A PACS-1, GGA3 and CK2 complex regulates CI-MPR trafficking
2006; Springer Nature; Volume: 25; Issue: 19 Linguagem: Inglês
10.1038/sj.emboj.7601336
ISSN1460-2075
AutoresGregory K. Scott, Hao Fei, Laurel Thomas, Guruprasad R. Medigeshi, Gary Thomas,
Tópico(s)RNA Research and Splicing
ResumoArticle14 September 2006free access A PACS-1, GGA3 and CK2 complex regulates CI-MPR trafficking Gregory K Scott Gregory K Scott Search for more papers by this author Hao Fei Hao FeiPresent address: Department of Psychiatry and Biobehavioral Sciences, UCLA, Los Angeles, CA 90095, USA Search for more papers by this author Laurel Thomas Laurel Thomas Search for more papers by this author Guruprasad R Medigeshi Guruprasad R MedigeshiPresent address: Department of Molecular Microbiology and Immunology, OHSU, Portland, OR, USA Search for more papers by this author Gary Thomas Corresponding Author Gary Thomas Vollum Institute, Portland, OR, USA Search for more papers by this author Gregory K Scott Gregory K Scott Search for more papers by this author Hao Fei Hao FeiPresent address: Department of Psychiatry and Biobehavioral Sciences, UCLA, Los Angeles, CA 90095, USA Search for more papers by this author Laurel Thomas Laurel Thomas Search for more papers by this author Guruprasad R Medigeshi Guruprasad R MedigeshiPresent address: Department of Molecular Microbiology and Immunology, OHSU, Portland, OR, USA Search for more papers by this author Gary Thomas Corresponding Author Gary Thomas Vollum Institute, Portland, OR, USA Search for more papers by this author Author Information Gregory K Scott, Hao Fei, Laurel Thomas, Guruprasad R Medigeshi and Gary Thomas 1 1Vollum Institute, Portland, OR, USA *Corresponding author. Vollum Institute, Oregon Health & Science University, Oregon Health Sciences University, L-474, 3181 SW Sam Jackson Park Road, Portland, OR 97239-3098, 97239, USA. Tel.: +1 503 494 6955; Fax: +1 503 494 1218; E-mail: [email protected] The EMBO Journal (2006)25:4423-4435https://doi.org/10.1038/sj.emboj.7601336 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The cation-independent mannose-6-phosphate receptor (CI-MPR) follows a highly regulated sorting itinerary to deliver hydrolases from the trans-Golgi network (TGN) to lysosomes. Cycling of CI-MPR between the TGN and early endosomes is mediated by GGA3, which directs TGN export, and PACS-1, which directs endosome-to-TGN retrieval. Despite executing opposing sorting steps, GGA3 and PACS-1 bind to an overlapping CI-MPR trafficking motif and their sorting activity is controlled by the CK2 phosphorylation of their respective autoregulatory domains. However, how CK2 coordinates these opposing roles is unknown. We report a CK2-activated phosphorylation cascade controlling PACS-1- and GGA3-mediated CI-MPR sorting. PACS-1 links GGA3 to CK2, forming a multimeric complex required for CI-MPR sorting. PACS-1-bound CK2 stimulates GGA3 phosphorylation, releasing GGA3 from CI-MPR and early endosomes. Bound CK2 also phosphorylates PACS-1Ser278, promoting binding of PACS-1 to CI-MPR to retrieve the receptor to the TGN. Our results identify a CK2-controlled cascade regulating hydrolase trafficking and sorting of itinerant proteins in the TGN/endosomal system. Introduction The localization and trafficking of itinerant membrane cargo proteins within the trans-Golgi network (TGN)/endosomal system relies upon canonical sorting motifs within their cytosolic domains, which are recognized by components of the vesicular trafficking machinery (Robinson, 2004). These motifs include tyrosine (Yxxϕ)- and dileucine ([D/E]xxxL[L/I])-based signals, which bind to the heterotetrameric adaptors (APs), acidic-dileucine (DxxLL)-based motifs, which bind to GGAs and