9 research outputs found
PIP30/FAM192A is a novel regulator of the nuclear proteasome activator PA28γ
PA28γ is a nuclear activator of the 20S proteasome involved in the regulation of several essential cellular processes, such as cell proliferation, apoptosis, nuclear dynamics, and cellular stress response. Unlike the 19S regulator of the proteasome, which specifically recognizes ubiquitylated proteins, PA28γ promotes the degradation of several substrates by the proteasome in an ATP- and ubiquitin-independent manner. However, its exact mechanisms of action are unclear and likely involve additional partners that remain to be identified. Here we report the identification of a cofactor of PA28γ, PIP30/FAM192A. PIP30 binds directly and specifically via its C-terminal end and in an interaction stabilized by casein kinase 2 phosphorylation to both free and 20S proteasome-associated PA28γ. Its recruitment to proteasome-containing complexes depends on PA28γ and its expression increases the association of PA28γ with the 20S proteasome in cells. Further dissection of its possible roles shows that PIP30 alters PA28γ-dependent activation of peptide degradation by the 20S proteasome in vitro and negatively controls in cells the presence of PA28γ in Cajal bodies by inhibition of its association with the key Cajal body component coilin. Taken together, our data show that PIP30 deeply affects PA28γ interactions with cellular proteins, including the 20S proteasome, demonstrating that it is an important regulator of PA28γ in cells and thus a new player in the control of the multiple functions of the proteasome within the nucleus
The stability of Fbw7α in M-phase requires its phosphorylation by PKC
<div><p>Fbw7 is a tumor suppressor often deleted or mutated in human cancers. It serves as the substrate-recruiting subunit of a SCF ubiquitin ligase that targets numerous critical proteins for degradation, including oncoproteins and master transcription factors. Cyclin E was the first identified substrate of the SCF<sup>Fbw7</sup> ubiquitin ligase. In human cancers bearing <i>FBXW7</i>-gene mutations, deregulation of cyclin E turnover leads to its aberrant expression in mitosis. We investigated Fbw7 regulation in <i>Xenopus</i> eggs, which, although arrested in a mitotic-like phase, naturally express high levels of cyclin E. Here, we report that Fbw7α, the only Fbw7 isoform detected in eggs, is phosphorylated by PKC (protein kinase C) at a key residue (S18) in a manner coincident with Fbw7α inactivation. We show that this PKC-dependent phosphorylation and inactivation of Fbw7α also occurs in mitosis during human somatic cell cycles, and importantly is critical for Fbw7α stabilization itself upon nuclear envelope breakdown. Finally, we provide evidence that S18 phosphorylation, which lies within the intrinsically disordered N-terminal region specific to the α-isoform reduces the capacity of Fbw7α to dimerize and to bind cyclin E. Together, these findings implicate PKC in an evolutionarily-conserved pathway that aims to protect Fbw7α from degradation by keeping it transiently in a resting, inactive state.</p></div
xFbw7α protein is expressed and xCyclin E can be degraded in an Fbw7α-dependent manner in MII-arrested eggs.
<p>A. Immunoblot analysis with anti-Fbw7 antibodies of protein extracts from MII-arrested oocytes. ivt: xFbw7α, β and γ isoforms translated <i>in vitro</i>. Asterisks denote non-specific immunoreactive bands. B. MII-arrested egg extracts immunoprecipitated with control IgG (C), or anti-Fbw7 antibodies. C. Stage VI oocytes were microinjected with mRNAs coding for hFbw7α or xFbw7α, or with H<sub>2</sub>O as a control. 12 hr later, they were induced to mature by addition of progesterone. The oocytes were collected at GVBD, 3 hr after GVBD (+3h) and during metaphase II arrest (MII). The equivalent of two oocytes was loaded in each lane. Oocyte extracts were analyzed with antibodies directed against Fbw7, xcyclin E, xCdc6 or tubulin. Cyclin E levels were quantified using ImageJ software and were normalized to tubulin. D. Immunoblot analysis of progesterone-treated oocytes microinjected with hFbw7α or hFbw7α-R465A mRNAs and collected during meiotic maturation.</p
The study of the determinants controlling Arpp19 phosphatase-inhibitory activity reveals an Arpp19/PP2A-B55 feedback loop
International audienceAbstract Arpp19 is a potent PP2A-B55 inhibitor that regulates this phosphatase to ensure the stable phosphorylation of mitotic/meiotic substrates. At G2-M, Arpp19 is phosphorylated by the Greatwall kinase on S67. This phosphorylated Arpp19 form displays a high affinity to PP2A-B55 and a slow dephosphorylation rate, acting as a competitor of PP2A-B55 substrates. The molecular determinants conferring slow dephosphorylation kinetics to S67 are unknown. PKA also phosphorylates Arpp19. This phosphorylation performed on S109 is essential to maintain prophase I-arrest in Xenopus oocytes although the underlying signalling mechanism is elusive. Here, we characterize the molecular determinants conferring high affinity and slow dephosphorylation to S67 and controlling PP2A-B55 inhibitory activity of Arpp19. Moreover, we show that phospho-S109 restricts S67 phosphorylation by increasing its catalysis by PP2A-B55. Finally, we discover a double feed-back loop between these two phospho-sites essential to coordinate the temporal pattern of Arpp19-dependent PP2A-B55 inhibition and Cyclin B/Cdk1 activation during cell division
Endogenous and ectopic Fbw7α are phosphorylated at Ser18 in M-phase.
