The phosphorylation state of an autoregulatory domain controls PACS-1-directed protein traffic
2003; Springer Nature; Volume: 22; Issue: 23 Linguagem: Inglês
10.1093/emboj/cdg596
ISSN1460-2075
AutoresGregory K. Scott, Feng Gu, Colin M. Crump, Laurel Thomas, Lei Wan, Yang Xiang, Gary Thomas,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoArticle1 December 2003free access The phosphorylation state of an autoregulatory domain controls PACS-1-directed protein traffic Gregory K. Scott Gregory K. Scott Vollum Institute, L-474, 3181 SW Sam Jackson Park Road, Portland, OR, 97239 USA Search for more papers by this author Feng Gu Feng Gu McGill Cancer Center, McGill University, 3655, Promenade Sir William Osler, Montreal, Quebec, H3G 1Y6 Canada Search for more papers by this author Colin M. Crump Colin M. Crump Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QP UK Search for more papers by this author Laurel Thomas Laurel Thomas Vollum Institute, L-474, 3181 SW Sam Jackson Park Road, Portland, OR, 97239 USA Search for more papers by this author Lei Wan Lei Wan Vollum Institute, L-474, 3181 SW Sam Jackson Park Road, Portland, OR, 97239 USA Search for more papers by this author Yang Xiang Yang Xiang Department of Molecular and Cellular Physiology, Stanford Medical Center, Palo Alto, CA, 94305 USA Search for more papers by this author Gary Thomas Corresponding Author Gary Thomas Vollum Institute, L-474, 3181 SW Sam Jackson Park Road, Portland, OR, 97239 USA Search for more papers by this author Gregory K. Scott Gregory K. Scott Vollum Institute, L-474, 3181 SW Sam Jackson Park Road, Portland, OR, 97239 USA Search for more papers by this author Feng Gu Feng Gu McGill Cancer Center, McGill University, 3655, Promenade Sir William Osler, Montreal, Quebec, H3G 1Y6 Canada Search for more papers by this author Colin M. Crump Colin M. Crump Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QP UK Search for more papers by this author Laurel Thomas Laurel Thomas Vollum Institute, L-474, 3181 SW Sam Jackson Park Road, Portland, OR, 97239 USA Search for more papers by this author Lei Wan Lei Wan Vollum Institute, L-474, 3181 SW Sam Jackson Park Road, Portland, OR, 97239 USA Search for more papers by this author Yang Xiang Yang Xiang Department of Molecular and Cellular Physiology, Stanford Medical Center, Palo Alto, CA, 94305 USA Search for more papers by this author Gary Thomas Corresponding Author Gary Thomas Vollum Institute, L-474, 3181 SW Sam Jackson Park Road, Portland, OR, 97239 USA Search for more papers by this author Author Information Gregory K. Scott1, Feng Gu2, Colin M. Crump3, Laurel Thomas1, Lei Wan1, Yang Xiang4 and Gary Thomas 1 1Vollum Institute, L-474, 3181 SW Sam Jackson Park Road, Portland, OR, 97239 USA 2McGill Cancer Center, McGill University, 3655, Promenade Sir William Osler, Montreal, Quebec, H3G 1Y6 Canada 3Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QP UK 4Department of Molecular and Cellular Physiology, Stanford Medical Center, Palo Alto, CA, 94305 USA ‡G.K.Scott and F.Gu contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:6234-6244https://doi.org/10.1093/emboj/cdg596 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info PACS-1 is a cytosolic sorting protein that directs the localization of membrane proteins in the trans-Golgi network (TGN)/endosomal system. PACS-1 connects the clathrin adaptor AP-1 to acidic cluster sorting motifs contained in the cytoplasmic domain of cargo proteins such as furin, the cation-independent mannose-6-phosphate receptor and in viral proteins such as human immunodeficiency virus type 1 Nef. Here we show that an acidic cluster on PACS-1, which is highly similar to acidic cluster sorting motifs on cargo molecules, acts as an autoregulatory domain that controls PACS-1-directed sorting. Biochemical studies show that Ser278 adjacent to the acidic cluster is phosphorylated by CK2 and dephosphorylated by PP2A. Phosphorylation of Ser278 by CK2 or a Ser278→Asp mutation increased the interaction between PACS-1 and cargo, whereas a Ser278→Ala substitution decreased this interaction. Moreover, the Ser278→Ala mutation yields a dominant-negative PACS-1 molecule that selectively blocks retrieval of PACS-1-regulated cargo molecules