Ubiquitin-mediated regulation of RIPK1 kinase activity independent of IKK and MK2

Tumor necrosis factor (TNF) can drive inflammation, cell survival, and death. While ubiquitylation-, phosphorylation-, and nuclear factor kappa B (NF-kappa B)-dependent checkpoints suppress the cytotoxic potential of TNF, it remains unclear whether ubiquitylation can directly repress TNF-induced death. Here, we show that ubiquitylation regulates RIPK1's cytotoxic potential not only via activation of downstream kinases and NF-kB transcriptional responses, but also by directly repressing RIPK1 kinase activity via ubiquitin-dependent inactivation. We find that the ubiquitin-associated (UBA) domain of cellular inhibitor of apoptosis (cIAP) 1 is required for optimal ubiquitin-lysine occupancy and K48 ubiquitylation of RIPK1. Independently of IKK and MK2, cIAP1-mediated and UBA-assisted ubiquitylation suppresses RIPK1 kinase auto-activation and, in addition, marks it for proteasomal degradation. In the absence of a functional UBA domain of cIAP1, more active RIPK1 kinase accumulates in response to TNF, causing RIPK1 kinase-mediated cell death and systemic inflammatory response syndrome. These results reveal a direct role for cIAP-mediated ubiquitylation in controlling RIPK1 kinase activity and preventing TNF-mediated cytotoxicity

Charakterisierung der Hefe-Proteine Neo1p und Sjl2p, zwei hochkonservierte Regulatoren der Phospholipid-Zusammensetzung in endosomalen Membranen