acidic cluster-based motifs, which bind to PACS proteins. The cytosolic domain of one membrane protein, the cation-independent mannose-6-phosphate receptor (CI-MPR), requires motifs that bind to each of these three groups of sorting molecules to localize to the TGN and to efficiently sort cathepsin D to lysosomes (Chen et al, 1997; Wan et al, 1998; Meyer et al, 2000; Puertollano et al, 2001a; Ghosh et al, 2003). The GGAs sort the CI-MPR into clathrin-coated vesicles at the TGN and may also mediate CI-MPR trafficking between endosomal compartments (Doray et al, 2002b; Mattera et al, 2003; Puertollano and Bonifacino, 2004). By contrast, PACS-1 and AP-1, which mediate endosome-to-TGN retrieval, are required to localize CI-MPR to the TGN (Wan et al, 1998; Meyer et al, 2000; Crump et al, 2001). In addition, other sorting molecules including TIP47, Retromer and EpsinR also function in the endosome-to-TGN retrieval of CI-MPR (Diaz and Pfeffer, 1998; Arighi et al, 2004; Saint-Pol et al, 2004; Seaman, 2004), supporting the highly regulated and complex trafficking pathway followed by this multifunctional receptor. We identified PACS-1 through its binding to the protein kinase CK2 (CK2)-phosphorylated acidic cluster (…EECPpSDpSEEDE…) on the furin cytosolic domain (Figure 1A and Wan et al, 1998). The 140 amino acid PACS-1 cargo-binding region (FBR, Figure 1A) contains an eight-amino-acid segment ETELQLTF175 that binds AP-1, and is required for correct subcellular localization of furin and CI-MPR to the TGN (Crump et al, 2001). PACS-1 also binds to acidic cluster motifs on several additional itinerant cellular proteins (Thomas, 2002), including proprotein convertase 6B (Xiang et al, 2000), polycystin-2 (Köttgen et al, 2005) and VAMP4 (Hinners et al, 2003), as well as the viral proteins HCMV gB (Crump et al, 2003) and HIV-1 Nef (Piguet et al, 2000). Studies using dominant negative-, siRNA- or antisense-based methods show PACS-1 is required for the TGN localization of each of these proteins, suggesting a broad role for PACS-1 in cellular homeostasis and disease. Figure 1.PACS-1 binds to GGA3. (A) Diagram of PACS-1 showing the atropin-1-related region (ARR), cargo-binding region (FBR), which interacts with cargo and AP-1/AP-3 adaptor complexes (Wan et al, 1998; Crump et al, 2001), the middle region (MR), which contains the autoregulatory acidic cluster and Ser278 (Scott et al, 2003), and the C-terminal region (CTR) and PACS-1 cargo. (B) Diagram of GGA3 showing the VHS (Vps27, Hrs, Stam) domain, which binds to cargo proteins, the GAT (GGA and TOM) domain, which binds to ARF1, the hinge segment, which contains the autoregulatory acidic-dileucine motif and Ser388, and the GAE (γ-adaptin ear) domain (Bonifacino, 2004) and GGA3 cargo. (C) Endogenous PACS-1 was immunoprecipitated from rat brain (lower panel) using anti-PACS-1 701 or 703, anti-PACS-2 834 or control IgG and co-precipitating GGA3 analyzed by SDS–PAGE and Western blot (upper panel). Immunoprecipitated PACS-1 and PACS-2 are shown by Western blot (bottom panel). (D–G) The indicated GST-fusion proteins were incubated with the indicated Trx-fusion proteins or with purified AP-1, isolated with glutathione sepharose and analyzed by Western blot using anti-Trx or anti-γ-adaptin antibody (upper panels). Each GST-protein is also shown (lower panel). The Trx-PACS-1FBR (residues 117–294) band is shifted lower in (E) because Trx-PACS-1FBR migrates at the same size as GST-GGA3VHS. GST-GGA3VHS−GAT captured ∼1% of Trx-PACS-1FBR input, and GST-PACS-1FBR (residues 117–294) captured ∼1%, 0.5% and 1% of the Trx-GGA3VHS−GAT, γ-adaptin and Trx-CI-MPRcd input, respectively. Binding assays were conducted as described in Materials and methods, except 