Didier Fesquet

Pin1 stabilizes Emi1 during G2 phase by preventing its association with SCFβtrcp

The anaphase‐promoting complex (APC) early mitotic inhibitor 1 (Emi1) is required to induce S‐ and M‐phase entries by stimulating the accumulation of cyclin A and cyclin B through APCCdh1/cdc20 inhibition. In this report, we show that Emi1 proteolysis can be induced by cyclin A/cdk (cdk for cyclin‐dependent kinase). Paradoxically, Emi1 is stable during G2 phase, when cyclin A/cdk, Plx1 and SCFβtrcp (SCF for Skp1‐Cul1‐Fbox protein)—which play a role in its degradation—are active. Here, we identify Pin1 as a new regulator of Emi1 that induces Emi1 stabilization by preventing its association with SCFβtrcp. We show that Pin1 binds to Emi1 and prevents its association with βtrcp in an isomerization‐dependent pathway. We also show that Emi1–Pin1 binding is present in vivo in XL2 cells during G2 phase and that this association protects Emi1 from being degraded during this phase of the cell cycle. We propose that S‐ and M‐phase entries are mediated by the accumulation of cyclin A and cyclin B through a Pin1‐dependent stabilization of Emi1 during G2.

Characterisation of PGs1, a subunit of a protein complex co-purifying with tubulin polyglutamylase.

Polyglutamylation is a post-translational modification initially discovered on tubulin. It has been implicated in multiple microtubule functions, including neuronal differentiation, axonemal beating and stability of the centrioles, and shown to modulate the interaction between tubulin and microtubule associated proteins. The enzymes catalysing this modification are not yet known. Starting with a partially purified fraction of mouse brain tubulin polyglutamylase, monoclonal antibodies were raised and used to further purify the enzyme by immunoprecipitation. The purified enzyme complex (Mr 360×103) displayed at least three major polypeptides of 32, 50 and 80×103, present in stochiometric amounts. We show that the 32×103 subunit is encoded by the mouse gene GTRGEO22, the mutation of which has recently been implicated in multiple defects in mice, including male sterility. We demonstrate that this subunit, called PGs1, has no catalytic activity on its own, but is implicated in the localisation of the enzyme at major sites of polyglutamylation, i.e. neurones, axonemes and centrioles.

Is Cdk7/cyclin H/MAT1 the genuine cdk activating kinase in cycling Xenopus egg extracts?

Formation of active cdk (cyclin dependent kinase)/cyclin kinases involves phosphorylation of a conserved threonine residue in the T loop of the cdk catalytic subunit by CAK (Cdk Activating Kinase). CAK was first purified biochemically from higher eukaryotes and identified as a trimeric complex containing a cdk7 catalytic subunit, cyclin H and MAT1 (Ménage à trois), a member of the RING finger family. The same trimeric complex is also part of basal transcription factor TFIIH. In budding yeast, the closest homologs of cdk7 and cyclin H, KIN28 and CCL1, respectively, also associate with TFIIH. However, the KIN28/CCL1 complex does not display CAK activity and a distinct protein kinase able to phosphorylate monomeric CDC28 and GST-cdk2 was recently identified, challenging the identification of cdk7 as the physiological CAK in higher eukaryotes. Here we demonstrate that immunodepletion of cdk7 suppresses CAK activity from cycling Xenopus egg extracts, and arrest them before M-phase. We also show that specific translation of mRNAs encoding Xenopus cdk7 and its associated subunits restores CAK activity in cdk7-immunodepleted Xenopus egg extracts. Hence, the cdk7 complex is necessary and sufficient for activation of cdk-cyclin complexes in cycling Xenopus egg extracts.

Human Mob1 proteins are required for cytokinesis by controlling microtubule stability

Summary The completion of cytokinesis requires abscission of the midbody, a microtubule-rich cytoplasmic bridge that connects the daughter cells before their final separation. Although it has been established that both the midbody structure and membrane fusion are essential for abscission, the biochemical machinery and the cellular processes of abscission remain ill-defined. Here we report that human Mob1A and Mob1B proteins are involved in the regulation of abscission of the intercellular bridge. The Mob family is a group of highly conserved proteins in eukaryotes, described as binding partners as well as co-activators of protein kinases of the Ndr family, and as members of the Hippo pathway. We show that depletion of Mob1A and Mob1B by RNAi causes abscission failure as a consequence of hyper-stabilization of microtubules in the midbody region. Interestingly, depleting Mob1 also increases cell motility after cytokinesis, and induces prolonged centriole separation in G1 phase. In contrast, centrosomes fail to split when either Mob1A or Mob1B is overexpressed. Our findings indicate that human Mob1 proteins are involved in the regulation of microtubule stability at the midbody. We conclude that Mob1A and Mob1B are needed for cell abscission and centriole re-joining after telophase and cytokinesis.

