Regulation of endosome sorting by a specific PP2A isoform - PubMed (original) (raw)
Regulation of endosome sorting by a specific PP2A isoform
S S Molloy et al. J Cell Biol. 1998.
Abstract
The regulated sorting of proteins within the trans-Golgi network (TGN)/endosomal system is a key determinant of their biological activity in vivo. For example, the endoprotease furin activates of a wide range of proproteins in multiple compartments within the TGN/endosomal system. Phosphorylation of its cytosolic domain by casein kinase II (CKII) promotes the localization of furin to the TGN and early endosomes whereas dephosphorylation is required for efficient transport between these compartments (Jones, B.G., L. Thomas, S.S. Molloy, C.D. Thulin, M.D. Fry, K.A. Walsh, and G. Thomas. 1995. EMBO [Eur. Mol. Biol. Organ.] J. 14:5869-5883). Here we show that phosphorylated furin molecules internalized from the cell surface are retained in a local cycling loop between early endosomes and the plasma membrane. This cycling loop requires the phosphorylation state-dependent furin-sorting protein PACS-1, and mirrors the trafficking pathway described recently for the TGN localization of furin (Wan, L., S.S. Molloy, L. Thomas, G. Liu, Y. Xiang, S.L. Ryback, and G. Thomas. 1998. Cell. 94:205-216). We also demonstrate a novel role for protein phosphatase 2A (PP2A) in regulating protein localization in the TGN/endosomal system. Using baculovirus recombinants expressing individual PP2A subunits, we show that the dephosphorylation of furin in vitro requires heterotrimeric phosphatase containing B family regulatory subunits. The importance of this PP2A isoform in directing the routing of furin from early endosomes to the TGN was established using SV-40 small t antigen as a diagnostic tool in vivo. The role of both CKII and PP2A in controlling multiple sorting steps in the TGN/endosomal system indicates that the distribution of itinerant membrane proteins may be acutely regulated via signal transduction pathways.
Figures
Figure 1
Phosphorylation-dependent routing of furin in early endosomes (top). Epitope-tagged furin showing the FLAG insertion (cross-hatched), the catalytic (shaded) and transmembrane domains (stippled), as well as the sequence of the cd with CKII phosphorylation site substitutions. (Bottom) BSC-40 cells were infected with vaccinia recombinants (m.o.i. = 10) expressing either fur/f with the native cytosolic domain (A and B) or with serine to aspartic acid substitutions within the CKII site (fur/f-DDD, C–E). At 6 h postinfection, the cells were incubated with mAb M1 (15 μg/ml) and TRITC-transferrin (40 ng/ml) (E) for 1 h before fixation and processing for immunofluorescence. The cells in B were treated with 100 nM tautomycin during the uptake period. Internalized mAb M1 was visualized using FITC goat anti–mouse IgG (A–D).
Figure 2
Internalization of furin is dependent upon dynamin function. BSC-40 cells were infected with vaccinia recombinants (m.o.i. = 5 each) expressing fur/f and either native dynamin I (A and C) or the dominant-negative dynamin construct K44E (B and D) in the presence of 10 μM hydroxyurea. At 16 h postinfection, cells were incubated with either mAb M1 (A and B) or TRITC-transferrin (C and D) for 1 h in culture before fixation and analysis by immunofluorescence. The extended culture time postinfection was required to allow sufficient expression levels for optimum K44E block.
Figure 3
Recycling of phosphorylated furin from endosomes to the cell surface. (A) HeLa TS-Dyn I cells were infected with vaccinia recombinants (m.o.i. = 10) expressing fur/f mutants mimicking either nonphosphorylated (fur/f ADA) or constitutively phosphorylated furin (fur/f DDD). After accumulation at the cell surface during incubation at nonpermissive temperature (37°C) for 6 h, surface proteins were biotinylated (refer to Materials and Methods) and then allowed to internalize for the indicated times at permissive temperature (31°C). The total (T) and internalized (I) pools of labeled furin were then determined by immunoprecipitation and Western analysis. (B) The percent of furin internalized at each time point was quantified by densitometric analysis (data averaged from four independent experiments, error bars = SEM). (Inset) Double-strip analyses were conducted in which the pool of internalized furin following an initial 20-min chase period was assessed for reexpression at the cell surface. After a second 20-min chase, total biotinylated furin was compared with surface localized furin using a second MesNa strip (− and +, respectively).