Endocytosis is a key membrane trafficking pathway by which all eukaryotic cells internalize extracellular material as well as portions of the plasma membrane. In the budding yeast Saccharomyces cerevisiae, the major fraction of the material internalized at the plasma membrane is transported through the early and late endosomal compartments to the vacuole. The endosomal compartments are highly dynamic and are connected to the plasma membrane, the late Golgi complex and the vacuole by several routes, which all involve vesicular transport. Thus, the formation of vesicles is essential to maintain the dynamic exchanges between these different compartments. In the present study, I analyzed two yeast proteins, Neo1p and Sjl2p, which are suggested to function as regulators of the phospholipid composition of endosomal organelles and thereby seem to participate in vesicle formation (Neo1p) and in vesicle uncoating (Sjl2p), two processes essential during vesicular membrane transport. Neo1p is an essential member of the Drs2 family of P-type ATPases with proposed function as aminophospholipid (APL) translocases. Genetic and biochemical data in the laboratory of B. Singer-Krüger indicated that Neo1p might function within the endosomal system. Consistent with these findings, I could confirm by indirect immunofluorescence that the major fraction of Neo1p localizes to the endosomal compartments, while a smaller part associates with late Golgi membranes. In agreement with this localization, two temperature-sensitive neo1 mutants were shown to be defective in endocytosis (B. Singer-Krüger's laboratory) and in vacuolar protein sorting (my studies). While these defects were already observed at permissive temperature, at nonpermissive temperature the neo1-ts mutants exhibited additional impairments in membrane transport between the ER and the early Golgi compartment. However, these defects were most likely a consequence of the accumulation of mutant Neo1 proteins in the ER under these conditions. In support of previous results in the laboratory of B. Singer-Krüger, I identified further links between Neo1p and the endosomal proteins Ysl2p and Arl1p. I could show that Neo1p interacts in vivo with Ysl2p and that the subcellular localization and the stability of Ysl2p are affected in the neo1-69 mutant. Furthermore, the subcellular distribution of Arl1p was also found to be impaired in neo1-69 cells at permissive temperature. Based on these findings and the work of B. Singer-Krüger, Neo1p was proposed to act together with Ysl2p and Arl1p in membrane trafficking within the endosomal/late Golgi system. In the second part of this work, the subcellular localization of Sjl2p, a polyphosphoinositide- and 5'-inositide phosphatase of the synaptojanin family, was examined. Based on genetic analyses, Sjl2p has been suggested to participate in early steps of endocytic transport. Here, I determined by indirect immunofluorescence that the Sjl2p-positive punctate structures were distinct from those containing typical early and late endosomal marker proteins and were not sensitive to mutations affecting the structure of either the endosomal compartments or the Golgi complex. Sjl2p did not show a typical plasma membrane staining pattern either. However, Sjl2p colocalized with cortical actin patch components found within clumps that accumulated in cells lacking the two actin-regulating kinases Prk1p and Ark1p. Within these aberrant structures, Sjl2p also colocalized with FM4-64 endocytosed for a short time, suggesting that Sjl2p localized to primary endocytic vesicles that interact with the cortical actin cytoskeleton. This interaction may at least to some extent be mediated by Bsp1p, a binding partner of Sjl2p isolated in B. Singer-Krüger's laboratory, which in my studies was found to be part of the cortical actin cytoskeleton. In cells deficient for the PtdIns(4)P kinase (pik1-ts), the staining pattern of Bsp1p was changed, suggesting that the subcellular distribution of Bsp1p is dependent on the levels of phosphoinositides. Consistent with that, Bsp1p was found to flotate with liposomes containing acidic phospholipids including phosphoinositides. Thus, Bsp1p may act as an adapter that directly connects Sjl2p-containing vesicles to the cortical actin cytoskeleton during early stages of endocytosis via interactions with Sjl2p (B. Singer-Krüger) and with a subset of phospholipids within the vesicle membrane (B. Singer-Krüger and my results). In summary, the results of my PhD thesis brought new insights into the localization and function of Neo1p and Sjl2p within the endosomal system. These data are relevant for future studies to elucidate the precise mechanism of Neo1p and Sjl2p during endocytosis.Endozytose ist ein Internalisierungsweg für extrazelluläres Material sowie für Proteine der