Localization of atypical protein kinase C isoforms into lysosome-targeted endosomes through interaction with p62 - PubMed (original) (raw)
Localization of atypical protein kinase C isoforms into lysosome-targeted endosomes through interaction with p62
P Sanchez et al. Mol Cell Biol. 1998 May.
Abstract
An increasing number of independent studies indicate that the atypical protein kinase C (PKC) isoforms (aPKCs) are critically involved in the control of cell proliferation and survival. The aPKCs are targets of important lipid mediators such as ceramide and the products of the PI 3-kinase. In addition, the aPKCs have been shown to interact with Ras and with two novel proteins, LIP (lambda-interacting protein; a selective activator of lambda/iotaPKC) and the product of par-4 (a gene induced during apoptosis), which is an inhibitor of both lambda/iotaPKC and zetaPKC. LIP and Par-4 interact with the zinc finger domain of the aPKCs where the lipid mediators have been shown to bind. Here we report the identification of p62, a previously described phosphotyrosine-independent p56(lck) SH2-interacting protein, as a molecule that interacts potently with the V1 domain of lambda/iotaPKC and, albeit with lower affinity, with zetaPKC. We also show in this study that ectopically expressed p62 colocalizes perfectly with both lambda/iotaPKC and zetaPKC. Interestingly, the endogenous p62, like the ectopically expressed protein, displays a punctate vesicular pattern and clearly colocalizes with endogenous lambda/iotaPKC and endogenous zetaPKC. P62 colocalizes with Rab7 and partially with lamp-1 and limp-II as well as with the epidermal growth factor (EGF) receptor in activated cells, but not with Rab5 or the transferrin receptor. Of functional relevance, expression of dominant negative lambda/iotaPKC, but not of the wild-type enzyme, severely impairs the endocytic membrane transport of the EGF receptor with no effect on the transferrin receptor. These findings strongly suggest that the aPKCs are anchored by p62 in the lysosome-targeted endosomal compartment, which seems critical for the control of the growth factor receptor trafficking. This is particularly relevant in light of the role played by the aPKCs in mitogenic cell signaling events.
Figures
FIG. 1
p62 interacts with the aPKC in vitro. (Upper panel) Purified MBP or MBP-p62 (400 nM) was immobilized on amylose beads and incubated with 1 mg of protein extracts from HeLa cells at 22°C for 1 h. After extensive washing, the recombinant proteins were fractionated by SDS-PAGE and the associated PKC isotypes were determined with the corresponding isotype-specific antibodies by immunoblotting. Ext., 50 μg of protein extracts was run in parallel as a control. MW, molecular weight (in thousands). (Lower panel) Amounts of MBP fusion proteins in each experiment were the same. Essentially identical results were obtained in two more experiments.
FIG. 2
p62 interacts with the aPKC in vivo. (Upper panel) Subconfluent cultures of HeLa cells in 100-mm-diameter plates were transfected with 20 μg of either pCDNA3 (control) or pCDNA3-HA-p62 (p62). Plasmid DNA was removed 4 h later, and the cells were incubated in DMEM containing 10% fetal calf serum for 36 h. Cell extracts (200 μg of protein) were immunoprecipitated with anti-HA antibody. Immunoprecipitates were resolved by SDS-PAGE and immunoblotted with isotype-specific anti-PKC antibodies. The extract (Ext.) lane contains 20 μg of cell protein extract. (Lower panel) Immunoblot analysis of the HA-p62 expressed protein in each assay. Essentially identical results were obtained in two more experiments.
FIG. 3
Endogenous p62 interacts with the aPKC in vivo. (A) Cell protein extracts from HeLa cells (20 μg) were separated by SDS-PAGE and immunoblotted with either preimmune (PI) or affinity-purified anti-p62 antiserum (I). MW, molecular weight (in thousands). (B) Protein extracts from HeLa cells (200 μg) were immunoprecipitated with either preimmune (PI) or affinity-purified immune anti-p62 antiserum (I), and the associated λ/ιPKC or ζPKC was determined by immunoblotting with isotype-specific antibodies. The extract (Ext.) lane contains 20 μg of cell protein extract. Essentially identical results were obtained in two more experiments.
FIG. 4
Activities of the aPKC are not regulated by recombinant p62. Immunopurified λ/ιPKC (upper panel) or ζPKC (lower panel) was incubated with 1 μg of either MBP, MBP-LIP (LIP), or MBP-Par-4 (Par-4) or with different amounts of MBP-p62 (p62) either with or without phosphatidylserine (50 μg/ml). Afterward, the activity of both PKCs toward MBP was determined as described in Materials and Methods. Essentially identical results were obtained in two more experiments.
FIG. 5
p62 is not a substrate of the aPKC. The ability of immunopurified λ/ιPKC or ζPKC to phosphorylate 1 μg of recombinant hnRNP A1, MyBP, or p62 was determined as described in Materials and Methods either in the absence or in the presence of phosphatidylserine (50 μg/ml). Essentially identical results were obtained in two more experiments.
FIG. 6
Cellular localization of ectopically expressed and endogenous p62. HeLa cells were transfected with 5 μg of an expression vector for the HA-tagged p62 (left panel) or left untransfected (right panel). Twenty hours posttransfection, the ectopically expressed HA-p62 (left panel) was immunostained with the monoclonal anti-HA antibody 12CA5, whereas the endogenous protein (right panel) was immunostained with the affinity-purified polyclonal anti-p62 antibody, and both were analyzed by confocal laser scanning microscopy. Bar, 5 μm. Essentially identical results were obtained in three more experiments.
