1. Volume 29, Issue 4, Pages 465-476 (February 2008)
Atypical Protein Kinase C Regulates Dual Pathways for Degradation of the Oncogenic Coactivator SRC-3/AIB1 
Ping Yi, Qin Feng, Larbi Amazit, David M. Lonard, Sophia Y. Tsai, Ming-Jer Tsai, Bert W. O'Malley 
Molecular Cell 
Volume 29, Issue 4, Pages (February 2008)
DOI: /j.molcel
Copyright © 2008 Elsevier Inc. Terms and Conditions

2. Figure 1 aPKC Stabilizes SRC-3 Protein
(A) Flag-SRC-3 and ERα expression vectors were cotransfected with HA-tagged kinase expression vectors into HeLa cells.
(B) Western blot analysis of Flag-SRC-3 protein level when cells were cotransfected with constitutively active mutant of PKCζ (A119E) or PKCι (A120D), or their kinase-dead mutants (K281W and K274W, respectively).
(C) RT-PCR was performed using Flag-specific primer and SRC-3-specific primer to detect the mRNA level of transfected Flag-SRC-3. An RT-PCR experiment without the addition of reverse transcriptase (RT) was included as a control.
(D) Pulse-chase experiment. HeLa cells were transfected with Flag-SRC-3 and ERα, with or without cotransfection of PKCζ. Shown is autoradiograph of Flag-SRC-3 immunoprecipitated from cell lysates.
(E) PKCζ stabilizes SRC-3 protein in proteasome-mediated degradation pathway. Cells were treated with proteasome inhibitor MG132 or vehicle DMSO for 16 hr.
(F) Endogenous SRC-3 and PKCζ protein levels in MCF-7 cells treated with siPKCζ.
(G) Real-time qRT-PCR was carried out to detect the mRNA level of endogenous SRC-3. The error bars represent the standard error of the means from at least three independent experiments.
(H) Overexpression of PKCι rescued the siRNA knockdown effect on SRC-3 protein level. Details were described in the Supplemental Experimental Procedures.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
Estrogen and Estrogen Receptor Are Required for the aPKC Stabilization Effect
(A) Effects of active PKCζ on SRCs in the presence of ERα.
(B) Effects of active PKCζ on Flag-SRC-3 protein level in the absence or presence of Flag-ERα, or Flag-PRB.
(C) E2 increases PKCζ-mediated stabilization effect. WT, wild-type PKCζ; CAT, constitutively active mutant; DM, kinase-dead mutant.
(D) PKCζ interacts with both SRC-3 and ERα. Cells were transfected with V5- PKCζ and Flag-SRC-3 or Flag-ERα. Cell lysates were immunoprecipitated by anti-Flag antibody or normal IgG, and western blot analysis was performed using anti-V5 antibody.
(E) Effects of different ERα mutants on the PKCζ-mediated stabilization effect. ERΔAF1, deletion of 1–178 amino acids; ERAF2m, K362D/V376D/L539A; ER(cyto)m, deletion of 250–303 amino acids.
(F) Coimmunoprecipitation experiment detecting the interaction between PKC and different ERα mutants.
(G) PKCζ has significantly reduced stabilization effect on SRC-3 AAA mutant.
(H) Autoradiograph of in vitro PKCζ kinase assay detecting phosphorylation of purified recombinant SRC-3 by incorporation of [γ-32P]ATP in the absence and presence of purified ERα and estrogen.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 PKCζ Enhances SRC-3 Coactivator Activity
(A) PKCζ enhances SRC-3-activated ER-targeted gene transcription. HeLa cells were transfected with ERα, SRC-3, PKCζ, or its mutants in the absence or presence of E2. The transcription of pS2 gene was measured by real-time qRT-PCR (left panel). The expression levels of PKCζ and its mutants were shown in the right panel.
(B) aPKC-stabilized SRC-3 is transcriptionally active in cell-free transcription assay. Plasmid ERE-E4 was assembled into chromatin and transcribed as described in the Experimental Procedures. ER and E2 were added in the reaction. Triangles represent increasing amounts of purified Flag-SRC-3 (1.5, 9, and 37.5 ng SRC-3 protein, respectively) from HeLa cells without (−PKCζ) or with (+PKCζ) cotransfection of active PKCζ. The relative level of E4 transcript was measured using real-time qPCR (left panel). Right panel: comparable amounts of Flag-SRC-3 (−PKCζ or +PKCζ, 9 ng protein) were used in the assay by western blot analysis. Error bars indicate the standard error of the means from three independent experiments.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Identification of an Important Region in SRC-3 for aPKC Stabilization Effect
(A) Effects of aPKC on SRC-3 A1–6 mutant.