<p>A. FLAG-Fbw7α was transfected or not in HeLa cells, as indicated. Total extracts of asynchronous cells (AS), cells synchronized in G1/S or arrested in pro-metaphase and recovered by shake-off (M) were analyzed by SDS-PAGE and immunoprobed for pS18-Fbw7α and FLAG-Fbw7α. The migration of Cdc27 was used as a marker of M phase (Cdc27<sup>P</sup>), and anti-tubulin as a loading control. B. HeLa cell extracts (500 μg) from cells arrested in prometaphase were subjected to immunoprecipitation with anti-Fbw7 or control antibodies and analyzed by immunoblotting to detect phosphorylated Ser18 (pS18). Input 10% (i), supernatant after IP (s). C. HeLa cells grown on coverslips were co-transfected with FLAG-Fbw7α and either GFP- PKCα, PKCβ1, PKCε or PKCδ, as indicated. Coverslips were fixed and stained with FLAG antibodies and DAPI. Scale bar, 10 μm. D. HeLa cells were co-transfected with FLAG-Fbw7α and the different GFP-PKC constructs, as indicated. At 24 hr post-tranfection, cells were harvested and the levels of GFP-PKC, pS18-Fbw7α, FLAG-Fbw7α and tubulin were monitored by immunoblotting.</p
xFbw7α is phosphorylated by PKC on S18.
<p>A. <i>In vitro</i> translated [<sup>35</sup>S]-xFbw7α was incubated in MII-egg extracts prepared with phosphatase inhibitors (left panel). [<sup>35</sup>S]-xFbw7α-wt was incubated 1 hr in MII-egg extracts prepared with phosphatase inhibitors and an equal amount of sample was subsequently incubated with an excess of lambda protein phosphatase (λP) and analyzed by phosphorimaging (right panel). (B) <i>In vitro</i> translated [<sup>35</sup>S]-xFbw7α-wt, -S18A, -S119A or -129A were incubated 1 hr in MII-egg extracts. C. Sequence alignment of the Fbw7α N-terminal region from <i>Xenopus</i> and human. D. GST-xFbw7α-wt or -S18A bound to magnetic beads were incubated with PKCα, activated (+) or not (-) and then submitted to immunoblot analysis with anti-pS18 antibodies. Total GST-Fbw7 was detected by Ponceau S staining. (E) GST-xFbw7α wt or S18A were incubated in MII-egg extracts plus phosphatase inhibitors, supplemented (+) or not (-) with Gö6976 and with [γ-<sup>33</sup>P]-ATP for 1 hr at 23°C and analyzed by immunoblotting and by phosphorimaging for quantification of <sup>33</sup>P incorporation (lower panel).</p
Fbw7 dimerization and cyclin E binding is reduced by a negative charge at Ser18.
<p>A. FLAG-and HA-tagged versions of Fbw7α either S18A or S18E were co-transfected in HeLa cells and analyzed for their interaction by immunoprecipitation with HA antibodies. Co-precipitated FLAG-tagged Fbw7α was detected by immunoblotting with an anti-FLAG antibody. The membrane was re-probed with HA antibodies. B. Fbw7 or control immunoprecipitates from HeLa cells transfected or not (-) with FLAG-Fbw7α-S18A or –S18E were mixed with <i>in vitro</i> translated [<sup>35</sup>S]-Fbw7α-18A or -18E (with no tag in N-terminal), as indicated. Complexes were analyzed by phosphorimaging and immunoblotting. C. <i>In vitro</i> translated [<sup>35</sup>S]-xCyclin E was first incubated with a recombinant Cdc25B phosphatase to activate its associated Cdk2 partner from the reticulocyte lysate and further incubated in MII-egg extracts with phosphatase inhibitors for 30 min. Phosphorylated cyclin E was mixed with FLAG-hFbw7α-18A or-18E immunoprecipitates. Complexes were analyzed by phosphorimaging and immunoblotting. D. <i>In vitro</i> translated [<sup>35</sup>S]-FLAG-hFbw7α-465A, -18A or -wt were incubated in MII-egg extracts with phosphatase inhibitors for 60 min and mixed with GST-cyclin E bound to magnetic beads. The mutant hFbw7α-465A that cannot bind cyclin E serves as a negative control. Input represents 10% of the total extract (i), total beads (B). Complexes were analyzed by phosphorimaging and immunoblotting.</p