to the TGN. These results suggest that coordinated signaling events regulate transport within the TGN/endosomal system through the phosphorylation state of both cargo and the sorting machinery. Introduction The control of homeostasis and disease requires that cellular or pathogen proteins are correctly modified and targeted to specific cellular compartments. Many of these modification and targeting steps rely on the communication between the dynamic and highly regulated network of late secretory pathway organelles that comprise the trans-Golgi network (TGN)/endosomal system (Gruenberg, 2001). In addition to housing several biochemical reactions, the TGN orchestrates the routing of proteins to lysosomes, secretory granules and, in polarized cells, the apical and basolateral surfaces. The TGN also receives molecules internalized from the cell surface via a series of complex and highly dynamic endosomal compartments. Precisely how the TGN/endosomal system controls the sorting and localization of proteins is incompletely understood, but numerous studies show the requirement for the orchestrated interaction of many cellular factors including various small molecules, lipids, cytosolic and membrane proteins and components of the cytoskeleton (Gu et al., 2001). The localization of many membrane proteins within the TGN/endosomal system relies upon specific sorting motifs contained within the cytoplasmic domain of these proteins (Bonifacino and Traub, 2003). One of these motifs is represented by clusters of acidic residues, often containing serine or threonine residues that can be phosphorylated by casein kinase 2 (CK2) or, less frequently, casein kinase 1 (CK1) (for reviews see Gu et al., 2001; Thomas, 2002; Bonifacino and Traub, 2003). Membrane proteins that contain acidic sorting motifs include processing enzymes such as furin (Jones et al., 1995), PC6B (Xiang et al., 2000) and carboxypeptidase D (CPD; Kalinina et al., 2002); receptors such as the cation-independent mannose-6-phosphate receptor (CI-MPR; Chen et al., 1997); transporters such as VMAT2 (Waites et al., 2001); SNAREs including VAMP-4 (Zeng et al., 2003); and a number of pathogenic molecules including the envelope glycoproteins of many herpesviruses such as varicella-zoster virus gE (VZV-gE; Alconada et al., 1999) and human cytomegalovirus gB (HCMV-gB; Norais et al., 1996), as well as human immunodeficiency virus type 1 (HIV-1) Nef (Piguet et al., 2000). Perhaps the best studied of these acidic cluster motifs is the one present in the cytoplasmic domain of furin (Thomas, 2002). The phosphorylation state of the furin acidic cluster controls in large part the dynamic sorting itinerary of the endoprotease including the localization of furin to the TGN (Jones et al., 1995), recycling of furin from early endosomes to the cell surface (Molloy et al., 1998) and, in neuroendocrine cells, removal of furin from immature secretory granules (Dittie et al., 1997). In contrast, dephosphorylation of the furin acidic cluster by specific isoforms of protein phosphatase 2A (PP2A) allows transport of furin between endosomal compartments (Molloy et al., 1998). Thus, the coordinated activities of CK2 and PP2A control the complex, acidic cluster-mediated trafficking of furin. The phosphorylated furin acidic cluster binds to the sorting protein PACS-1 (phosphofurin acidic cluster sorting protein-1), which is a sorting connector that links furin to the AP-1 clathrin adaptor and is required for localizing furin to the TGN (Wan et al., 1998; Crump et al., 2001). PACS-1 is not dedicated exclusively to the localization of furin, as it is also required for the TGN localization of a number of membrane proteins that contain acidic cluster sorting motifs. These include cellular proteins, such as CI-MPR (Wan et al., 1998), PC6B (Xiang et al., 2000) and VMAT2 (Waites et al., 2001), as well as several viral proteins including HCMV-gB (Crump et al., 2003) and HIV-1 Nef (Piguet et al., 2000). PACS-1 binding to HIV-1 Nef is required for the ability of this viral protein to downregulate cell surface major histocompatibility class-I (MHC-I) complexes (Piguet et al., 2000; Blagoveshchenskaya et al., 2002). While the importance of PACS-1 for localizing acidic