Figure 4
Endocytic sorting of furin requires the phosphorylated cd binding protein PACS-1. Fur/f was expressed in control cells (A, B, E, and F) and PACS-1–deficient antisense cells (C, D, G, and H) using vaccinia recombinants (m.o.i. = 10). At 4 h postinfection, the cells were exposed to mAb M1 as a marker for endocytosed furin and TRITC-labeled transferrin (B and D) for either 1 h (A–D, F, and H) or 10 min. (E and G) before fixation and processing for immunofluorescence microscopy. The cells in A–D were exposed to 100 nM tautomycin during the 1-h incubation period with mAb M1 and transferrin. Internalized mAb M1 was visualized using FITC-conjugated goat anti–mouse IgG2b (A, C, and E–H).
Figure 5
Dephosphorylation of furin by purified phosphatase. PP1 and PP2A catalytic subunits were incubated with either 32P-labeled GST-Furcd fusion protein (GST–furincd) phosphorylated with CKII, or a 32P-labeled control substrate (phosphorylase a, Phos. a) for the indicated times (in min). Aliquots from the dephosphorylation reactions were then separated by SDS-PAGE and analyzed by autoradiography. Parallel experiments analyzed by quantitative filter paper assays also indicated no measurable dephosphorylation of the furin-cd by either enzyme (data not shown).
Figure 6
Endogenous furin-directed phosphatase activity. (A) Extracts from bovine brain (shown) and cultured cells were incubated with 32P-labeled GST–Furcd in vitro in the absence (Control) or presence of 100 nM tautomycin for the indicated times (in min). Aliquots of the reactions were separated by SDS-PAGE and analyzed by autoradiography. (B) Affinity chromatography of endogenous furin-directed phosphatase activity in BSC-40 cells. Cell extract was applied to a microcystin affinity column and the bound phosphatase eluted with 3M NaSCN. Aliquots of the original extract (Load), the unbound protein (FT), and eluted material (E1–3) were assayed for total PP1/PP2A activity using 32P-labeled phosphorylase a (open bars) and furin-directed phosphatase activity using 32P-labeled GST–Furcd (hatched bars). The total activities were calculated based on fraction volume and expressed as pmol/min. Assays were performed in triplicate with relative error (one standard deviation) of less than 5%. Similar results were obtained in several independent experiments.
Figure 7
Isoform-specific dephosphorylation of furin by PP2A. (A) Baculovirus recombinants (m.o.i. = 2) were used to express PP2A subunits, either alone or in combination, in Sf9 cells. At 64–72 h postinfection, cells were harvested and lysates were assayed for phosphatase activity with both 32P-labeled phosphorylase a (open bars) and phosphorylated 32P-labeled GST–Furcd (hatched bars). Noninfected Sf9 cells (data not shown) expressed a detectable level of endogenous furin-directed phosphatase activity which was not increased by expression of the catalytic subunit either alone or in combination with the A subunit (C, and A+C, respectively). The C and A subunits were also expressed with either the α or β isoforms of the B family of regulatory subunits (A+C+Bα and A+C+Bβ, respectively), as well as the α subunit from the unrelated B′ family (A+C+B′α). As a control, lysates from A+C+_Bβ–_expressing cells were exposed to 100 nM tautomycin and okadaic acid (+Inhib.) during the phosphatase assays. Assays were performed in triplicate with relative error <5%. The data shown are representative of several independent experiments. (B) Western analysis of PP2A subunit expression. Lysates of Sf9 cells infected with baculovirus recombinants encoding the indicated PP2A subunits were resolved by SDS-PAGE and screened by Western blot using antisera specific for the catalytic subunit or the Bα, Bβ, or B′α regulatory subunits.
Figure 8
Disruption of endogenous PP2A affects furin trafficking in vivo. BSC-40 cells were infected with vaccinia recombinants (m.o.i. = 5 each) expressing fur/f alone (A and B) or in combination with viruses expressing either SV-40 small t (C) or truncated and inactive small t mut3. At 6 h (D) postinfection, the cells were incubated with mAb M1 for 1 h in culture before fixation. The cells in B were incubated in the presence of 100 nM tautomycin during mAb M1 uptake The cells were then processed for immunofluorescence to localize internalized furin.
Figure 9
Model of furin trafficking in the endosomal system. Furin molecules are internalized via dynamin-sensitive, clathrin-dependent endocytosis. Within early endosomes shared by TfR, furin phosphorylated by CKII is directed toward a cell surface recycling pathway by virtue of its selective interaction with PACS-1, which links phosphorylated furin to components of the clathrin sorting machinery (Wan et al., 1998). This endosome to plasma membrane recycling pathway mirrors the local cycling loop which localizes furin to the TGN (Wan et al., 1998). Furin molecules dephosphorylated by PP2A isoforms containing B regulatory subunits are sorted from the plasma membrane/early endosome cycling loop to a TGN retrieval pathway. Black shading of either the tyrosine or AC motifs denotes their “active” state whereas gray shading reflects a “silencing” of these signals.
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