4% NP40 was used. (H) A7 cells infected with wild-type (WT) AV or AV expressing Myc-GGA3, Myc-GGA3 and HA-PACS-1, or Myc-GGA3 and HA-PACS-1GGAmut were harvested and HA-tagged proteins immunoprecipitated and co-precipitating myc-GGA analyzed by Western blot (upper panel). Lower panels show myc-GGA3 and HA-PACS-1 expression. Download figure Download PowerPoint Similar to furin, binding of PACS-1 to the CI-MPR cytosolic domain (CI-MPRCD) requires the CK2 phosphorylatable acidic cluster …DDpSDEDLLHI, located at the CI-MPRCD C-terminus (Wan et al, 1998). Interestingly, the three GGA family members (1–3) also bind to this phosphorylated motif on the CI-MPRCD but require the dileucine motif for binding, which furin lacks (Puertollano et al, 2001a). The GGAs contain three principal domains including the VHS domain, which binds to cargo proteins, the GAT domain, which binds to ARF1, a hinge segment, which binds clathrin and contains an autoregulatory acidic-dileucine motif (GGA1 and 3 only) and the GAE domain, which binds to several accessory proteins (Figure 1B and Bonifacino, 2004). Through these interactions, the GGAs function as monomeric clathrin adaptors that link itinerant cargo directly to clathrin (Puertollano et al, 2001b). However, why GGAs and PACS-1 share overlapping binding sites on the CI-MPRCD is not known. The functional similarities shared by PACS-1 and GGAs extend to regulation of their cargo binding. The sorting activity of PACS-1 is regulated by the CK2- and PP2A-controlled phosphorylation of an autoregulatory domain (Scott et al, 2003). Phosphorylation of PACS-1Ser278 within the PACS-1 autoregulatory domain activates cargo binding and is required for the endosome-to-TGN transport of furin, CI-MPR and HIV-1 Nef. Similar to PACS-1, GGA1 and GGA3 binding to cargo proteins is regulated by CK2 phosphorylation of an autoregulatory domain within the GGA1 and GGA3 hinge segment (Doray et al, 2002a; Ghosh and Kornfeld, 2003). Phosphorylation of GGA1Ser355 (which corresponds to GGA3Ser388, see Figure 1B) within the GGA1 autoregulatory domain inhibits binding to CI-MPR. Therefore, CK2 phosphorylation of the PACS-1 autoregulatory domain promotes cargo binding (Scott et al, 2003), whereas CK2 phosphorylation of GGA1 or three autoregulatory domains inhibits cargo binding (Doray et al, 2002a). CK2 is a ubiquitous protein kinase with more than 300 putative polypeptide substrates and is a heterotetramer composed of two catalytic subunits (αα, αα′, or α′α′) and two regulatory β subunits (Meggio and Pinna, 2003). The regulation of this basally active kinase has long remained enigmatic, although the binding of the regulatory β subunit to polyamines or substrate proteins can increase kinase activity three-fold (Litchfield, 2003). The requirement for CK2 phosphorylation for the regulation of PACS-1, GGA1 and GGA3 action led us to determine how this kinase may control the PACS-1 and GGA3-mediated trafficking of CI-MPR. We report that PACS-1 binds to GGA3 and recruits CK2, forming a multimeric complex, which regulates PACS-1/GGA3-mediated sorting of CI-MPR between the TGN and early endosomes. Together our results describe a novel cellular mechanism for the phospho-regulation of membrane protein traffic through the TGN/endosomal system. Results PACS-1 binds to GGA3 Despite regulating opposing CI-MPR trafficking steps, PACS-1 and GGAs share several biochemical functions. These include binding to the CI-MPRCD at a C-terminal acidic cluster and the regulation of their binding to membrane cargo by the CK2 phosphorylation of an autoregulatory domain (Wan et al, 1998; Puertollano et al, 2001a; Doray et al, 2002a; Scott et al, 2003). These common properties led us to ask if GGA3 and PACS-1 