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Binding to Syntenin-1 Protein Defines a New Mode of Ubiquitin-based Interactions Regulated by Phosphorylation

Rajesh, Sundaresan, Bago, Ružica, Odintsova, Elena, Muratov, Gayrat, Baldwin, Gouri, Sridhar, Pooja, Rajesh, Sandya, Overduin, Michael, Berditchevski, Fedor·2011·Journal of Biological Chemistry
DOI 10.1074/jbc.m111.262402
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FIGURE 1 Syntenin-1 links its transmembrane partners to ubiquitylated proteins. A , lysates prepared from HeLa cells transfected with either the siRNAtargetingsyntenin-1orcontrolsiRNAwereincubatedwithbiotinylated peptides corresponding to the cytoplasmic tails of syndecan-2 ( SYND-C ), TGF 𝛂 ( TGF 𝛂 -C ), and Tspan-12-C ( TSP12-C ). Complexes were collected on NeutrAvidin-agarose and resolved on SDS-PAGE. Pulled-down ubiquitylated proteins interacting with the peptides were visualized with anti-Ub mAb (FK2). Shownareresults of the pulldown experiments. Lower panel showsefficiency of syntenin-1 knockdown. Note, knockdown of syntenin-1 markedly disrupted the interaction of the peptides with ubiquitylated proteins. WB , Western blotting. B , lysates prepared from HEK293T cells expressing FLAG-syntenin-1 and HA-Ub were incubated with either biotinylated CD63-C and Tspan12-C (control) peptides or anti-FLAG mAb. Protein complexes were collected on NeutrAvidin-agarose, resolved on SDS-PAGE, and subsequently probed with anti-HA and anti-FLAG polyclonal Abs in Western blotting. Arrows point to ubiquitylated proteins from the syntenin-1 immunoprecipitate ( IP ; lane 1 ) that are distinct from the ubiquitylated species of syntenin-1 (characteristic ladder, lane 7 ). Note that CD63-C interacts with both ubiquitylated syntenin-1 and the associated proteins. Two exposures are shown to compare the patterns of proteins precipitated with anti-FLAG mAb and CD63-C.
FIGURE 1 Syntenin-1 links its transmembrane partners to ubiquitylated proteins. A , lysates prepared from HeLa cells transfected with either the siRNAtargetingsyntenin-1orcontrolsiRNAwereincubatedwithbiotinylated peptides corresponding to the cytoplasmic tails of syndecan-2 ( SYND-C ), TGF 𝛂 ( TGF 𝛂 -C ), and Tspan-12-C ( TSP12-C ). Complexes were collected on NeutrAvidin-agarose and resolved on SDS-PAGE. Pulled-down ubiquitylated proteins interacting with the peptides were visualized with anti-Ub mAb (FK2). Shownareresults of the pulldown experiments. Lower panel showsefficiency of syntenin-1 knockdown. Note, knockdown of syntenin-1 markedly disrupted the interaction of the peptides with ubiquitylated proteins. WB , Western blotting. B , lysates prepared from HEK293T cells expressing FLAG-syntenin-1 and HA-Ub were incubated with either biotinylated CD63-C and Tspan12-C (control) peptides or anti-FLAG mAb. Protein complexes were collected on NeutrAvidin-agarose, resolved on SDS-PAGE, and subsequently probed with anti-HA and anti-FLAG polyclonal Abs in Western blotting. Arrows point to ubiquitylated proteins from the syntenin-1 immunoprecipitate ( IP ; lane 1 ) that are distinct from the ubiquitylated species of syntenin-1 (characteristic ladder, lane 7 ). Note that CD63-C interacts with both ubiquitylated syntenin-1 and the associated proteins. Two exposures are shown to compare the patterns of proteins precipitated with anti-FLAG mAb and CD63-C.CC-BY · From the PDF
FIGURE 2 Syntenin-1 interacts with Ub. A , syntenin-1 is retained by immobilized mono-Ub from cellular lysates. Cellular lysates prepared from HeLa cells were incubated with mono-Ub covalently attached to agarose beads ( mUbi ); interacting proteins were eluted from the beads and equal volumes of the eluate (controlled by Western blotting with mAb to actin) were resolved on SDS-PAGE. Syntenin-1, Hrs, and CASK were detected by Western blotting ( WB ). B , upper , recombinant syntenin-1 (0.1 and 0.5 𝛍 g) was incubated with immobilized His-Ub or His-SmpA (negative control) and His-poly-Ub (2-7) chains (Lys 48 -linked) or His-poly-Ub (2-7) chains (Lys 63 -linked). Interactions between the proteins were visualized by Western blotting using anti-syntenin-1 mAb. Lower , loading of His-Ub species was controlled using anti-His HRP mAb antibody.