Plasmamembran, den alle eukaryotischen Zellen verwenden. Während der Endozytose wird ein Großteil des internalisierten Materials über frühe und späte Endosomen zur Vakuole transportiert. Die endosomale Kompartimente sind hochdynamisch und verbunden mit der Plasmamembran, dem späten Golgi-Komplex und der Vakuole über mehrere Transportwege, welche alle durch Membranvesikel vermittelt werden. Somit sind Vesikelbildungsprozesse essentiell, um den dynamischen Austausch zwischen den Kompartimenten zu gewährleisten. In der vorliegenden Arbeit habe ich zwei Hefe-Proteine analysiert, Neo1p und Sjl2p. Es sollte deren potentielle Funktion als Regulatoren der Phospholipid-Zusammensetzung in endosomalen Membranen untersucht werden, wo sie dadurch wahrscheinlich an der Vesikelbildung (Neo1p) und der Dissoziation von Vesikel-Umhüllungen (Sjl2p) beteiligt sind. Diese beiden Prozesse sind essentiell für den vesikulären Membrantransport. Neo1p ist ein essentielles Mitglied der Drs2-Familie von P-Typ ATPasen, dessen Funktion wahrscheinlich die einer Aminophospholipid-Translokase ist. Genetische und biochemische Daten aus dem Labor von B. Singer-Krüger deuteten darauf hin, dass Neo1p im endosomalen System funktioniert. Diesen Befund konnte ich durch indirekte Immunfluoreszenz bestätigen, da ein Großteil von Neo1p an endosomalen Kompartimenten lokalisiert, während ein kleinerer Teil mit späten Golgi-Membranen assoziiert. In Übereinstimmung mit dieser Lokalisierung zeigten zwei temperaturempfindliche neo1-Mutanten Defekte in der Endozytose (Labor von B. Singer-Krüger) und bei der Sortierung von vakuolären Proteinen (eigene Daten). Während diese Defekte schon bei permissiver Temperatur beobachtet werden konnten, zeigten die neo1-ts-Mutanten bei nicht-permissiven Temperaturen noch zusätzliche Schädigungen im Membrantransport zwischen ER und dem frühen Golgi-Kompartiment. Jedoch waren diese Defekte unter diesen Bedingungen höchstwahrscheinlich eine Konsequenz aus der Akkumulierung von mutierten Neo1-Proteinen im ER. Aufgrund früherer Ergebnisse aus dem Labor von B. Singer-Krüger sollte es weiterhin Verbindungen zwischen Neo1p und den endosomalen Proteinen Ysl2p und Arl1p geben. Ich konnte zeigen, dass Neo1p mit Ysl2p in vivo interagiert, und dass sowohl die subzelluläre Lokalisierung, als auch die Stabilität von Ysl2p in der neo1-69-Mutante beeinflusst sind. In dieser Mutante war, bei permissiver Temperatur, auch die subzelluläre Verteilung von Arl1p verändert. Aufgrund dieser Ergebnisse und den Daten von B. Singer-Krüger wurde Neo1p mit Ysl2p und Arl1p eine Funktion bei Membranbewegungen im endosomalen/späten Golgi-System zugeordnet. Im zweiten Teil der vorliegenden Arbeit wurde die Lokalisierung von Sjl2p untersucht, eine Inositolphospholipid-Phosphatase der Synaptojanin-Familie. Aufgrund genetischer Analysen wurde für Sjl2p eine Funktion in frühen Schritten des endozytotischen Transports vermutet. Ich konnte durch indirekte Immunfluoreszenz zeigen, dass die Sjl2-positiven, punktartigen Strukturen keine Kolokalisation mit typischen frühen und späten endosomalen Marker-Proteinen aufwiesen, und nicht empfindlich auf Mutationen reagieren, welche die Strukturen von endosomalen Kompartimenten oder die des Golgi-Komplexes beeinflussen. Sjl2p zeigte auch kein typisches Färbungsmuster für Plasmamembranen. Jedoch kolokalisierte Sjl2p mit kortikalen Aktinflecken, welche in Zellen zu Klumpen akkumulieren, in denen die zwei Aktin-regulierenden Kinasen Prk1p und Ark1p deletiert waren. In diesen anormalen Strukturen kolokalisierte Sjl2p während einer kurzen Phase auch mit FM4-64, was ein Hinweis dafür sein könnte, dass Sjl2p an primären endozytotischen Vesikeln lokalisiert, die mit dem Aktin-Zytoskelett interagieren. Diese Interaktion könnte teilweise über Bsp1p vermittelt sein, ein Bindungspartner von Sjl2p, welcher im Labor von B. Singer-Krüger identifiziert wurde. Dieser konnte in meiner Arbeit als Komponente des kortikalen Aktin-Zytoskeletts aufgezeigt werden. In PtdIns(4)P-Kinase (pik1-ts)-defizienten Zellen veränderte sich das Färbungsmuster von Bsp1p, was auf eine subzelluläre Verteilung hinwies, die abhängig von Inositolphospholipiden ist. Des weiteren flotierte Bsp1 mit Liposomen, welche saure Phospholipide, wie Inositolphospholipide, enthielten. Das lässt schließen, dass Bsp1p während der frühen Stadien der Endozytose als Adapter für Sjl2-enthaltende Vesikel fungieren könnte, da er diese über die Interaktion mit Sjl2p (B. Singer-Krüger) und mit einer Untergruppe von Phospholipiden innerhalb der Vesikelmembran (B. Singer-Krüger und eigene Ergebnisse), direkt mit dem kortikalen Aktin-Zytokelett verbinden könnte. Zusammenfassend, die Ergebnisse meiner Doktorarbeit brachten neue Einblicke in die Lokalisierung und Funktion von Neo1p und Sjl2p innerhalb des endosomalen Systems