(B) Effects of aPKC on SRC-3 deletion mutants.
(C) Effects of aPKC on SRC-3 mutants with deletion within the region of 1031–1130.
(D) Effects of aPKC on SRC-3 mutants with Ser/Thr-Ala mutations within the region of 1031–1130. Details of the mutants were described in the Supplemental Experimental Procedures.
(E) Protein stability of SRC-3 wild-type and the mutants as assayed by treating cells with 200 μg/ml cycloheximide at indicated time period. Shown in the left panel is the western blot analysis of Flag-SRC-3 protein. The right panel shows the quantification of protein intensity.
(F) Amino acid sequences of SRC –1097 (bolded) and the surrounding sequences. Acidic residues are in larger fonts. Ser/Thr residues are in italic.
(G) L5A mutation (shown underlined in [A]) significantly reduced aPKC stabilization effect.
(H) D/E8K/R mutation (shown underlined in [A]) significantly reduced aPKC stabilization effect.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
aPKC Stabilizes SRC-3 by Preventing Interaction with the 20S Proteasome C8
(A) SRC-3 interacts with GST-C8. 293T cells were transfected with Flag-SRC-3 and ERα. Cell lysates were incubated with GST fusion proteins, and the bound Flag-SRC-3 was detected by western blot. Upper panel, western blot analysis of Flag-SRC-3 pulled down by GST fusion proteins. Lower panel, Coomassie staining of GST and GST fusion proteins.
(B) GST-PSMA7 is a functional protein. In vitro-transcribed and -translated HA-HIF-1α was incubated with GST or GST-PSMA7, and the bound HA-HIF-1α was detected by anti-HA antibody.
(C) PKCζ decreases the interaction between SRC-3 and GST-C8.
(D) D/E8K/R mutation or deletion of 1031–1130 in SRC-3 abolishes the effect of PKCζ on the interaction between SRC-3 and C8.
(E) PKCζ decreases the interaction between endogenous SRC-3 and C8 in 293T cells.
(F) Knocking down of C8 protein in MCF-7 cells increases the SRC-3 protein level.
(G) p21-SRC-3(1028–1099) has weaker interaction with C8. GST-C8 pull-down experiment was carried out using in vitro-synthesized p21-SRC-3(1028–1099) and p21-SRC-3(1101–1172) fusion proteins. The bound proteins were detected using anti-p21 antibody.
(H) SRC –1099 region increased the stability of p21. HeLa cells were transfected with p21, p21-SRC-3(1028–1099) and p21-SRC-3(1101–1172), respectively, and cells were treated with 200 μg/ml cycloheximide at an indicated time period 1 day after transfection. Shown are western blot analyses using anti-p21 antibody.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6 PKCζ Protects SRC-3 from Proteasome-Mediated Degradation
(A) In vitro 26S proteasome degradation assay. Details were described in the Experimental Procedures.
(B) PKCζ protects SRC-3 from REGγ-mediated proteasome degradation.
(C) PKCζ protects SRC-3 from REGγ-mediated proteasome degradation in an in vitro-purified system. Purified baculovirus-expressed recombinant SRC-3 was phosphorylated by PKCζ in vitro in the presence of ERα and E2. Purified REGγ and 20S proteasome were then added into the reaction and were incubated for an indicated time period.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7
Effects of aPKC on ER-Mediated Gene Expression and Estrogen-Dependent Cell Growth
(A) SRC-3 protein level was decreased in MCF-7 (shPKCζ) stable cell line.
(B) Knocking down of PKCζ reduces estrogen-dependent ER target gene transcription. Cells were treated with 10−8 M E2 for 2 hr (c-myc, cyclin D1) or overnight (pS2) before being harvested. The transcription was quantified by real-time qRT-PCR and normalized against a cyclophilin mRNA level. ∗p < ∗∗p < Error bars indicate the standard error of the means from three independent experiments.
(C) MTT assay for detecting estrogen-dependent growth of MCF-7. ∗p < 0.01 (control versus shPKCζ cells in the presence of E2). Error bars indicate the standard error of the means from triplicate experiments.
(D) Estrogen-dependent cell growth by crystal violet staining. Cells were stained with crystal violet after 9 days of growth in medium with or without 10−8 M E2 (left panel). The right panel shows the relative cell densities estimated by Scion Image. Shown is the representative result from three independent experiments.
(E) Flow cytometry analysis on cell-cycle distribution of MCF-7 cells after 2 days of growth in the presence of 10−7 M E2.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions