Journal: Molecular Biology of the Cell

Article Title: The Rac-GAP Bcr is a novel regulator of the Par complex that controls cell polarity

doi: 10.1091/mbc.e13-06-0333

Figure Lengend Snippet: FIGURE 3: Bcr is a novel interaction partner of the Par-Tiam1 complex. (A) Schematic depicting the role of the Par-Tiam1 complex in polarized cell migration. Tiam1-mediated Rac1 activation promotes cytoskeletal remodeling important for protrusion formation, whereas PKCζ regulates the reorientation of the centrosome. (B) Bcr interacts with Par3 in COS7 cells. Lysates from COS7 cells expressing Par3 in the presence or absence of Flag-tagged Bcr or Tiam1 (positive control) were immunoprecipitated (IP) with an α-Flag antibody, and then immunoblotted with an α-Par3 antibody. Lysates were also immunoblotted with α-Par3 and α-Flag antibodies to demonstrate protein expression. N = 3. (C) Bcr interacts with Par3 in astrocytes. Lysates from cultured rat cortical astrocytes (DIV21) were immunoprecipitated with control immunoglobulin G (IgG; nonimmune: NI) or α-Bcr antibodies, and then immunoblotted with the α-Par3 or α-Bcr antibody. N = 3. (D) Bcr interacts with PKCζ in COS7 cells. Lysates from COS7 cells expressing Tiam1 (positive control) or Bcr alone or in combination with Flag-PKCζ were immunoprecipitated with α-Flag antibody, and then immunoblotted with α-Tiam1 or α-Bcr antibodies. Lysates were also blotted with α-Tiam1, α-Bcr, or α-Flag antibodies to confirm protein expression. N = 3. (E) Bcr interacts with PKCζ in cortical astrocytes. Lysates from cultured rat cortical astrocytes (DIV21) were immunoprecipitated with control IgG (NI) or α-Bcr antibodies, and then immunoblotted with α-PKCζ or α-Bcr antibodies. N = 3. (F) Bcr and Tiam1 require PKCζ to interact with Par6 in COS7 cells. Flag-tagged Tiam1 or Bcr was coexpressed alone or in combination with myc-Par6 in the absence or presence of Flag-PKCζ. Tiam1 and Bcr were immunoprecipitated with α-Tiam1 or α-Bcr antibodies, respectively, and then immunoblotted with anti-myc antibody to assess Par6 association. Lysates were blotted with α-Flag antibody to confirm protein expression. N = 3. (G) Domain structures of full-length Bcr and Abr as well as truncated Bcr constructs used in

Article Snippet: PKCζ pseudosubstrate inhibitor was from Santa Cruz Biotechnology.

Techniques: Migration, Activation Assay, Expressing, Positive Control, Immunoprecipitation, Cell Culture, Control, Construct

Journal: Molecular Biology of the Cell

Article Title: The Rac-GAP Bcr is a novel regulator of the Par complex that controls cell polarity