CARD-MEDIATED AUTOINHIBITION OF CIAP1'S E3 LIGASE ACTIVITY SUPPRESSES CELL PROLIFERATION AND MIGRATION

E3 ligases mediate the covalent attachment of ubiquitin to target proteins thereby enabling ubiquitin-dependent signaling. Unraveling how E3 ligases are regulated is important because miscontrolled ubiquitylation can lead to disease. Cellular inhibitor of apoptosis (cIAP) proteins are E3 ligases that modulate diverse biological processes such as cell survival, proliferation, and migration. Here, we have solved the structure of the caspase recruitment domain (CARD) of cIAP1 and identified that it is required for cIAP1 autoregulation. We demonstrate that the CARD inhibits activation of cIAP1's E3 activity by preventing RING dimerization, E2 binding, and E2 activation. Moreover, we show that the CARD is required to suppress cell proliferation and migration. Further, CARD-mediated autoregulation is also necessary to maximally suppress caspase-8-dependent apoptosis and vascular tree degeneration in vivo. Taken together, our data reveal mechanisms by which the E3 ligase activity of cIAP1 is controlled, and how its deregulation impacts on cell proliferation, migration and cell survival.status: publishe

MK2 Phosphorylates RIPK1 to Prevent TNF-Induced Cell Death

TNF is an inflammatory cytokine that upon binding to its receptor, TNFR1, can drive cytokine production, cell survival, or cell death. TNFR1 stimulation causes activation of NF-kappa B, p38 alpha, and its downstream effector kinase MK2, thereby promoting transcription, mRNA stabilization, and translation of target genes. Here we show that TNF-induced activation of MK2 results in global RIPK1 phosphorylation. MK2 directly phosphorylates RIPK1 at residue S321, which inhibits its ability to bind FADD/caspase-8 and induce RIPK1-kinase-dependent apoptosis and necroptosis. Consistently, a phospho-mimetic S321D RIPK1 mutation limits TNF-induced death. Mechanistically, we find that phosphorylation of S321 inhibits RIPK1 kinase activation. We further show that cytosolic RIPK1 contributes to complex-II-mediated cell death, independent of its recruitment to complex-I, suggesting that complex-II originates from both RIPK1 in complex-I and cytosolic RIPK1. Thus, MK2-mediated phosphorylation of RIPK1 serves as a checkpoint within the TNF signaling pathway that integrates cell survival and cytokine production

Ubiquitylation of MLKL at lysine 219 positively regulates necroptosis-induced tissue injury and pathogen clearance

Necroptosis is a form of cell death characterized by membrane rupture via MLKL oligomerization, although mechanistic details remain unclear. Here, the authors show that MLKL ubiquitylation of K219 facilitates high-order membrane assembly and subsequent rupture, promoting cytotoxicity. Necroptosis is a lytic, inflammatory form of cell death that not only contributes to pathogen clearance but can also lead to disease pathogenesis. Necroptosis is triggered by RIPK3-mediated phosphorylation of MLKL, which is thought to initiate MLKL oligomerisation, membrane translocation and membrane rupture, although the precise mechanism is incompletely understood. Here, we show that K63-linked ubiquitin chains are attached to MLKL during necroptosis and that ubiquitylation of MLKL at K219 significantly contributes to the cytotoxic potential of phosphorylated MLKL. The K219R MLKL mutation protects animals from necroptosis-induced skin damage and renders cells resistant to pathogen-induced necroptosis. Mechanistically, we show that ubiquitylation of MLKL at K219 is required for higher-order assembly of MLKL at membranes, facilitating its rupture and necroptosis. We demonstrate that K219 ubiquitylation licenses MLKL activity to induce lytic cell death, suggesting that necroptotic clearance of pathogens as well as MLKL-dependent pathologies are influenced by the ubiquitin-signalling system

The Zds proteins control entry into mitosis and target protein phosphatase 2A to the Cdc25 phosphatase

Protein phosphatase 2A (PP2A) is a key regulator of mitosis, but the roles that it plays are poorly understood. New evidence in budding yeast shows that the Zds proteins form a tight stoichiometric complex with PP2A and target its activity to the Cdc25 phosphatase, which is a key regulator of entry into mitosis