Binding of Kif23-iso1/CHO1 to 14-3-3 Is Regulated by Sequential Phosphorylations at Two LATS Kinase Consensus Sites

Kif23 kinesin is an essential actor of cytokinesis in animals. It exists as two major isoforms, known as MKLP1 and CHO1, the longest of which, CHO1, contains two HXRXXS/T NDR/LATS kinase consensus sites. We demonstrate that these two sites are readily phosphorylated by NDR and LATS kinases in vitro, and this requires the presence of an upstream -5 histidine residue. We further show that these sites are phosphorylated in vivo and provide evidence revealing that LATS1,2 participate in the phosphorylation of the most C-terminal S814 site, present on both isoforms. This S814 phosphosite was previously reported to constitute a 14-3-3 binding site, which plays a role in Kif23 clustering during cytokinesis. Surprisingly, we found that phosphorylation of the upstream S716 NDR/LATS consensus site, present only in the longest Kif23 isoform, is required for efficient phosphorylation at S814, thus revealing sequential phosphorylation at these two sites, and differential regulation of Kif23-14-3-3 interaction for the two Kif23 isoforms. Finally, we provide evidence that Kif23 is largely unphosphorylated on S814 in post-abscission midbodies, making this Kif23 post-translational modification a potential marker to probe these structures.

PIP30/FAM192A is a novel regulator of the nuclear proteasome activator PA28γ

Significance The 20S proteasome is a key actor of the control of protein levels and integrity in cells. To perform its multiple functions, it works with a series of regulators, among which is a nuclear complex called PA28γ. In particular, PA28γ participates in the regulation of cell proliferation and nuclear dynamics. We describe here the characterization of a protein, PIP30/FAM192A, which binds tightly to PA28γ and favors its interaction with the 20S proteasome while inhibiting its association with coilin, a central component of nuclear Cajal bodies. Thus, PIP30/FAM192A critically controls the interactome and, consequently, the functions of PA28γ, and appears to be a previously unidentified player in the fine regulation of intracellular proteostasis in the cell nucleus. 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.

FAM192A/PIP30 regulates Cajal body dynamics by antagonizing PA28γ/coilin interaction

PA28γ, a nuclear regulator of the 20S proteasome, is involved in the control of several essential cellular processes, such as cell proliferation and nuclear organization, including Cajal body dynamics. However, the mechanisms controlling PA28γ function in the regulation of nuclear architecture are not known. Here we identify through a SILAC-based proteomics approach a specific and prominent interaction partner of PA28γ, called FAM192A/PIP30. We show that the PA28γ/PIP30 complex is stabilized by Casein Kinase 2-dependent phosphorylation of the PIP30 C-terminal region. PIP30 depletion reduces the number of Cajal bodies in human cells similar to PA28γ overexpression. Importantly, PIP30 depletion also results in the accumulation of PA28γ in residual Cajal body structures, which correlates with an increased interaction between PA28γ and coilin. Altogether our data identify the first regulator of PA28γ, which plays a critical role in Cajal body dynamics by antagonizing the formation of PA28γ/coilin complexes.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 to involve additional partners that remain to be identified. Here we report the identification of the first 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. Altogether, our data show that PIP30 deeply affects PA28γ interactions with cellular proteins, including 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. Significance Statement The 20S proteasome is a key actor of the control of protein levels and integrity in cells. To perform its multiple functions, it works with a series of regulators, among which a nuclear complex called PA28γ. In particular, PA28γ participates in the regulation of cell proliferation and nuclear dynamics. We describe here the characterization of a novel protein, PIP30/FAM192A, which binds tightly to PA28γ and favors its interaction with the 20S proteasome while inhibiting its association with coilin, a central component of nuclear Cajal bodies. Thus PIP30/FAM192A critically controls the interactome and consequently the functions of PA28γ, and appears to be a new player in the fine regulation of intracellular proteostasis in the cell nucleus.