doi: 10.1091/mbc.e13-06-0333

Figure Lengend Snippet: FIGURE 4: Bcr negatively regulates PKCζ signaling by facilitating its degradation. (A) Western blot analysis of lysates obtained from WT and Bcr−/− mouse cortical astrocytes. Bcr-deficient astrocytes showed an increase in total PKCζ levels. Lysates were also immunoblotted with α-GAPDH antibodies for a loading control. (B) Quantification of PKCζ levels. N = 3. (C) Representative images showing the localization of PKCζ in WT and Bcr−/− astrocytes migrating in a scratch assay. PKCζ is localized to the leading edge (white arrows) in WT cells, whereas PKCζ immunostaining was increased and more diffuse in Bcr−/− cells. Dashed black line shows scratch location. Scale bar: 10 μm. (D) Western blot analysis of lysates obtained from WT and Bcr−/− mouse cortical astrocytes. Bcr-deficient astrocytes showed an increase in p-GSK-3β levels. Lysates were also blotted with total GSK-3β antibodies for a loading control. (E) Quantification of p-GSK-3β levels. N = 3. (F) Western blot analysis of lysates obtained from WT and Bcr−/− mouse cortical astrocytes. Bcr-deficient astrocytes showed an increase in β-catenin levels. Lysates were also blotted with α-GAPDH antibodies for a loading control. (G) Quantification of β-catenin levels. N = 3. (H) Western blot analysis of lysates from COS7 cells expressing myc (control) or myc-Bcr and treated with DMSO (control) or 10 μM of the proteasomal inhibitor MG132. Bcr overexpression reduces PKCζ levels, which is blocked by treating cells with MG132. Lysates were blotted with α-GAPDH antibodies for a loading control. (I) Quantification of PKCζ levels from (H). N = 3. (J) The ability of Bcr to reduce PKCζ levels depends on its Rac-GAP activity. Lysates from COS7 cells expressing myc (control) or myc-tagged Bcr, GAP-dead Bcr (BcrGD), constitutively active Rac (RacV12), or RacV12 plus myc-Bcr were immunoblotted with an α-PKCζ antibody. Lysates were

Article Snippet: PKCζ pseudosubstrate inhibitor was from Santa Cruz Biotechnology.

Techniques: Western Blot, Control, Wound Healing Assay, Immunostaining, Expressing, Over Expression, Activity Assay

Journal: Molecular Biology of the Cell

Article Title: The Rac-GAP Bcr is a novel regulator of the Par complex that controls cell polarity

doi: 10.1091/mbc.e13-06-0333

Figure Lengend Snippet: FIGURE 5: Bcr regulates polarity by restricting PKCζ function. (A) PKCζ inhibitor can partially rescue protrusion formation in Bcr−/− cortical mouse astrocytes. Representative images showing WT and Bcr−/− astrocytes treated overnight with PBS (control) or 10 μM of PKCζ pseudosubstrate inhibitor and then fixed and immunostained for acetylated tubulin. Dashed white line represents the scratch. Scale bar: 10 μm. n = ∼100; N = 3. (B) PKCζ inhibitor can partially rescue centrosome reorientation in Bcr−/− cortical mouse astrocytes. Quantification of polarized centrosomes in WT and Bcr−/− cortical mouse astrocytes treated overnight with PBS (control) or 10 μM of PKCζ pseudosubstrate inhibitor. n = ∼100; N = 3. (C) Overexpression of Bcr, but not Abr, negatively regulates PKCζ levels. Western blot analysis was performed on lysates from COS7 cells expressing control (empty vector) or myc-tagged Tiam1, Bcr, or Abr. Lysates were immunoblotted with α-PKCζ antibodies to assess PKCζ levels and α-GAPDH for a loading control. N = 3. (D) Quantification of PKCζ levels from (C). N = 3. (E) Bcr and Abr overexpression reduces Pak phosphorylation in COS7 cells. Western blot analysis of lysates from COS7 cells expressing control (myc) or myc-tagged Tiam1, Tiam1 and Bcr, or Tiam1 and Abr, immunoblotted for PKCζ. Lysates were blotted with an α-GAPDH antibody for a loading control. (F) Quantification of PKCζ levels from (E). N = 3. (G) Expression of Bcr, but not Abr, rescues protrusion defects in Bcr−/− cortical mouse astrocytes. WT astrocytes were transfected with eGFP as a positive control. Bcr−/− astrocytes were transfected with eGFP alone or in combination with Bcr or Abr expression plasmids, and then astrocytes were subjected to scratch assays. Cells were then fixed 18 h postscratch and immunostained for acetylated tubulin (red). Yellow dashed line represents scratch. Scale bar: 10 μm. (H) Quantification of the protrusion assay. n = ∼100; N = 3. (I) Expression of Bcr, but not Abr, can rescue centrosome reorientation defects in Bcr−/− cortical mouse astrocytes. Quantification of polarized centrosomes in WT astrocytes transfected with eGFP (positive control) or Bcr−/− cortical mouse astrocytes transfected with eGFP, eGFP and Bcr, or eGFP and Abr. n = ∼100; N = 3. Data are shown ± SEM.

Article Snippet: PKCζ pseudosubstrate inhibitor was from Santa Cruz Biotechnology.

Techniques: Control, Over Expression, Western Blot, Expressing, Plasmid Preparation, Phospho-proteomics, Transfection, Positive Control

Journal: Biochemical Journal

Article Title: Effects of transiently expressed atypical (ζ, λ), conventional (α, β) and novel (δ, ɛ) protein kinase C isoforms on insulin-stimulated translocation of epitope-tagged GLUT4 glucose transporters in rat adipocytes: specific interchangeable effects of protein kinases C-ζ and C-λ

doi: 10.1042/bj3370461

Figure Lengend Snippet: Figure 4 Effects of KI forms of PKC-α, PKC-β2, PKC-δ, PKC-ε and PKC-ζ on HAA-GLUT4 translocation in rat adipocytes

Article Snippet: Antibodies used for Western analyses included mouse monoclonal anti-HAA antibody (Berkeley Antibody Corp.), polyclonal antisera recognizing PKC-α, PKC-β1, PKC-β2, PKCδ, the nearly identical C-termini of PKC-ζ and PKC-λ, the Nterminus of PKC-ζ and the N-terminus of PKC-λ (Santa Cruz Biotechnologies, Santa Cruz, CA, U.S.A.), polyclonal anti(PKC-ε serum) (Life Technologies, Gaithersburg, MD, U.S.A.), polyclonal anti-(Myc serum) (Upstate Biotechnologies Inc., Lake Placid, NY, U.S.A.) and mouse monoclonal anti-(PKC-λ) antibody (raised against an internal site : amino acids 397–558) (Transduction Laboratories, Lexington, KY, U.S.A.).

Techniques: Translocation Assay

Journal: Biochemical Journal

Article Title: Effects of transiently expressed atypical (ζ, λ), conventional (α, β) and novel (δ, ɛ) protein kinase C isoforms on insulin-stimulated translocation of epitope-tagged GLUT4 glucose transporters in rat adipocytes: specific interchangeable effects of protein kinases C-ζ and C-λ

doi: 10.1042/bj3370461

Figure Lengend Snippet: Figure 5 Effects of WT forms of PKC-α, PKC-β1, PKC-β2, PKC-δ, PKC-ε and PKC-ζ on HAA-GLUT4 translocation in rat adipocytes

Article Snippet: Antibodies used for Western analyses included mouse monoclonal anti-HAA antibody (Berkeley Antibody Corp.), polyclonal antisera recognizing PKC-α, PKC-β1, PKC-β2, PKCδ, the nearly identical C-termini of PKC-ζ and PKC-λ, the Nterminus of PKC-ζ and the N-terminus of PKC-λ (Santa Cruz Biotechnologies, Santa Cruz, CA, U.S.A.), polyclonal anti(PKC-ε serum) (Life Technologies, Gaithersburg, MD, U.S.A.), polyclonal anti-(Myc serum) (Upstate Biotechnologies Inc., Lake Placid, NY, U.S.A.) and mouse monoclonal anti-(PKC-λ) antibody (raised against an internal site : amino acids 397–558) (Transduction Laboratories, Lexington, KY, U.S.A.).

Techniques: Translocation Assay

Journal: Biochemical Journal

Article Title: Effects of transiently expressed atypical (ζ, λ), conventional (α, β) and novel (δ, ɛ) protein kinase C isoforms on insulin-stimulated translocation of epitope-tagged GLUT4 glucose transporters in rat adipocytes: specific interchangeable effects of protein kinases C-ζ and C-λ

doi: 10.1042/bj3370461

Figure Lengend Snippet: Figure 6 Effects of Constit forms of PKC-α, PKC-β2 and PKC-ζ on HAA-GLUT4 translocation in rat adipocytes

Article Snippet: Antibodies used for Western analyses included mouse monoclonal anti-HAA antibody (Berkeley Antibody Corp.), polyclonal antisera recognizing PKC-α, PKC-β1, PKC-β2, PKCδ, the nearly identical C-termini of PKC-ζ and PKC-λ, the Nterminus of PKC-ζ and the N-terminus of PKC-λ (Santa Cruz Biotechnologies, Santa Cruz, CA, U.S.A.), polyclonal anti(PKC-ε serum) (Life Technologies, Gaithersburg, MD, U.S.A.), polyclonal anti-(Myc serum) (Upstate Biotechnologies Inc., Lake Placid, NY, U.S.A.) and mouse monoclonal anti-(PKC-λ) antibody (raised against an internal site : amino acids 397–558) (Transduction Laboratories, Lexington, KY, U.S.A.).

Techniques: Translocation Assay

Journal: Biochemical Journal

Article Title: Effects of transiently expressed atypical (ζ, λ), conventional (α, β) and novel (δ, ɛ) protein kinase C isoforms on insulin-stimulated translocation of epitope-tagged GLUT4 glucose transporters in rat adipocytes: specific interchangeable effects of protein kinases C-ζ and C-λ

doi: 10.1042/bj3370461

Figure Lengend Snippet: Figure 7 Effects of WT and KI forms of PKC-λ on HAA-GLUT4 translocation in rat adipocytes

Article Snippet: Antibodies used for Western analyses included mouse monoclonal anti-HAA antibody (Berkeley Antibody Corp.), polyclonal antisera recognizing PKC-α, PKC-β1, PKC-β2, PKCδ, the nearly identical C-termini of PKC-ζ and PKC-λ, the Nterminus of PKC-ζ and the N-terminus of PKC-λ (Santa Cruz Biotechnologies, Santa Cruz, CA, U.S.A.), polyclonal anti(PKC-ε serum) (Life Technologies, Gaithersburg, MD, U.S.A.), polyclonal anti-(Myc serum) (Upstate Biotechnologies Inc., Lake Placid, NY, U.S.A.) and mouse monoclonal anti-(PKC-λ) antibody (raised against an internal site : amino acids 397–558) (Transduction Laboratories, Lexington, KY, U.S.A.).

Techniques: Translocation Assay

Journal: Biochemical Journal

Article Title: Effects of transiently expressed atypical (ζ, λ), conventional (α, β) and novel (δ, ɛ) protein kinase C isoforms on insulin-stimulated translocation of epitope-tagged GLUT4 glucose transporters in rat adipocytes: specific interchangeable effects of protein kinases C-ζ and C-λ

doi: 10.1042/bj3370461

Figure Lengend Snippet: Figure 8 Effects of combined treatment with KI forms of PKC-λ and PKC-ζ on HAA-GLUT4 translocation in rat adipocytes

Article Snippet: Antibodies used for Western analyses included mouse monoclonal anti-HAA antibody (Berkeley Antibody Corp.), polyclonal antisera recognizing PKC-α, PKC-β1, PKC-β2, PKCδ, the nearly identical C-termini of PKC-ζ and PKC-λ, the Nterminus of PKC-ζ and the N-terminus of PKC-λ (Santa Cruz Biotechnologies, Santa Cruz, CA, U.S.A.), polyclonal anti(PKC-ε serum) (Life Technologies, Gaithersburg, MD, U.S.A.), polyclonal anti-(Myc serum) (Upstate Biotechnologies Inc., Lake Placid, NY, U.S.A.) and mouse monoclonal anti-(PKC-λ) antibody (raised against an internal site : amino acids 397–558) (Transduction Laboratories, Lexington, KY, U.S.A.).

Techniques: Translocation Assay

Journal: Biochemical Journal

Article Title: Effects of transiently expressed atypical (ζ, λ), conventional (α, β) and novel (δ, ɛ) protein kinase C isoforms on insulin-stimulated translocation of epitope-tagged GLUT4 glucose transporters in rat adipocytes: specific interchangeable effects of protein kinases C-ζ and C-λ

doi: 10.1042/bj3370461

Figure Lengend Snippet: Figure 9 Interchangeable reversing effects of WT forms of PKC-ζ and PKC-λ on HAA-GLUT4 translocation in adipocytes inhibited by KI forms of PKC-λ and PKC-ζ

Article Snippet: Antibodies used for Western analyses included mouse monoclonal anti-HAA antibody (Berkeley Antibody Corp.), polyclonal antisera recognizing PKC-α, PKC-β1, PKC-β2, PKCδ, the nearly identical C-termini of PKC-ζ and PKC-λ, the Nterminus of PKC-ζ and the N-terminus of PKC-λ (Santa Cruz Biotechnologies, Santa Cruz, CA, U.S.A.), polyclonal anti(PKC-ε serum) (Life Technologies, Gaithersburg, MD, U.S.A.), polyclonal anti-(Myc serum) (Upstate Biotechnologies Inc., Lake Placid, NY, U.S.A.) and mouse monoclonal anti-(PKC-λ) antibody (raised against an internal site : amino acids 397–558) (Transduction Laboratories, Lexington, KY, U.S.A.).

Techniques: Translocation Assay

Journal: Biochemical Journal

Article Title: Effects of transiently expressed atypical (ζ, λ), conventional (α, β) and novel (δ, ɛ) protein kinase C isoforms on insulin-stimulated translocation of epitope-tagged GLUT4 glucose transporters in rat adipocytes: specific interchangeable effects of protein kinases C-ζ and C-λ

doi: 10.1042/bj3370461

Figure Lengend Snippet: Figure 10 Comparative effects of PMA (TPA), wortmannin, RO 31-8220, myristylated PKC-ζ/λ pseudosubstrate and GO 6976 on HAA-GLUT4 translocation (A) and 2-DOG uptake (B) in the rat adipocyte

Article Snippet: Antibodies used for Western analyses included mouse monoclonal anti-HAA antibody (Berkeley Antibody Corp.), polyclonal antisera recognizing PKC-α, PKC-β1, PKC-β2, PKCδ, the nearly identical C-termini of PKC-ζ and PKC-λ, the Nterminus of PKC-ζ and the N-terminus of PKC-λ (Santa Cruz Biotechnologies, Santa Cruz, CA, U.S.A.), polyclonal anti(PKC-ε serum) (Life Technologies, Gaithersburg, MD, U.S.A.), polyclonal anti-(Myc serum) (Upstate Biotechnologies Inc., Lake Placid, NY, U.S.A.) and mouse monoclonal anti-(PKC-λ) antibody (raised against an internal site : amino acids 397–558) (Transduction Laboratories, Lexington, KY, U.S.A.).

Techniques: Translocation Assay

Journal: Biochemical Journal

Article Title: Effects of transiently expressed atypical (ζ, λ), conventional (α, β) and novel (δ, ɛ) protein kinase C isoforms on insulin-stimulated translocation of epitope-tagged GLUT4 glucose transporters in rat adipocytes: specific interchangeable effects of protein kinases C-ζ and C-λ

doi: 10.1042/bj3370461

Figure Lengend Snippet: Figure 11 Effects of the cell-permeant myristylated (Myr) PKC-ζ/λ pseudosubstrate (PS) on insulin-stimulated GLUT4 translocation in rat adipocytes down-regulated or non-down-regulated by prolonged phorbol ester treatment

Article Snippet: Antibodies used for Western analyses included mouse monoclonal anti-HAA antibody (Berkeley Antibody Corp.), polyclonal antisera recognizing PKC-α, PKC-β1, PKC-β2, PKCδ, the nearly identical C-termini of PKC-ζ and PKC-λ, the Nterminus of PKC-ζ and the N-terminus of PKC-λ (Santa Cruz Biotechnologies, Santa Cruz, CA, U.S.A.), polyclonal anti(PKC-ε serum) (Life Technologies, Gaithersburg, MD, U.S.A.), polyclonal anti-(Myc serum) (Upstate Biotechnologies Inc., Lake Placid, NY, U.S.A.) and mouse monoclonal anti-(PKC-λ) antibody (raised against an internal site : amino acids 397–558) (Transduction Laboratories, Lexington, KY, U.S.A.).

Techniques: Translocation Assay

Journal: Scientific Reports

Article Title: TLR4 counteracts BVRA signaling in human leukocytes via differential regulation of AMPK, mTORC1 and mTORC2

doi: 10.1038/s41598-019-43347-8

Figure Lengend Snippet: Our working model for biliverdin/BVRA and TLR4 signaling in leukocytes. Biliverdin induced responses are shown in black. Biliverdin acts on BVRA. Our data suggest that biliverdin facilitates formation of BVRA signaling complex(s) that include mTORC2. mTORC2 induces phosphorylation of two targets: PKCζ at Thr410 and Akt at Ser473. PKCζ is involved in LKB1 Ser431 and AMPKα Thr172 phosphorylation. AMPK phosphorylates TSC1/2 at Ser1387 and Raptor at Ser792. Therefore, BVRA activation may contribute to mTORC1 inhibition via two SI: phosphorylated Raptor, which inhibits mTORC1 complex formation, and TSC1/2 mediated inhibition of mTORC1 activity. Because mTORC1 is central to TLR4 signaling in leukocytes , BVRA inhibition of mTORC1 suppresses TLR4. Pharmacological inhibitors are shown in blue. Torin and PP242, but not rapamycin, inhibited BVRA-mediated protein-protein interactions. TLR4 induced response are shown in red. Once activated, TLR4 down-regulates BVRA and PKCζ expression (dashed lines). TLR4 upregulates expression of MMP9 , which then triggers AMPKα cleavage (dashed line), leading to Raptor Ser792 dephosphorylation . This enables mTORC1 activation, which then triggers an increase in HIF-1α expression and S6K1 phosphorylation at Thr389. TLR4 also triggers an increase in haptoglobin expression, thus limiting heme availability. Using these parallel mechanisms TLR4 ensures biliverdin/BVRA and mTORC2 signaling inhibition, and on the other hand, mTORC1 activation.

Article Snippet: HIF-1α (sc-10790; 1:250), AMPKα (sc-25792; 1:1000), MMP9 (sc-10737; 1:1000), TLR4 (sc-10741; 1:500), BVRA (sc-393385; 1:1000), Akt1 (sc-5928; 1:500), p-LKB1 Ser431 (sc-271924; 1:500), LKB1 (sc-32245; 1:500), p-CaMKKβ Thr286 (sc-32289; 1:750), CaMKKβ (sc-100364; 1:1000), p-mTOR Ser2481 (sc-293132; 1:1000), mTOR (sc-517464; 1:1000), p-PKCζ Thr410 (sc-271962; 1:500), PKCζ (sc-393218; 1:500), heme oxygenase 1 (HO-1) (sc-136960; 1:1000), heme oxygenase 2 (HO-2) (sc-17786; 1:1000), tuberin (TSC1/2)(sc-271314; 1:500), haptoglobin β (sc-390962; 1:500), p-NOS3 Ser1177 (sc-81510; 1:500) and NOS3 (sc-376751; 1:500) were all from Santa Cruz Biotechnology. p-Raptor Ser792 (#2083; 1:1000), Raptor (#2280; 1:200), p-p70 S6 kinase Thr389 (#9205; 1:1000), p-AMPKα Thr172 (#2535; 1:1000), p-Akt Ser473 (#4051; 1:1000), p-Akt Thr308 (#9275; 1:1000), tuberin/TSC2 (#4308; 1:1000) and p-tuberin/TSC2 Ser1387 (#5584; 1:1000) were all from Cell Signaling Technology.

Techniques: Phospho-proteomics, Activation Assay, Inhibition, Activity Assay, Protein-Protein interactions, Expressing, De-Phosphorylation Assay