cluster-containing membrane cargo to the TGN is well established, no information pertaining to the regulation of its sorting activity has been reported. For many cargo molecules including furin, CI-MPR, VMAT2 and VZV-gE, phosphorylation of specific residues within their acidic clusters enhances binding to PACS-1 (Wan et al., 1998; Waites et al., 2001), whereas others including PC6B and HIV-1 Nef contain non-phosphorylatable acidic clusters (Piguet et al., 2000; Xiang et al., 2000). Together, these results suggest that binding of PACS-1 to cargo acidic clusters may be regulated by more than just the phosphorylation state of the cargo protein. Interestingly, PACS-1 itself contains an acidic cluster with a potential CK2 phosphorylation site, -S278EEEEE-, located C-terminal to the cargo-binding domain (named the FBR, see Figure 1A). The striking similarity between the acidic clusters in PACS-1 and those in cargo molecules such as furin raises the possibility that the PACS-1 acidic cluster may control PACS-1 sorting activity. Figure 1.PACS-1 Ser278 is phosphorylated by CK2 and dephosphorylated by PP2A. (A) Schematic diagram of PACS-1 showing the atrophin-related region (ARR), the furin-binding region (FBR) which interacts with cargo molecules and AP-1/AP-3 adaptor complexes, the middle region (MR) which contains the autoregulatory acidic cluster and Ser278, and the C-terminal region (CTR). The acidic cluster in the PACS-1 MR as well as acidic cluster sequences contained in membrane cargo molecules, which bind to the PACS-1 FBR, are indicated. (B) In vivo phosphorylation of expressed PACS-1. BSC40 cells infected with VV recombinants expressing either epitope (HA)-tagged PACS-1 or PACS-1S278A were labeled with 32Pi, and the immunoprecipitated PACS-1 proteins were separated by SDS–PAGE and analyzed by autoradiography (upper panel). A western blot using the anti-HA mAb HA.11 shows equal expression and loading of the two proteins (lower panel). (C) In vitro CK2 phosphorylation of GST–MR. GST–MR or GST–MRS278A was incubated with CK2 and [γ-32P]ATP, separated by SDS–PAGE and analyzed by autoradiography. (D) PACS-1 MR is phosphorylated at amino acid 278. GST–MR or GST–MRS278T was phosphorylated by CK2 with [γ-32P]ATP, separated by SDS–PAGE, transferred to PVDF, acid hydrolyzed, and subjected to two-dimensional thin-layer chromatography. The positions of phosphoserine (pS), phosphothreonine (pT) and phosphotyrosine (pY) standards are shown. (E) PP2A dephosphorylates GST–PACS-1 MR. In vitro CK2-phosphorylated [32P]GST–MR was incubated without (control) or with recombinant PP2A in the presence or absence of 10 nM OA, separated by SDS–PAGE and analyzed by autoradiography. (F) CK2 and PP2A regulate phosphorylation of endogenous PACS-1 in vivo. A7 cells were labeled with 32Pi, treated or not with either 100μM DRB or 20 nM OA, and endogenous PACS-1 was immunoprecipitated, separated by SDS–PAGE and analyzed by autoradiography. All autoradiography was quantified using NIH image 1.61 software. 32P incorporation was normalized to PACS-1 protein loading and is presented relative to control PACS-1 phosphorylation. Bar graphs represent the mean ± SE of at least three separate experiments. Download figure Download PowerPoint In this report, we show that the PACS-1 acidic cluster acts as an autoregulatory domain for PACS-1-directed protein trafficking and is itself a target of CK2 phosphorylation and PP2A dephosphorylation. We demonstrate that the PACS-1 acidic cluster associates with the cargo-binding region of PACS-1 in a phosphorylation-dependent manner. Using biochemical, cellular and cell-free studies, we show that the phosphorylation state of the PACS-1 acidic cluster regulates the ability of PACS-1 to bind to and sort cargo proteins to the TGN. Disruption of PACS-1 phosphorylation by a Ser278→Ala substitution results in an interfering mutant that inhibits PACS-1-directed endosome to TGN sorting. These results provide new insight into how a coordinated signaling mechanism controlling the phosphorylation of both the cargo and a sorting connector can regulate TGN/endosomal sorting. Results Inspection of the PACS-1 protein sequence reveals an acidic cluster, -S278EEEEE-, C-terminal to the cargo-binding region (FBR, residues 117–266) that is similar to the acidic cluster sorting motifs on many membrane cargo proteins that bind to the PACS-1 FBR (Figure 1A). Interestingly, the serine residue within the PACS-1 acidic cluster forms a consensus sequence for CK2 phosphorylation. To determine if Ser278 is a major site of phosphorylation in PACS-1, cells expressing wild-type PACS-1 or PACS-1 with a Ser278→Ala substitution (PACS-1S278A) were incubated with 32Pi and the immunoprecipitated PACS-1 proteins were resolved by SDS–PAGE. Quantification of the incorporated radioactivity showed that PACS-1S278A contained 50% less 32P than wild-type PACS-1, indicating that Ser278 is a major PACS-1 phosphorylation site in vivo (Figure 1B). Next we conducted a series of in vitro studies to characterize the reversible phosphorylation of PACS-1 at Ser278. First, we showed that Ser278 could be phosphorylated by CK2 (Figure 1C). GST–PACS-1240–479 (GST–MR, which contains the PACS-1 acidic cluster) or GST–MRS278A was incubated with purified CK2 and [γ-32P]ATP. GST–MRS278A incorporated significantly less 32P than did GST–MR, suggesting that Ser278 within the PACS-1 acidic cluster is a substrate for CK2. Moreover, neither protein kinase A nor protein kinase C could phosphorylate GST–MR despite their ability to phosphorylate a control substrate efficiently (data not shown). Secondly, to identify Ser278 unequivocally as a CK2 phosphorylation site, we conducted phosphoamino acid analysis on GST–MRS278T following CK2 phosphorylation (Figure 1D). The Ser278→Thr substitution was chosen because GST–MR lacks a phosphorylatable threonine residue. Thus, the formation of pThr in GST–MRS278T following incubation with CK2 must be due to phosphorylation at residue 278. Replicate samples of GST–MR, GST–MRS278A and GST–MRS278T were incubated with recombinant CK2 and [γ-32P]ATP. Phosphoamino acid analysis of GST–MR and GST–MRS278A showed a prominent signal of only pSer. In contrast, analysis of GST–MRS278T revealed pThr in addition to pSer. These data strongly indicate that Ser278 is a major CK2 phosphorylation site in PACS-1. Thirdly, because many cargo acidic cluster motifs that require CK2 phosphorylation for binding to PACS-1 are dephosphorylated by PP2A (Molloy et al., 1998; Varlamov et al., 2001), we investigated whether PP2A can similarly dephosphorylate the CK2-phophorylated GST–MR (Figure 1E). Consistent with a potential role for PP2A at this step, [32P]GST–MR was dephosphorylated efficiently by PP2A and this reaction was blocked by okadaic acid (OA), a PP2A-specific inhibitor. In contrast, protein phosphatase 1 (PP1) failed to dephosphorylate [32P]GST–MR despite its ability to dephosphorylate a control substrate efficiently (data not shown), suggesting a role for PP2A as the PACS-1 Ser278 phosphatase. We conducted metabolic labeling studies to determine whether endogenous PACS-1 is phosphorylated in vivo. Cells were incubated with 32Pi and endogenous PACS-1 was immunoprecipitated with affinity-purified anti-PACS-1 antibodies but not with an IgG control (Figure 1F). In agreement with our in vitro studies, incubation of the cells with OA increased the amount of 32P incorporated into PACS-1 by 2-fold. In contrast, and in agreement with the analysis of PACS-1S278A (Figure 1B), incubation of the cells with the CK2 inhibitor 5,6-dichlorobenzimidazole riboside (DRB) decreased the amount of 32P incorporated into endogenous PACS-1 by ∼50% relative to control conditions. Together with our in vitro studies, these data strongly implicate CK2 and PP2A as the enzymes that control the phosphorylation state of PACS-1 Ser278. The reversible phosphorylation of Ser278 raised the possibility that, similarly to PACS-1 cargo proteins, the PACS-1 acidic cluster may interact with the PACS-1 FBR. To test this possibility, non-phosphorylated or CK2-phosphorylated GST–MR was incubated with thioredoxin (Trx)–PACS-1 FBR and captured using glutathione–agarose (Figure 2A). We found that GST–PACS-1 MR indeed bound to Trx–PACS-1 FBR. Surprisingly, however, whereas phosphorylation of most PACS-1 cargo proteins enhanced binding to the PACS-1 FBR, the phosphorylation of GST–PACS-1 MR by CK2 diminished binding to Trx–PACS-1 FBR. To test whether this reduced