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, 107 (45), 19314-9

Acetylation Modulates Prolactin Receptor Dimerization

Affiliations

Acetylation Modulates Prolactin Receptor Dimerization

Li Ma et al. Proc Natl Acad Sci U S A.

Abstract

Cytokine-activated receptors undergo extracellular domain dimerization, which is necessary to activate intracellular signaling pathways. Here, we report that in prolactin (PRL)-treated cells, PRL receptor (PRLR) undergoes cytoplasmic loop dimerization that is acetylation-dependent. PRLR-recruited CREB-binding protein (CBP) acetylates multiple lysine sites randomly distributed along the cytoplasmic loop of PRLR. Two PRLR monomers appear to interact with each other at multiple parts from the membrane-proximal region to the membrane-distal region, relying on the coordination among multiple lysine sites neutralized via acetylation. Cytoplasmic loop-dimerized PRLR activates STAT5, which is also acetylated by CBP and undergoes acetylation-dependent dimerization. PRLR dimerization and subsequent signaling are enhanced by treating the cells with deacetylase sirtuin (SIRT) inhibitor nicotinamide or histone deacetylase (HDAC) inhibitor trichostatin A but inhibited by expressing exogenous deacetylase SIRT2 or HDAC6. Our results suggest that acetylation and deacetylation provide the rheostat-like regulation for the cytokine receptor PRLR in its cytoplasmic loop dimerization and subsequent STAT5 activation.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
PRLR cytoplasmic loop is acetylated by CBP. (A) T47D cells received no treatment, PRL treatment for 30 min, or NAM (5 mM) pretreatment for 5.5 h, followed by PRL treatment for 30 min. Immunoprecipitated (IP) PRLR was analyzed for acetylated-PRLR (aK-PRLR) with polyclonal antibody to acetyl-lysine. (B) In T47D cells, CBP-specific siRNA or control (Ctl) siRNA was transiently transfected, followed by PRL treatment for 30 min. PRLR acetylation was analyzed as above. (C) PRLR was cotransfected with CBP in 293T cells, followed by no treatment or treatment with NAM or TSA (1 μM). To test PRLR deacetylation, PRLR and CBP were transfected along with Flag-tagged SIRT1, SIRT2, HDAC6, or HDAC3 in 293T cells, followed by acetylation blotting analysis. (D) Myc-PRLR cytoplasmic loop truncation variants (from K259 to a different C-terminal end as illustrated in Fig. S1D) were transfected along with CBP in 293T cells, followed by monoclonal anti-acetyl-lysine blotting. (E) Endogenous PRLR was immunoprecipitated from T47D cells treated with PRL for different times for acetylation analysis, with specific antibodies recognizing indicated acetylation sites of PRLR. (F) GST-CBP-HAT (amino acids 1,196–1,718)–catalyzed PRLR-CD acetylation in vitro was detected with anti-acetyl-K277-PRLR.
Fig. 2.
Fig. 2.
CBP nuclear exportation on PRL stimulation. (A) In T47D cells treated with PRL for different times (as indicated), CBP was recovered from PRLR immunoprecipitate (IP) for Western blot analysis. (B) Cytosolic (Cyt) and nuclear (Nuc) fractions were prepared from T47D cells treated with PRL for different times, as indicated, and analyzed for the localization of CBP, histone H1, and IκBα. Dynamic CBP subcellular localization was also analyzed with the cytosolic and nuclear fractions prepared from MCF7 cells receiving PRL treatment. (C) CBP was immunostained with monoclonal anti-CBP in T47D cells treated with or without PRL for 60 min. (D) YFP-tagged CBP of WT, NLS1 deletion (NLS1Δ), NSL2 deletion (NLS2Δ), or nuclear export signal deletion (NESΔ) form was transiently expressed in 293T cells and visualized with a confocal microscope. (E) Empty vector (EV) and Myc-tagged PRLR cytoplasmic loop truncation mutants were cotransfected with HA-CBP and coimmunoprecipitated with HA polyclonal antibody. (F) Mass spectrum of the peptide-bearing phospho-T539 of PRLR. (G) Immunoprecipitated PRLR from T47D cells treated with PRL for different times, as indicated, was analyzed by Western blotting with anti-pT539-PRLR. (H) Purified GST-CBP protein was incubated with whole-cell extracts prepared from 293T cells transfected with empty vector (EV) or PRLR-HA (WT, T539A, and T539D). PRLR proteins were recovered from GST-CBP precipitates in Western blots. (I) Indicated GST-CBP domain proteins were purified from bacteria and incubated with Myc-tagged PRLR cytoplasmic domain prepared from 293T transfectants. The CBP-precipitated PRLR cytoplasmic domain was detected by blotting with anti-Myc.
Fig. 3.
Fig. 3.