FIGURE 2 Syntenin-1 interacts with Ub. A , syntenin-1 is retained by immobilized mono-Ub from cellular lysates. Cellular lysates prepared from HeLa cells were incubated with mono-Ub covalently attached to agarose beads ( mUbi ); interacting proteins were eluted from the beads and equal volumes of the eluate (controlled by Western blotting with mAb to actin) were resolved on SDS-PAGE. Syntenin-1, Hrs, and CASK were detected by Western blotting ( WB ). B , upper , recombinant syntenin-1 (0.1 and 0.5 𝛍 g) was incubated with immobilized His-Ub or His-SmpA (negative control) and His-poly-Ub (2-7) chains (Lys 48 -linked) or His-poly-Ub (2-7) chains (Lys 63 -linked). Interactions between the proteins were visualized by Western blotting using anti-syntenin-1 mAb. Lower , loading of His-Ub species was controlled using anti-His HRP mAb antibody.CC-BY · From the PDF
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FIGURE 4 Both the N- and C-terminal regions are important for the interaction of syntenin-1 with Ub: role of dimerization. A , protein lysates prepared from HEK293T cells expressing syntenin-1WT-HA, syntenin𝚫 N-HA ( SYNT𝚫 N ), and syntenin𝚫 C-HA ( SYNT -𝚫 C ) were incubated with mUb covalently attached to agarose beads, and interacting proteins were resolved on SDS-PAGE. Syntenin-1 proteins were detected by Western blotting ( WB ) with anti-HA Ab. In the control experiments cells were transfected with pcDNA ( lanes 1 and 5 ). B , Protein lysates prepared from HEK293T cells expressing HA-tagged syntenin-1WT or dimerization mutant (F195A/L233A, referred to as SYNTmono ), were incubated with mUb covalently attached to agarose beads, and interacting proteins were resolved on SDS-PAGE. Syntenin-1 was detected by Western blotting with anti-HA Ab. C and D , protein lysates prepared from HEK293T cells expressing syntenin-1WT-FLAG, and various FLAG-tagged mutants of syntenin-1 were incubated with mUb covalently attached to agarose beads. Interacting proteins were resolved on SDS-PAGE. Syntenin-1 was detected by Western blotting with anti-FLAG Ab. In the control experiments cells were transfected with pcDNA ( lanes 1 and 5 for B and D ; lanes 1 and 4 for C ). E , recombinant wild-type syntenin-1 ( SYNTwt ) and syntenin-1 point mutants (P5A, SYNT P5A; S6A, SYNT S6A; and S6E, SYNT S6E) (0.5 𝛍 g each) were incubated with 30 𝛍 l of His-Ub-immobilized nickel-Sepharose beads in 50 mM NaPO4, pH 7.4, 500 mM NaCl, 0.5 mM tris(2-carboxyethyl)phosphine, 20 mM imidazole, 0.2% Triton X-100, 0.02% BSA. Bound proteins were eluted with 50 mM EDTA. The protein samples were resolved using SDS-PAGE and visualized with Coomassie Brilliant Blue staining. Note, additional low molecular mass protein bands correspond to proteolytic products of full-length syntenin. PD , pulldown.
FIGURE 4 Both the N- and C-terminal regions are important for the interaction of syntenin-1 with Ub: role of dimerization. A , protein lysates prepared from HEK293T cells expressing syntenin-1WT-HA, syntenin𝚫 N-HA ( SYNT𝚫 N ), and syntenin𝚫 C-HA ( SYNT -𝚫 C ) were incubated with mUb covalently attached to agarose beads, and interacting proteins were resolved on SDS-PAGE. Syntenin-1 proteins were detected by Western blotting ( WB ) with anti-HA Ab. In the control experiments cells were transfected with pcDNA ( lanes 1 and 5 ). B , Protein lysates prepared from HEK293T cells expressing HA-tagged syntenin-1WT or dimerization mutant (F195A/L233A, referred to as SYNTmono ), were incubated with mUb covalently attached to agarose beads, and interacting proteins were resolved on SDS-PAGE. Syntenin-1 was detected by Western blotting with anti-HA Ab. C and D , protein lysates prepared from HEK293T cells expressing syntenin-1WT-FLAG, and various FLAG-tagged mutants of syntenin-1 were incubated with mUb covalently attached to agarose beads. Interacting proteins were resolved on SDS-PAGE. Syntenin-1 was detected by Western blotting with anti-FLAG Ab. In the control experiments cells were transfected with pcDNA ( lanes 1 and 5 for B and D ; lanes 1 and 4 for C ). E , recombinant wild-type syntenin-1 ( SYNTwt ) and syntenin-1 point mutants (P5A, SYNT P5A; S6A, SYNT S6A; and S6E, SYNT S6E) (0.5 𝛍 g each) were incubated with 30 𝛍 l of His-Ub-immobilized nickel-Sepharose beads in 50 mM NaPO4, pH 7.4, 500 mM NaCl, 0.5 mM tris(2-carboxyethyl)phosphine, 20 mM imidazole, 0.2% Triton X-100, 0.02% BSA. Bound proteins were eluted with 50 mM EDTA. The protein samples were resolved using SDS-PAGE and visualized with Coomassie Brilliant Blue staining. Note, additional low molecular mass protein bands correspond to proteolytic products of full-length syntenin. PD , pulldown.CC-BY · From the PDF
FIGURE 5 Co-localization of syntenin-1 and ubiquitylated proteins. HeLa cells expressing FLAG-tagged syntenin-1 WT or syntenin-1 S6A ( S6A ) and syntenin-1 P5A ( P5A ) mutants were fixed with 2% paraformaldehyde and permeabilized with 0.1% Triton X-100. Cells were co-stained with anti-FLAG rabbit pAb and anti-Ub mouse mAb (clone FK2). Staining was visualized with Alexa Fluor 488-conjugated goat anti-mouse antibody and labeled with Alexa Fluor 594-conjugated anti-rabbit antibody. Overlay panels and insets show partial co-localization of wild-type syntenin-1 ( red ) with ubiquitylated cellular proteins ( green ) and lack of co-localization in cells expressing mutants of syntenin-1 ( red ). Scale bars , 10 𝛍 m.
FIGURE 5 Co-localization of syntenin-1 and ubiquitylated proteins. HeLa cells expressing FLAG-tagged syntenin-1 WT or syntenin-1 S6A ( S6A ) and syntenin-1 P5A ( P5A ) mutants were fixed with 2% paraformaldehyde and permeabilized with 0.1% Triton X-100. Cells were co-stained with anti-FLAG rabbit pAb and anti-Ub mouse mAb (clone FK2). Staining was visualized with Alexa Fluor 488-conjugated goat anti-mouse antibody and labeled with Alexa Fluor 594-conjugated anti-rabbit antibody. Overlay panels and insets show partial co-localization of wild-type syntenin-1 ( red ) with ubiquitylated cellular proteins ( green ) and lack of co-localization in cells expressing mutants of syntenin-1 ( red ). Scale bars , 10 𝛍 m.CC-BY · From the PDF
FIGURE 6 Ulk1 modifies syntenin via phosphorylation. A , protein lysates prepared from HEK293T cells expressing syntenin-1WT-FLAG, Ulk1-Myc, and the Ulk1-KD-Myc mutant were resolved by SDS-PAGE. B , samples prepared as aboveweresubjectedtotreatment with alkaline phosphatase ( AP ) for 40 min at 37 °C ( lanes 4 -6 ) and subsequently resolved on 10% SDS-PAGE. Note, alkalkine phosphatase treatment resulted in the loss of modification indicating that syntenin-1 is phosphorylated in the presence of Ulk1. C , protein lysates prepared from 293T cells expressing syntenin-1WT-FLAG, syntenin-1 S6A-FLAG mutant, and Ulk1-Myc were resolved on SDS-PAGE. D , protein lysates prepared from 293T cells expressing syntenin-1WT-FLAG and Ulk1Myc were incubated with immobilized monoubiquitin ( mUbi ), and proteins were resolved on SDS-PAGE. In the control experiments, cells were transfected with pcDNA ( lanes 1 and 4 ). Syntenin-1 and Ulk1 were detected by Western blotting ( WB ) with anti-FLAG and anti-Myc Abs, respectively ( A-D ). Actin was used as a loading control in all blots.