binding was due specifically to phosphorylation of Ser278, we measured the binding of the PACS-1 FBR to GST–MR constructs containing either a Ser278→Ala or a Ser278→Asp substitution. Consistent with the results using CK2-phosphorylated molecules, GST–MRS278A exhibited stronger binding to Trx–PACS-1 FBR than the GST–MRS278D. Figure 2.Phosphorylation of PACS-1 Ser278 promotes PACS-1 binding to cargo proteins. (A) Phosphorylation of PACS-1 Ser278 inhibits PACS-1 MR binding to the PACS-1 FBR. GST–MR, GST–MRS278A, GST–MRS278D or CK2-phosphorylated GST–MR were incubated with Trx–PACS-1 FBR and captured using glutathione–agarose. Bound Trx–PACS-1 FBR was analyzed by western blot (upper panel). Incomplete phosphorylation of GST–MR may explain the slightly greater binding of Trx–PACS-1 FBR to CK2-phosphorylated GST–MR compared with GST–MRS278D. (B) The PACS-1 MR acidic cluster competes with cargo proteins for binding to the PACS-1 FBR. GST capture assays were performed as in (A), with the addition of varying concentrations of Trx–furinS773,775D (Trx-furin S→D) or Trx alone to the binding reaction. (C) Ser278 regulates binding to cargo proteins. Epitope-(HA)-tagged PACS-1, PACS-1S278A and PACS-1S278D were expressed in replicate plates of A7 cells using VV recombinants. Cell lysates were incubated with GST–Nef, and GST–Nef was captured using glutathione–agarose. PACS-1 proteins bound to GST–Nef were analyzed by western blot using the anti-HA mAb HA.11 (top panel). GST–Nef input and expression of PACS-1 proteins is shown (lower panels). (D) Mutation of PACS-1 Ser278 does not affect binding to AP-1. Epitope-(HA)-tagged PACS-1, PACS-1S278A, PACS-1S278D or PACS-1Admut were expressed in A7 cells and immunoprecipitated with mAb HA.11. Precipitated proteins were separated by SDS–PAGE and analyzed by western blot using mAb 100/3 (AP-1) or mAb PACS-1. All data were quantified as described in the legend to Figure 1. Download figure Download PowerPoint We performed competitive binding assays to test whether GST–MR, which contains the PACS-1 acidic cluster, competes with cargo protein acidic clusters for binding to the PACS-1 FBR (Figure 2B). GST–PACS-1 MR was pre-incubated with Trx–PACS-1 FBR and then mixed with increasing concentrations of either Trx or Trx–furinS773,775D, which contains the CK2 phosphomimic furin cytoplasmic domain that binds to the PACS-1 FBR (Wan et al., 1998). GST–PACS-1 MR was captured using glutathione–agarose, and bound Trx–PACS-1 FBR was detected by western blot. Using this assay, we found that Trx–furinS773,775D competed in a dose-dependent manner with GST–MR for binding to the PACS-1 FBR, whereas Trx alone had no effect. Together, the data in Figure 2A and B indicate that binding of the PACS-1 MR to the PACS-1 FBR precludes cargo binding to PACS-1. To examine the importance of Ser278 phosphorylation in the context of full-length PACS-1, we tested the ability of PACS-1S278A or PACS-1S278D to bind to the PACS-1 cargo protein HIV-1 Nef (Figure 2C). We previously showed that the Nef acidic cluster, EEEE65, is required for binding to PACS-1 and for PACS-1-dependent sorting of HIV-1 Nef reporter proteins to the TGN (Piguet et al., 2000; Blagoveshchenskaya et al., 2002). Because the Nef acidic cluster cannot be phosphorylated by CK2, only CK2 phosphorylation of the putative PACS-1 autoregulatory domain may regulate Nef–PACS-1 binding. Cells expressing PACS-1, PACS-1S278A or PACS-1S278D were harvested and incubated with GST–Nef. Quantification of proteins binding to HIV-1 Nef showed that the S278D substitution enhanced PACS-1 binding to GST–Nef by >50%, whereas the S278A substitution inhibited PACS-1 binding to GST–Nef by nearly 80%. Thus, the ability of PACS-1 to bind cargo is regulated by the phosphorylation state of Ser278. The Ser278→Ala/Asp substitutions could potentially inhibit the interaction of PACS-1 with adaptor complexes due to some indirect structural deformation. To test this possibility, PACS-1 proteins were immunoprecipitated from cells expressing PACS-1, PACS-1S278A, PACS-1S278D or PACS-1Admut, which contains an alanine substitution of E168TELQLTF175 within the PACS-1 FBR that blocks binding to adaptor complexes but not cargo (Crump et al., 2001). Co-immunoprecipitation analysis showed that each PACS-1 construct, except PACS-1Admut, associated with AP-1 (Figure 2D). Together, the data in Figure 2 show that phosphorylation at Ser278 terminates the autoinhibitory domain to the cargo-binding domain, thereby promoting binding of PACS-1 to cargo proteins. The inhibitory effect of the Ser278→Ala substitution on the ability of PACS-1 to bind cargo proteins suggested that expression of PACS-1S278A in cells might interfere with PACS-1-dependent sorting. Therefore, we examined the effect of PACS-1S278A and PACS-1S278D on the TGN sorting of the HIV-1 Nef reporter construct 44Nef (Figure 3A). 44Nef is a chimera composed of the CD4 extracellular and transmembrane domains fused to cytoplasmic HIV-1 Nef. Using this construct, CD4 antibody uptake assays showed that co-expression of the reporter with either PACS-1 or PACS-1S278D had no effect on the efficient delivery of internalized 44Nef to the TGN. In contrast, co-expression of PACS-1S278A blocked the sorting of internalized 44Nef to the TGN and caused the reporter to be mislocalized to a dispersed, punctate endosomal population. The interfering effect of PACS-1S278A on the sorting of 44Nef to the TGN was indistinguishable from the missorting of 44NefAla, which contains an EEEE65→AAAA65 substitution that cannot bind to PACS-1 (Piguet et al., 2000). Figure 3.Phosphorylated PACS-1 directs endosome to TGN transport. (A) PACS-1S278A expression disrupts the TGN localization of 44Nef. A7 cells were infected with recombinant virus expressing 44Nef as well as PACS-1, PACS-1S278A or PACS-1S278D. Localization of 44Nef was determined by CD4 antibody uptake. The cells were fixed, permeabilized and co-stained with the TGN marker anti-TGN46 followed by fluorescent secondary antisera. As a control, a mutant Nef reporter, 44NefAla, which does not bind PACS-1, was also expressed. (B) PACS-1 directs endosome to TGN sorting in a cell-free assay. A7 cells were infected with AV expressing 44Nef-Y, after which membranes from these cells were harvested and quenched with cold PAPS. The quenched membranes were incubated in the absence (background, bkg) or presence of cytosol from C6 (control), AS19 (PACS-1 antisense) or from AS19 cells expressing PACS-1 (AS19 + PACS-1), PACS-1S278A (AS19 + S278A) or PACS-1S278D (AS19 + S278D) and with the sulfate donor [35S]PAPS. 44Nef-Y was immunoprecipitated and 35S incorporation was determined by autoradiography, and protein load by western blot. 35S incorporation was quantified using NIH image 1.61 software, normalized for protein load and presented relative to 44Nef-Y labeling with AS19 cytosol. The bar graph represents the mean ± SE of three separate experiments. Download figure Download PowerPoint Next, we used a cell-free assay to establish unequivocally the role of PACS-1, and in particular the phosphorylation state of Ser278, in endosome to TGN sorting (Figure 3B). This assay utilizes the activity of a TGN resident tyrosylprotein sulfotransferase that sulfates tyrosine residues in the lumenal domain of cargo proteins to demonstrate sorting to the TGN. A 44Nef mutant, 44Nef-Y, was constructed containing the cholecystokinin tyrosine O-sulfation motif within the CD4 lumenal domain. Cells expressing 44Nef-Y were treated with cycloheximide to block protein synthesis and promote accumulation of the nascently synthesized 44Nef-Y in late secretory pathway compartments. The membrane fraction from these cells was incubated with the unlabeled sulfate donor PAPS (3′-phosphoadenosine 5′-phosphosulfate) to quench any 44Nef-Y present at the TGN but not in endosomal compartments, which lack the sulfotransferase. The quenched membrane preparation was then incubated with cytosol from control C6 cells, AS19 PACS-1 antisense cells or AS19 cells expressing PACS-1, PACS-1S278D or PACS-1S278A. Each sample was incubated with an ATP-regenerating system in the presence of [35S]PAPS to promote endosome to TGN transport. Using this assay, we found that addition of cytosol from control cells (C6) but not cytosol from PACS-1 antisense cells (AS19), which contain reduced levels of PACS-1, supported endosome to TGN transport as measured by an increase in 35S-labeled 44Nef-Y. In contrast, cytosol from AS19 cells