Acetylation modulates PRLR cytoplasmic loop dimerization. (A) HA-PRLR-Full-Length (FL) and Myc-PRLR-Cytoplasmic Domain (C) were cotransfected in 293T cells, followed by no treatment or treatment with PRL, TSA, NAM, PRL plus NAM, or PRL plus TSA for 30 min. Anti-Myc immunoprecipitates (IP) were analyzed with anti-HA, anti-Myc, or anti-aK277-PRLR. (B) In 293T cells, HA-PRLR-FL and Myc-PRLR-C were cotransfected with empty vector, Flag-SIRT2, or Flag-HDAC6, followed by no treatment or PRL treatment for 30 min. The Myc immunoprecipitates were analyzed with anti-HA in Western blots (C) CBP depletion with siRNA on PRLR-FL and PRLR-C dimerization was analyzed with coimmunoprecipitation. (D) In 293T cells, HA-PRLR-FL was cotransfected with Myc-PRLR-C truncates, followed by NAM and PRL cotreatment. Anti-Myc immunoprecipitates were analyzed with anti-HA, anti-aK277-PRLR, or anti-aK505-PRLR. (E) FRET images of the indicated pairs of CFP-PRLR and YFP-PRLR expressed in CHO cells, followed by PRL treatment or no treatment. Images of CFP and YFP fluorescence for individual CHO cells before and after YFP bleaching are shown in the upper two rows. The disappearance of YFP fluorescence after bleaching and the increase in CFP fluorescence for WT and K1–15A mutant but not for K1–15R mutant after PRL treatment were observed. (Left) Merging of YFP before and after bleach and CFP before and after bleach are shown in the third row. (Right) Ratios of CFPpost/CFPpre are shown in the third row. (F) FRET efficiency (y axis) from the indicated CFP/YFP-tagged constructs in CHO cells treated with or without PRL. For pre- and postphotobleaching image sets of CFP, the cell of interest was selected and the background values were subtracted from the donor pre- and postbleaching. The spectrally corrected FRET efficiency (E) was calculated using the equation E = 1 − (FCFP(d)Pre/FCFP(d)Post) (23), where FCFP(d)Pre and FCFP(d)Post are the mean CFP emission intensity of pre- and postphotobleaching. Data represent the mean ± SEM for three to six cells. When compared with WT, (−), or PRL, *P < 0.05 for K1–3R, K4–6R, K1–6R, K7–12R, K1–12R, and K13–15R; **P < 0.01 for K1–15R; NS (not significant) for K1–15A. (G) HA-tagged and Myc-tagged PRLR-FL of WT, K1–15R, or K1–15A was cotransfected in pairs in 293T cells, followed by PRL treatment for 30 min. Anti-Myc immunoprecipitates were analyzed with anti-HA for PRLR dimerization.
Fig. 4.
Fig. 4.
STAT5 dimerization requires acetylation. (A) Mass spectra of acetylated peptides recovered from STAT5b prepared from 293T cells transfected with STAT5 and CBP. (B) STAT5b WT and STAT5b with a K→R mutation were transiently transfected along with CBP in 293T cells. Immunoprecipitated (IP) STAT5b proteins were analyzed with pan–anti-acetyl-lysine. (C) STAT5 acetylation in T47D cells received PRL treatment for different times and was analyzed with specific polyclonal antibodies against ak359-STAT5b, aK694-STAT5b, and aK701-STAT5b, respectively. (D) In T47D, CBP down-regulation with siRNA on STAT5 acetylation (pan–anti-acetyl-lysine) in response to PRL treatment for 30 min. (E) In PC3 cells, Myc-tagged and HA-tagged STAT5b (WT or Lysine-to-Arginine (KR) mutant) was cotransfected, followed by treatment with or without PRL for 30 min. Anti-Myc immunoprecipitates were blotted with anti-HA. (F) In PC3 cells, empty vector (EV), STAT5b WT, or STAT5b KR mutants were transfected along with pLHRE-luciferase reporter, followed by treatment with PRL for 6–12 h before luciferase activity assay. (G) Myc-STAT5b was transfected along with EV, PRLR WT, or PRLR K1–15R in 293T cells, followed by PRL treatment for 30 min. Anti-Myc immunoprecipitates were analyzed with indicated antibodies. (H) 293T cells were transiently transfected with PRLR and JAK2, followed by PRL treatment for 60 min. Immunoprecipitated JAK2 was analyzed for PRLR association or phospho-JAK2. (I) (Left) In 293T cells, PRLR WT and indicated PRLR mutants were transfected along with pLHRE-luciferase reporter, followed by PRL treatment for 6–12 h before luciferase activity assay. In 293T cells, EV, CBP, or SIRT2 was cotransfected with PRLR and luciferase reporter or siRNA of control (CTL), CBP, or SIRT was cotransfected with PRLR and luciferase reporter. (Right) Luciferase activity assay was performed under the same conditions as above.
Fig. 5.
Fig. 5.
Model of acetylation-dependent PRLR-STAT5 route activation. The cytoplasmic CBP (basal and CBP exported from nuclei on PRL treatment) is responsible for PRLR cytoplasmic loop acetylation on multiple sites. Acetylated PRLR undergoes dimerization between two monomers within their cytoplasmic loops, followed by STAT5 activation. CBP is also responsible for STAT5 acetylation-dependent dimerization and transcriptional activation. SIRT (SIRT2) can reverse this CBP-dependent PRLR-STAT5b route activation. cCBP, CBP in cytoplasm; nCBP, CBP in nucleus.

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