reaction. (B) Kinases utilize γ-modified ATP analogs, such as ATP-biotin, to covalently modify substrate proteins. The γ-modified phosphoryl group once attached is resistant to phosphatase degradation. (C) The K-BIPS method. Phosphatases present in lysates remove phosphoryl groups from already phosphorylated substrates (i, left path). However, when a phosphatase is inactivated by inhibitor treatment, the substrates are maintained in the phosphorylated form (ii, right path). (i) Phosphatase substrates in the untreated lysates are biotinylated by ATP-biotin and active kinases. After avidin purification of biotinylation proteins, LC-MS/MS analysis will identify the captured substrates. (ii) Phosphorylated substrates in the phosphatase-inhibited sample are immune to biotinylation by ATP-biotin. After avidin purification and LC-MS/MS analysis, quantitative comparison of proteins present at elevated levels in the untreated sample versus the phosphatase inhibitor-treated sample will reveal possible substrates.

Figure 2

K-BIPS study with OA. (A) Avidin purification of biotinylated proteins after kinase-catalyzed biotinylation. Biotinylation was carried out with lysates from HeLa cells untreated (ethanol only; lane 1) or treated with OA (1 or 0.01 μM in ethanol; lanes 2–3). After reaction, biotinylated proteins were enriched with avidin resin (elution in lanes 4–6). Proteins in each sample were separated by SDS-PAGE and stained for total proteins using SYPRO^® Ruby stain. Full gel image shown in Figure S1. (B) Known substrates identified by K-BIPS (Substrate), along with the corresponding PPP family member. Refer to Table S1 for primary literature. (C) Phosphatase interacting proteins (Gene name) from the K-BIPS hits. Interaction information was obtained from Uniprot and BioGrid databases, or literature (see Table S1). The catalytic and regulatory subunits known to interact with the hit are indicated. For example, PPP1CA refers to PP1 alpha isoform of the catalytic subunit, whereas PPP1R8 refers to the regulatory subunit 8 of PP1 phosphatase.

Figure 3

K-BIPS study with Gb treatment. (A) Biotinylation of proteins after Gb treatment. Untreated or Gb-treated HeLa lysates were labeled by ATP-biotin. Proteins were separated by SDS-PAGE and visualized with Streptavidin-Cy5 (top) and Sypro®Ruby (bottom). The arrows and brackets indicate proteins that are reduced in the GB-treated sample (lane 3) compared to the untreated samples (lanes 1–2). The full gel image is shown in Figure S6. (B) Known physical protein-protein interactions among the 130 K-BIPS hits and the PP1-Gadd34 complex were mapped using GeneMANIA in Cytoscape. The inner circles of hexagons include known direct interacting proteins of PP1 catalytic subunits (PPP1CA, PPP1CB, PPP1CC, red rectangles) or Gadd34 (PPP1R15A, red rectangle), including the known PP1-Gadd34 substrate, EIF2S1 (eIF2α, red star). The outer circles represent indirect interacting proteins of PP1-Gadd34. Protein colors indicate the biological function of the proteins: yellow - translation initiation; pink – ribosome; dark blue – UPR; orange - ER stress; light blue – protein ubiquitination; purple - stress granules; grey - oxidative stress. Except when cited (Table S2), functional information was obtained from Uniprot database. Grey lines indicate interactions among the enriched proteins. The other line colors indicate the proteins that directly interact with PPP1CA (blue lines), PPP1CB (green lines), PPP1CC (black lines) or Gadd34 (PPP1R15A, red lines). Line thickness and length were adjusted arbitrary to improve clarity. The black star highlights the proteins validated in Figure 4. An enlarged version of this figure is available as Figure S9. The Cytoscape file is also available as a supplementary document. (C) The interaction of COPS5 with stress granule proteins. (D) K-BIPS hits not reported to interact with PP1-Gadd34 are also shown.

Figure 4

Validation of K-BIPS hits. (A) Enrichment of K-BIPS hits with Gb treatment. HeLa cells were treated with tunicamycin (Tm) alone, or Tm + guanabenz (Gb) before lysis and kinase-catalyzed labeling with ATP-biotin. After avidin enrichment of biotinylated proteins, the presence of the four candidate substrates (COPS5, WDR5, CAPRIN, and G3BP1) was assessed by Western blot analysis with specific antibodies. Full gel images and repetitive trials are shown in Figures S10–S13. (B) COPS5 enrichment after Gadd34 knockdown. HeLa cells were treated with a pool of siRNA targeting Gadd34 or a pool of control siRNA (Figure S15), followed by treatment with (Tm). Cells were lysed and labeled with ATP-biotin. COPS5 enrichment was monitored by Western blot after avdin enrichment. Full gel image and repetitive trials are shown in Figure S16. (C) Phos-tag^™ SDS-PAGE of COPS5 phosphorylation. HeLa cells were treated with Tm and Gb, or Tm alone. After lysis, proteins were separated using Phos-tag^™ SDS-PAGE. COPS5 was visualized by Western blot analhysis (anti-COPS5). Total proteins were visualized as a load control by Sypro®Ruby total protein stain. Bands affected by Gb treatment are indicated with a bracket. Full image and repetitive trials was shown in Figure S17.