replete with PACS-1 rescued the endosome to TGN transport of 44Nef-Y, demonstrating the importance of PACS-1 in the retrieval of membrane cargo from endosomes to the TGN. Next, we analyzed the PACS-1 phosphorylation state of mutants and found that only PACS-1S278D but not PACS-1S278A rescued the endosome to TGN sorting of 44Nef-Y, supporting a role for the phosphorylation of Ser278 as a key regulator of PACS-1 sorting activity. We suspect that the less efficient rescue of 44Nef-Y sorting by PACS-1S278D compared with PACS-1 may reflect a requirement for the temporal regulation of PACS-1 phosphorylation in directing protein sorting. Nonetheless, these results demonstrate that PACS-1 directs the endosome to TGN transport and that its sorting activity is regulated by the phosphorylation state of Ser278. The immunofluorescence and in vitro transport studies showed that phosphorylation of PACS-1 controls the endosome to TGN transport of the 44Nef reporter and suggested that PACS-1S278A may act as an interfering mutant to block the sorting of PACS-1 cargo proteins selectively. To test this possibility, we first examined the effect of PACS-1S278D and PACS-1S278A on the ability of HIV-1 Nef to downregulate MHC-I molecules (Figure 4). Cells expressing HIV-1 Nef alone or together with either PACS-1 or PACS-1S278D caused the redistribution of cell surface MHC-I to the TGN, whereas co-expression of PACS-1S278A blocked MHC-I downregulation. Secondly, we determined whether PACS-1S278A could affect the TGN localization of CI-MPR and furin (Figure 5A). Expression of PACS-1 or PACS-1S278D in A7 cells had no effect on the paranuclear localization of either endogenous CI-MPR or co-expressed Flag-tagged furin (fur/f), both of which overlapped with the staining pattern of TGN46. In contrast, expression of PACS-1S278A caused both CI-MPR and fur/f to redistribute to an endosomal population that no longer overlapped with TGN46. Finally, to establish that PACS-1S278A selectively blocked the sorting of PACS-1 cargo, we examined the effects of PACS-1S278A and PACS-1S278D on the distribution of several secretory compartment markers (Figure 5B). In addition to their lack of effect on the localization of TGN46, which does not require PACS-1 to localize to the TGN, we found that neither PACS-1 mutant affected the localization of AP-1 or markers for early endosomes (internalized transferrin), late endosomes (LBPA) or the Golgi cisternae (mannosidase II). Together, these analyses show that the sorting activity of PACS-1 is controlled by the phosphorylation state of an autoregulatory domain and that interference with this autoregulatory mechanism specifically blocks PACS-1-directed protein traffic in the TGN/endosomal system. Figure 4.Expression of PACS-1S278A blocks HIV-1 Nef-mediated MHC-1 downregulation. A7 cells were infected with VV:WT or co-infected with VV recombinants expressing Nef and either PACS-1, PACS-1S278A or PACS-1S278D. Cells were fixed, permeabilized and incubated with anti-MHC-I and anti-TGN46 followed by fluorescently labeled secondary antisera. The expression of PACS-1 alone has no effect on MHC-I localization (Blagoveshchenskaya et al., 2002; and data not shown). Download figure Download PowerPoint Figure 5.Expression of PACS-1S278A disrupts furin and CI-MPR localization (A) A7 cells were co-infected with VV co-expressing flag-tagged furin (fur/f) and PACS-1, PACS-1S278A or PACS-1S278D. The cells were fixed, permeabilized and incubated with anti-TGN46 and either M1 (fur/flag) or anti-CI-MPR, followed by fluorescently labeled secondary antisera. (B) A7 cells infected with VV expressing PACS-1, PACS-1S278A or PACS-1S278D were fixed, permeabilized and stained with anti-γ-adaptin, anti-LBPA and anti-mannosidase II. A replicate plate of cells was incubated with rhodamine–transferrin prior to fixation. Download figure Download PowerPoint Discussion In this study, we have identified a biochemical mechanism that controls the sorting activity of PACS-1. We discovered that the phosphorylation state of an autoregulatory domain within PACS-1 controls binding of this sorting connector to cargo proteins. CK2 phosphorylation of Ser278 within this autoregulatory domain weakens the interaction
Referência(s)