associate in vivo. Accordingly, we immunoprecipitated PACS-1 from rat brain using two different PACS-1 antibodies and found that GGA3 co-precipitated with PACS-1 (Figure 1C). By contrast, GGA3 did not co-precipitate with PACS-2, which is a PACS-1 homologue that mediates ER/mitochondria trafficking (Simmen et al, 2005). To determine if PACS-1 bound directly to GGA3 and to identify the GGA3-binding region of PACS-1, we used glutathione-S-transferase (GST)-tagged PACS-1 fusion proteins corresponding to predicted domains of PACS-1 (Figure 1A) to capture Thioredoxin (Trx)-tagged GGA3VHS+GAT (Figure 1D). Only GST-PACS-1FBR, which binds to cargo including CI-MPRCD, was able to precipitate Trx-GGA3VHS+GAT. Reciprocal mapping experiments using purified GST-GGA3 constructs (Figure 1B) showed that the GGA3 VHS domain, which binds the CI-MPRCD, was sufficient to bind Trx-PACS-1FBR (Figure 1E). These results demonstrate a direct interaction between PACS-1 and GGA3 through their cargo-binding regions. To further define the GGA3-binding site on the PACS-1 FBR, we took advantage of the fact that through the FBR, PACS-1 and PACS-2 are 75% identical and 83% homologous. Serial mutation of nonhomologous amino acids was used to identify residues in the PACS-1 FBR required for binding GGA3. Using this approach, we found that mutation of PACS-1 FBR residues K249IY to the corresponding PACS-2 residues (W171IA; hereafter PACS-1 FBR-GGAmut) disrupted GGA3 binding (Figure 1F). In addition, we found that protein-binding studies showed that the K249Y251 → WA substitution had no effect on GST-PACS-1FBR binding to purified AP-1 or Trx-tagged CI-MPRCD (Figure 1G). Therefore, we introduced the K249Y251 → WA mutation into full-length PACS-1 (hereafter PACS-1GGAmut), and compared the ability of hemagglutinin (HA)-tagged PACS-1 and HA-PACS-1GGAmut to co-immunoprecipitate co-expressed myc-GGA3 (Figure 1H). In agreement with our in vitro binding studies, we found that myc-GGA3 co-immunoprecipitated with HA-PACS-1, but not with HA-PACS-1GGAmut. Thus, we identified a PACS-1 mutant that fails to bind GGA3 but is unaffected for binding cargo and AP-1. Blocking the PACS-1/GGA3 interaction disrupts CI-MPR and GGA3 localization We expressed PACS-1GGAmut in cells to determine if PACS-1 binding to GGA3 is required for the steady-state localization of their mutual cargo protein: CI-MPR. In control cells or PACS-1-expressing cells, CI-MPR demonstrated a paranuclear staining pattern that overlapped with TGN46 (Figure 2A). However, in PACS-1GGAmut-expressing cells, CI-MPR showed a pronounced redistribution to an endosomal population that overlapped with the early endosomal marker EEA1. As a control, we asked whether PACS-1GGAmut disrupted the localization of furin, which requires PACS-1 for endosome-to-TGN retrieval (Wan et al, 1998), but lacks the canonical D/ExxLL GGA-binding motif (Figure 2B). We found that expression of PACS-1 or PACS-1GGAmut failed to affect the TGN localization of FLAG-furin, suggesting that PACS-1GGAmut selectively disrupts the trafficking of itinerant cargo that depend on binding to both PACS-1 and GGAs. Because GGA3 distributes between the TGN and early endosomes (Puertollano and Bonifacino, 2004), we also examined the localization of GGA3 in PACS-1GGAmut-expressing cells (Figure 2C). We found that expression of PACS-1GGAmut, but not PACS-1, caused a striking redistribution of GGA3 from a paranuclear localization to a dispersed endosome population that overlapped with the redistributed CI-MPR. In addition, we tested the effect of a interfereing mutant PACS-1 molecule, PACS-1Admut, that fails to bind AP-1 and redistributes the CI-MPR and furin from the TGN (Crump et al, 2001), on the localization of GGA3. We observed no effect of PACS-1Admut expression on the localization of GGA3 (Figure 2C), suggesting that redistribution of GGA3 to endosomal compartments induced by PACS-1GGAmut results from the inability of PACS-1GGAmut to interact with GGA3. These findings suggest the PACS-1/GGA3 interaction is required for CI-MPR retrieval and for release of GGA3 from endosomal membranes. Figure 2.PACS-1GGAmut disrupts CI-MPR trafficking. (A) A7 cells infected with WT vaccinia virus (VV) or VV recombinants expressing PACS-1 or PACS-1GGAmut were stained with antibodies to detect CI-MPR, TGN46 or EEA1 as indicated. Inset: colocalization of CI-MPR (green) and EEA1 (red) from the boxed area. CI-MPR staining outside the TGN area increased from 11±5 and 9±7% in the WT and PACS-1-expressing cells, respectively, to 40±10% for PACS-1GGAmut-expressing cells. (B) A7 cells expressing FLAG-furin were treated as in (A) and stained with anti-FLAG and anti-TGN46. (C) A7 cells infected with VV:WT or with VV expressing PACS-1, PACS-1Admut or PACS-1GGAmut and then costained with anti-GGA3 and anti-TGN46 or anti-CI-MPR. Inset: Colocalization of GGA3 (green) and CI-MPR (red) from the boxed area. GGA3 staining outside the TGN area increased from 11±6, 8±4 and 9±5% in the WT, PACS-1- and PACS-1Admut- expressing cells, respectively, to 38±7% for PACS-1GGAmut-expressing cells. Scale bars=20 μm. Download figure Download PowerPoint PACS-1 is required for CI-MPR function To better understand how PACS-1 and GGA3 might cooperate to direct trafficking of CI-MPR, we conducted protein–protein binding assays to define the PACS-1-binding site on the CI-MPRCD. Previously, we found that truncation of the last 10 amino acids (…DDpS2484DEDLLHI) of the CI-MPRCD, which contain a CK2 phosphorylatable acidic cluster and constitute a DxxLL GGA-binding motif, abolished binding to the FBR region of PACS-1 (Wan et al, 1998). First, we sought to determine if, similar to the interaction of PACS-1 and furin (Wan et al, 1998), as well as the CI-MPR with GGA3 (Kato et al, 2002), phosphorylation of CI-MPR Ser2484 would enhance binding to the PACS-1 FBR (Figure 3A). We tested the binding of Trx-PACS-1FBR to GST-CI-MPRCD phosphorylated by CK2 or to GST-CI-MPRCD mutants containing a phosphomimic Ser2484 → Asp or nonphosphorylatable Ser2484 → Ala substitution. We found that both preincubation of GST-CI-MPRCD with CK2 and the Ser2484 → Asp substitution enhanced binding to Trx-PACS-1FBR, indicating that like other PACS-1 cargo proteins, CK2 phosphorylation of Ser2484 within the CI-MPR acidic cluster enhanced binding to PACS-1. Second, we conducted an alanine scan of each of the acidic residues from Asp2482 to Asp2487 and found that alanine mutation of any of the acidic residues reduced binding to Trx-PACS-1FBR (Figure 3B). Finally, we found that Leu2488 → Ala and Leu2489 → Ala mutations had no effect on Trx-PACS-1FBR binding, whereas these mutations completely blocked Trx-GGA3VHS−GAT binding, as previously reported (Figure 3C and Puertollano et al, 2001a). Thus, PACS-1 and GGA3 share overlapping but not identical CI-MPR-binding sites. Figure 3.PACS-1 is required for CI-MPR function. (A–C) GST, GST-CI-MPRCD preincubated or not with CK2, or GST-CI-MPRCD containing the indicated mutations was incubated with Trx-PACS-1FBR (residues 117–294) or Trx-GGA3VHS+GAT, isolated with glutathione sepharose, washed three times with GST-binding buffer containing 4% NP-40 and analyzed by Western blot using anti-Trx (upper panels). Input of each GST-protein is shown (lower panel). GST-CI-MPRCD pulled down ∼1% of the Trx-PACS-1FBR. (D) A7 cells were treated with scrambled (scr.) or PACS-1 siRNAs and cell lysates analyzed by Western blot using anti-PACS-1 or anti-tubulin. (E) A7 cells were treated with the indicated siRNA and Cathepsin D pulse chase experiments performed. Cellular and secreted fractions were immunoprecipitated with anti-cathepsin D and analyzed by fluorography. Precursor (P), intermediate (I) and mature (M) forms of cathepsin D are shown (lower panel). The percentage of missorted (secreted) cathepsin D compared to the processed form is shown (n=3, P=0.01). Download figure Download PowerPoint The importance of CI-MPR Asp2485, which is required for GGA binding and sorting of lysosomal enzymes (Chen et al, 1997; Puertollano et al, 2001a), for binding to PACS-1, as well as the requirement of PACS-1 for the TGN localization of CI-MPR (Wan et al, 1998; Simmen et al, 2005), led us to determine if PACS-1 is required for CI-MPR function. Therefore, we investigated the effect of PACS-1 depletion on the sorting of lysosomal enzymes by CI-MPR. The sorting and maturation of cathepsin D, a ligand of CI-MPR, to lysosomes was followed in metabolically labeled cells. The intracellular (C) and extracellular (E) forms of cathepsin D were immunoprecipitated from both the cells and medium after pulse-chase in the presence of mannose-6-phosphate. We found that siRNA depletion of PACS-1 (Figure 3D), which redistributes CI-MPR from the TGN (Simmen et al, 2005), caused an ∼20% increase in secreted cathepsin D and a corresponding ∼20% decrease in intracellular cathepsin D compared to control cells (Figure 3E). As a positive control, and in agreement with previous studies, we found that siRNA depletion of the μ1A subunit of AP-1 caused ∼50% of the newly synthesized procathepsin D to be released into the culture medium. Additionally, we observed no change in the half-life of CI-MPR in PACS-1-depleted cells (data not shown), indicating that this increased secretion of cathepsin D does not result from decreased CI-MPR stability. PACS-1 binds to and activates CK2 The overlapping PACS-1- and GGA3-binding sites on CI-MPR, as well as the requirement for binding of PACS-1 to GGA3 to control the TGN localization of CI-MPR, suggested that the interaction between PACS-1, GGA3 and CI-MPR is tightly regulated. One clue to the underlying mechanism controlling the PACS-1/GGA3-dependent sorting of CI-MPR is the prominent role CK2 phosphorylation plays in the regulation of each protein (Doray et al, 2002a; Scott et al, 2003). Although earlier studies demonstrated that an AP-1-associated CK2 activity could phosphorylate GGA1 (Doray et al, 2002b), we speculated that a more direct association of CK2 with PACS-1 and GGA3 might afford greater signaling efficacy. Accordingly, we immunoprecipitated PACS-1 from rat brain and assayed the bound material for co-precipitating CK2 activity (Figure 4A). We observed a ∼14-fold increase in PACS-1-associated CK2 activity compared to the control, which was blocked by the CK2-specific inhibitor TBB, but not the PKA inhibitor PKI. To identify the region of PACS-1 that associates with CK2, we used GST-PACS-1 segments (see Figure 1A) to capture CK2α from rat brain cytosol (Figure 4B). Similar to our analysis of GGA3 binding (Figure 1), we found that CK2α was captured solely by GST-PACS-1FBR. We more precisely identified PACS-1 FBR residues required for CK2 binding by testing a battery of GST-PACS-1FBR truncations and substitutions for their ability to capture CK2α from rat brain cytosol (Figure 4C). We found that an 18-amino acid segment of the PACS-1 FBR between L194 and A212 was required to capture CK2α (Figure 4D). Next, we conducted an alanine scan of this PACS-1 segment and found that an R196RKRY → AAAAA substitution (hereafter called PACS-1FBR–CKmut) blocked CK2α association with GST-PACS-1FBR, whereas alanine substitution of adjacent five-amino-acid segments, including K201NRTI → AAAAA and L206GYKT → AAAAA, did not. As a control, we observed no difference between the binding of GST-PACS-1FBR or GST-PACS-1FBR−CKmut to purified AP-1, Trx-CI-MPRCD or Trx-GGA3VHS+GAT (Figure 4E). Thus, PACS-1 associates with CK2 in vivo and the PACS-1 FBR-CKmut substitution specifically blocks the CK2/PACS-1 interaction. Figure 4.PACS-1 associates with CK2. (A) Rat brain cytosol was incubated with affinity-purified anti-PACS-1 or control IgG to immunoprecipitate endogenous PACS-1, and co-precipitating CK2 activity was measured with an in vitro kinase assay in the absence or presence of 40 μM TBB (CK2 inhibitor) or 400 μM PKI (PKA inhibitor). Error bars represent mean and s.d. of three independent experiments. (B–D) GST-PACS-1 ARR, FBR (residues 117–266), MR or CTR, or the indicated PACS-1 FBR truncations or alanine substitutions (see Figure 1A and D) were incubated with rat brain cytosol, captured with glutathione sepharose and analyzed by Western blot using anti-CK2α (panel B, D, upper panel). Input of each GST-protein is also shown (panel B, D, lower panel). GST-PACS-1FBR captured ∼3% of the input CK2α. Relative to GST-PACS-1FBR, the interaction of CK2 with GST-Δ2 and GST-Δ6 was reduced 60 and 75%, respectively (n=3). (E) GST, GST-PACS-1FBR (residues 117–266) or GST-PACS-1FBR−CKmut was incubated with Trx-GGA3VHS+GAT, Trx-CI-MPRCD or purified AP-1, captured with glutathione sepharose and analyzed by Western blot using anti-Trx or anti-γ-Adaptin antibody (upper panels). GST-PACS-1FBR (residues 117–266) pulled down 9% of the Trx-CI-MPRCD. Input of each GST-protein is shown (lower panel). Download figure Download PowerPoint To determine which CK2 subunit associates with PACS-1, we conducted a yeast-two-hybrid analysis (Figure 5A). We found that yeast expressing PACS-1 FBR and CK2β, but not CK2α or CK2α′, supported growth under histidine selection. Moreover, cotransformation of PACS-1FBR–CKmut with CK2β failed to support cell growth, further indicating that PACS-1 R196RKRY is required for the interaction between the PACS-1 FBR and CK2β. To determine if the PACS-1 FBR binds directly to CK2β, we conducted a protein–protein binding assay, and found that Trx-CK2β bound directly to GST-PACS-1FBR but not GST-PACS-1FBR–CKmut (Figure 5B). Finally, to confirm the effect of the CKmut substitution in the context of full-length PACS-1, we expressed full-length HA-PACS-1 or HA-PACS-1CKmut in cells, immunoprecipitated the PACS-1 proteins and examined co-precipitating endogenous CK2α and β by Western blot (Figure 5C). In agreement with the in vitro protein capture studies, we found that HA-PACS-1, but not HA-PACS-1CKmut, co-precipitated CK2. Figure 5.PACS-1 binding to CK2β activates CK2. (A) Yeast transformed with the indicated Gal4 activation and DNA-binding domain (Gal4ad and Gal4bd) constructs were screened for growth on His+ and His− media. (B) GST, GST-PACS-1FBR (residues 117–266) or GST-PACS-1FBR–CKmut was incubated with Trx-CK2β, isolated with glutathione sepharose, washed twice with GST-binding buffer, once with GST-binding buffer containing 1% deoxycholate and analyzed by Western blot using anti-Trx (upper panel). Input of each GST-protein is shown (lower panel). GST-PACS-1FBR captured 2.5% of the input Trx-CK2β. (C) A7 cells infected with VV:WT or VV expressing HA-PACS-1 or HA-PACS-1CKmut were lysed, immunoprecipitated with HA antibody and any co-immunoprecipitating CK2α and CK2β detected by Western blot using subunit-specific antisera (upper panels). HA-PACS-1 expression is shown (bottom panel). (D) In vitro CK2 holoenzyme activity assayed in the absence or presence of purified GST, GST-PACS-1FBR (residues 