FIGURE 6 Ulk1 modifies syntenin via phosphorylation. A , protein lysates prepared from HEK293T cells expressing syntenin-1WT-FLAG, Ulk1-Myc, and the Ulk1-KD-Myc mutant were resolved by SDS-PAGE. B , samples prepared as aboveweresubjectedtotreatment with alkaline phosphatase ( AP ) for 40 min at 37 °C ( lanes 4 -6 ) and subsequently resolved on 10% SDS-PAGE. Note, alkalkine phosphatase treatment resulted in the loss of modification indicating that syntenin-1 is phosphorylated in the presence of Ulk1. C , protein lysates prepared from 293T cells expressing syntenin-1WT-FLAG, syntenin-1 S6A-FLAG mutant, and Ulk1-Myc were resolved on SDS-PAGE. D , protein lysates prepared from 293T cells expressing syntenin-1WT-FLAG and Ulk1Myc were incubated with immobilized monoubiquitin ( mUbi ), and proteins were resolved on SDS-PAGE. In the control experiments, cells were transfected with pcDNA ( lanes 1 and 4 ). Syntenin-1 and Ulk1 were detected by Western blotting ( WB ) with anti-FLAG and anti-Myc Abs, respectively ( A-D ). Actin was used as a loading control in all blots.CC-BY · From the PDF
FIGURE 7 Role of Ub binding in cellular localization of syntenin-1. HeLa cells expressing FLAG-tagged syntenin-1 (WT or syntenin-1 S6A ( S6A ) and syntenin-1 P5A ( P5A ) mutants) were fixed with 2% paraformaldehyde and permeabilized with 0.1% Triton X-100. A , cells were co-stained with anti-FLAG mouse mAb (M2) and anti-STAM rabbit pAb. Staining was visualized with Alexa Fluor 488-conjugated goat anti-mouse antibody and labeled with Alexa Fluor 594-conjugated anti-rabbit antibody. Overlay panels show minimal co-localization syntenin-1 proteins ( green ) with STAM ( red ). B , cells were co-stainedwithanti-FLAGrabbitpAbandanti-CD63mousemAb(6H1).Staining was visualized with Alexa Fluor 594-conjugated goat anti-mouse antibody and labeled with Alexa Fluor 488-conjugated anti-rabbit antibody. Shownare only overlay panels . Co-localization profiles (along the lines on the images directed toward the arrows ) are presented as the fluorescence intensity distribution for each channel (shown underneath each of the panels). Note the differences in degree of syntenin-1/CD63 co-localization in cells expressing the wild-type syntenin-1 ( top ) and syntenin-1 mutants ( middle and bottom ). Scale bars , 10 𝛍 m.
FIGURE 7 Role of Ub binding in cellular localization of syntenin-1. HeLa cells expressing FLAG-tagged syntenin-1 (WT or syntenin-1 S6A ( S6A ) and syntenin-1 P5A ( P5A ) mutants) were fixed with 2% paraformaldehyde and permeabilized with 0.1% Triton X-100. A , cells were co-stained with anti-FLAG mouse mAb (M2) and anti-STAM rabbit pAb. Staining was visualized with Alexa Fluor 488-conjugated goat anti-mouse antibody and labeled with Alexa Fluor 594-conjugated anti-rabbit antibody. Overlay panels show minimal co-localization syntenin-1 proteins ( green ) with STAM ( red ). B , cells were co-stainedwithanti-FLAGrabbitpAbandanti-CD63mousemAb(6H1).Staining was visualized with Alexa Fluor 594-conjugated goat anti-mouse antibody and labeled with Alexa Fluor 488-conjugated anti-rabbit antibody. Shownare only overlay panels . Co-localization profiles (along the lines on the images directed toward the arrows ) are presented as the fluorescence intensity distribution for each channel (shown underneath each of the panels). Note the differences in degree of syntenin-1/CD63 co-localization in cells expressing the wild-type syntenin-1 ( top ) and syntenin-1 mutants ( middle and bottom ). Scale bars , 10 𝛍 m.CC-BY · From the PDF

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Authors
Rajesh, S., Bago, R., Odintsova, E., Muratov, G., Baldwin, G., Sridhar, P., Rajesh, S., Overduin, M., & Berditchevski, F.
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
2011-11
Volume
286
Issue
45
Pages
39606-39614
ISSN
0021-9258
DOI
10.1074/jbc.m111.262402
Cited by
42
References
0
Language
en
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