FIG. 7
Colocalization of ectopically expressed p62 with transfected λ/ιPKC and ζPKC but not with αPKC or ɛPKC. HeLa cells were transfected with 5 μg of an expression vector for myc-tagged p62 along with 5 μg of expression plasmids for either HA-λ/ιPKC or HA-ζPKC (A) or His-αPKC or His-ɛPKC (B). Twenty hours posttransfection, cells were analyzed by confocal laser scanning microscopy with the monoclonal antibody 12CA5 and Texas red-conjugated anti-mouse IgG (red fluorescence) to detect λ/ιPKC or ζPKC or monoclonal anti-His and FITC-conjugated anti-mouse IgG (green fluorescence), αPKC or ɛPKC and rabbit polyclonal anti-myc antibody and either FITC-conjugated (green fluorescence) or Texas red-conjugated (red fluorescence) anti-rabbit IgG, and myc-p62. Bar, 5 μm. Essentially identical results were obtained in three more experiments.
FIG. 8
Colocalization of endogenous p62 with endogenous λ/ιPKC and ζPKC. HeLa cells were analyzed by confocal laser scanning microscopy with monoclonal anti-λ/ιPKC and monoclonal anti-ζPKC (A) and monoclonal anti-αPKC or anti-ɛPKC (B) and Texas red-conjugated anti-mouse IgG (red fluorescence) and affinity-purified anti-p62 polyclonal antibody and FITC-conjugated anti-rabbit IgG (green fluorescence). Bar, 5 μm. Essentially identical results were obtained in three more experiments.
FIG. 9
Colocalization of p62 with different endosomal and lysosomal markers. HeLa cells were transfected with 5 μg of an expression vector for HA-tagged p62 and analyzed 20 h posttransfection by confocal laser scanning microscopy with monoclonal anti-HA antibody and FITC-conjugated anti-mouse IgG (green fluorescence) (A) or polyclonal anti-HA antibody (B and C) and Texas red-conjugated anti-rabbit IgG (red fluorescence) (B) or FITC-conjugated anti-rabbit IgG (green fluorescence) (C). Rab7 (A) was detected with an anti-rab 7 polyclonal antibody and Texas red-conjugated anti-rabbit IgG (red fluorescence). Lamp-1 (B) was detected with a monoclonal anti-lamp-1 antibody and FITC-conjugated anti-mouse IgG (green fluorescence). Limp II (C) was detected with a monoclonal anti-Limp II antibody and Texas red-conjugated anti-mouse IgG (red fluorescence). Bar, 5 μm. Essentially identical results were obtained in three more experiments.
FIG. 10
Association of p62 and λ/ιPKC with endosome-enriched fraction in HeLa cells. HeLa cells loaded with HRP at 37°C for 30 min were homogenized, and the postnuclear supernatant was collected and loaded at the bottom of a sucrose step gradient as described in Materials and Methods. After centrifugation, fractions were analyzed by immunoblotting (with p62, Rab7, and λ/ιPKC) and assayed for enzymatic markers (HRP and β-
d
-glucuronidase activities). Essentially identical results were obtained in three more experiments.
FIG. 11
Colocalization of p62 with the EGF receptor. HeLa cells were made quiescent by 24 h of serum starvation, after which they were incubated with 100 ng of EGF per ml for 1 h at 4°C. Afterward, unbound EGF was extensively washed, and the cells were incubated for different times at 37°C and analyzed by confocal laser scanning microscopy with monoclonal anti-EGF receptor and Texas red-conjugated anti-mouse IgG (red fluorescence) and affinity-purified anti-p62 polyclonal antibody and FITC-conjugated anti-rabbit IgG (green fluorescence). Bar, 5 μm. Essentially identical results were obtained in three more experiments.
FIG. 12
Lack of colocalization of p62 with the transferrin receptor. HeLa cells were transfected with 5 μg of HA-tagged p62 expression vector and analyzed 20 h posttransfection by confocal laser scanning microscopy with a polyclonal anti-HA antibody and Texas red-conjugated anti-rabbit IgG (red fluorescence) to detect the p62 and monoclonal anti-transferrin receptor (TfR) antibody and FITC-conjugated anti-mouse IgG (green fluorescence). Bar, 5 μm. Essentially identical results were obtained in three more experiments.
FIG. 13
Role of λ/ιPKC in the endocytic membrane transport of the EGF receptor. HeLa cells were transfected with 5 μg of either HA-tagged λ/ιPKC or HA-tagged λ/ιPKCMUT expression vectors. Twenty hours posttransfection, cells were made quiescent by serum starvation for 24 h. Afterward, cells were incubated for 1 h at 4°C with 100 ng of EGF per ml. After extensive washing of the unbound EGF, cells were incubated for 60 min at 37°C. Cells were analyzed by confocal microscopy with monoclonal antibodies specific for either EGF receptor (EGFR) or transferrin receptor (TfR) and FITC-conjugated anti-mouse IgG (green fluorescence) and a polyclonal anti-HA antibody and Texas red-conjugated anti-rabbit IgG (red fluorescence) to detect the transfected λ/ιPKC constructs. Bar, 5 μm. Essentially identical results were obtained in three more experiments.
References
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