117–266) or GST-PACS-1FBR–CKmut. Activity is normalized to a parallel sample assayed in the absence of added protein. Error bars represent mean and s.d. of three independent experiments. (E) A7 cells were infected with VV:WT or VV expressing HA-PACS-1, HA-PACS-1CKmut, HA-PACS-1S278A or HA-PACS-1S278A/CKmut and metabolically labeled with 32Pi. HA-proteins were immunoprecipitated with mAb HA.11, resolved by SDS–PAGE and analyzed by autoradiography (upper panel). HA-PACS-1 expression is shown (bottom panel). Error bars represent mean and s.d. of three independent experiments. Download figure Download PowerPoint One characteristic property of CK2 is the three-fold activation observed upon binding of polycationic molecules or proteins containing clusters of basic amino acids to a patch of acidic residues in the regulatory β subunit (Bonnet et al, 1996; Leroy et al, 1997). As the R196RKRY cluster of basic amino acids in the PACS-1 FBR is required for binding to CK2β, we tested the effect of PACS-1 on CK2 activity levels using an in vitro kinase assay. Purified bovine CK2 holoenzyme was preincubated with increasing concentrations of GST, GST-PACS-1FBR or GST-PACS-1FBR–CKmut, and CK2 activity was scored as incorporation of 32P into a peptide substrate (Figure 5D). GST-PACS-1FBR stimulated CK2 activity ∼2.5-fold, whereas GST or GST-PACS-1FBR–CKmut had a lesser (∼0.5-fold) effect on CK2 activity. Thus, PACS-1 FBR binding stimulates the activity of the CK2 holoenzyme. We previously determined that CK2 phosphorylation of Ser278 within the PACS-1 autoregulatory domain activates cargo binding and accounts for ∼50% of the incorporated phosphate on PACS-1 (Scott et al, 2003). Thus, our finding that PACS-1 bound and activated CK2 suggested that this interaction may be critical for regulating the phosphorylation state of PACS-1. To test this possibility, we metabolically labeled replicate plates of cells expressing full-length HA-PACS-1, HA-PACS-1CKmut or HA-PACS-1S278A with 32Pi, and quantified the amount of radiolabel incorporated into each protein (Figure 5E). We observed ∼40% less 32P incorporation into HA-PACS-1CKmut compared to HA-PACS-1, whereas HA-PACS-1S278A exhibited ∼60% less 32P incorporation compared to HA-PACS-1. This indicated that the PACS-1/CK2 interaction is required for efficient PACS-1 phosphorylation, but does not reduce PACS-1 phosphorylation to the level observed by Ser278 → Ala substitution. Therefore, to gauge whether the CKmut substitution affects PACS-1 Ser278 phosphorylation, we examined the 32P incorporation into a PACS-1S278A/CKmut double mutant. We predicted that if CK2 that is bound to PACS-1 phosphorylates only Ser278, then PACS-1S278A/CKmut would exhibit equal 32P incorporation compared to PACS-1S278A. Conversely, if CK2 bound to PACS-1 primarily phosphorylates residues other than Ser278, then PACS-1S278A/CKmut would incorporate less 32P than PACS-1S278A. We observed no difference between the 32P incorporation of HA-PACS-1S278A and HA-PACS-1S278A/CKmut, suggesting that CK2 binding to PACS-1 is required for efficient phosphorylation of Ser278 and thus the ability of PACS-1 to bind cargo. PACS-1-bound CK2 inactivates GGA3 to retrieve CI-MPR to the TGN The inhibitory effects of the CKmut substitution suggested that PACS-1CKmut may interfere with the PACS-1-dependent sorting of membrane cargo. To test this possibility, we expressed PACS-1CKmut in cells and determined any effect on the TGN localization of CI-MPR and furin. Similar to PACS-1GGAmut, PACS-1CKmut caused CI-MPR to redistribute to an EEA1-positive compartment (Figure 6A). We also found that PACS-1CKmut caused furin to redistribute from the TGN, sugge
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