The incredible ULKs

Macroautophagy (commonly abbreviated as autophagy) is an evolutionary conserved lysosome-directed vesicular trafficking pathway in eukaryotic cells that mediates the lysosomal degradation of intracellular components. The cytoplasmic cargo is initially enclosed by a specific double membrane vesicle, termed the autophagosome. By this means, autophagy either helps to remove damaged organelles, long-lived proteins and protein aggregates, or serves as a recycling mechanism for molecular building blocks. Autophagy was once invented by unicellular organisms to compensate the fluctuating external supply of nutrients. In higher eukaryotes, it is strongly enhanced under various stress conditions, such as nutrient and growth factor deprivation or DNA damage. The serine/threonine kinase Atg1 was the first identified autophagy-related gene (ATG) product in yeast. The corresponding nematode homolog UNC-51, however, has additional neuronal functions. Vertebrate genomes finally encode five closely related kinases, of which UNC-51-like kinase 1 (Ulk1) and Ulk2 are both involved in the regulation of autophagy and further neuron-specific vesicular trafficking processes. This review will mainly focus on the vertebrate Ulk1/2-Atg13-FIP200 protein complex, its function in autophagy initiation, its evolutionary descent from the yeast Atg1-Atg13-Atg17 complex, as well as the additional non-autophagic functions of its components. Since the rapid nutrient- and stress-dependent cellular responses are mainly mediated by serine/threonine phosphorylation, it will summarize our current knowledge about the relevant upstream signaling pathways and the altering phosphorylation status within this complex during autophagy induction

The mycotoxin phomoxanthone A disturbs the form and function of the inner mitochondrial membrane.

Mitochondria are cellular organelles with crucial functions in the generation and distribution of ATP, the buffering of cytosolic Ca2+ and the initiation of apoptosis. Compounds that interfere with these functions are termed mitochondrial toxins, many of which are derived from microbes, such as antimycin A, oligomycin A, and ionomycin. Here, we identify the mycotoxin phomoxanthone A (PXA), derived from the endophytic fungus Phomopsis longicolla, as a mitochondrial toxin. We show that PXA elicits a strong release of Ca2+ from the mitochondria but not from the ER. In addition, PXA depolarises the mitochondria similarly to protonophoric uncouplers such as CCCP, yet unlike these, it does not increase but rather inhibits cellular respiration and electron transport chain activity. The respiration-dependent mitochondrial network structure rapidly collapses into fragments upon PXA treatment. Surprisingly, this fragmentation is independent from the canonical mitochondrial fission and fusion mediators DRP1 and OPA1, and exclusively affects the inner mitochondrial membrane, leading to cristae disruption, release of pro-apoptotic proteins, and apoptosis. Taken together, our results suggest that PXA is a mitochondrial toxin with a novel mode of action that might prove a useful tool for the study of mitochondrial ion homoeostasis and membrane dynamics

EBV Negative Lymphoma and Autoimmune Lymphoproliferative Syndrome Like Phenotype Extend the Clinical Spectrum of Primary Immunodeficiency Caused by STK4 Deficiency

Serine/threonine kinase 4 (STK4) deficiency is an autosomal recessive genetic condition that leads to primary immunodeficiency (PID) typically characterized by lymphopenia, recurrent infections and Epstein Barr Virus (EBV) induced lymphoproliferation and -lymphoma. State-of-the-art treatment regimens consist of prevention or treatment of infections, immunoglobulin substitution (IVIG) and restoration of the immune system by hematopoietic stem cell transplantation. Here, we report on two patients from two consanguineous families of Turkish (patient P1) and Moroccan (patient P2) decent, with PID due to homozygous STK4 mutations. P1 harbored a previously reported frameshift (c.1103 delT, p.M368RfsX2) and P2 a novel splice donor site mutation (P2; c.525+2 T>G). Both patients presented in childhood with recurrent infections, CD4 lymphopenia and dysregulated immunoglobulin levels. Patient P1 developed a highly malignant B cell lymphoma at the age of 10 years and a second, independent Hodgkin lymphoma 5 years later. To our knowledge she is the first STK4 deficient case reported who developed lymphoma in the absence of detectable EBV or other common viruses. Lymphoma development may be due to the lacking tumor suppressive function of STK4 or the perturbed immune surveillance due to the lack of CD4+ T cells. Our data should raise physicians' awareness of [1] lymphoma proneness of STK4 deficient patients even in the absence of EBV infection and [2] possibly underlying STK4 deficiency in pediatric patients with a history of recurrent infections, CD4 lymphopenia and lymphoma and unknown genetic make-up. Patient P2 experienced recurrent otitis in childhood, but when she presented at the age of 14, she showed clinical and immunological characteristics similar to patients suffering from Autoimmune Lymphoproliferative Syndrome (ALPS): elevated DNT cell number, non-malignant lymphadenopathy and hepatosplenomegaly, hematolytic anemia, hypergammaglobulinemia. Also patient P1 presented with ALPS-like features (lymphadenopathy, elevated DNT cell number and increased Vitamin B12 levels) and both were initially clinically diagnosed as ALPS-like. Closer examination of P2, however, revealed active EBV infection and genetic testing identified a novel STK4 mutation. None of the patients harbored typically ALPS-associated mutations of the Fas receptor mediated apoptotic pathway and Fas-mediated apoptosis was not affected. The presented case reports extend the clinical spectrum of STK4 deficiency

Eine molekulare Grundlage für differentielle Ca2+ Signale in B-Lymphozyten

Stork B. A molecular basis for differential Ca2+ signalling in B lymphocytes. Bielefeld (Germany): Bielefeld University; 2006.Die Generierung Antigen-spezifischer B-Lymphozyten ist essentiell für die humorale Immunantwort. Die Aktivierung von B-Zellen ist von Signalen abhängig, die über den B-Zell-Antigenrezeptor (BCR) vermittelt werden. Cytoplasmatische Protein-Tyrosinkinasen (PTKs) sind BCR-proximale Effektorproteine, die cytoplasmatische und transmembrane Adapterproteine wie SLP-65 oder NTAL phosphorylieren. Phosphoryliertes SLP-65 bildet das Gerüst für die Assemblierung und Aktivierung eines Proteinkomplexes, der die Ca2+-Freisetzung aus intrazellulären Speichern initiiert. Dieser Ca2+-Initiationskomplex besteht mindestens aus Brutons Tyrosinkinase (Btk) und Phospholipase C-[gamma]2 (PLC-[gamma]2). Die BCR-induzierte Ca2+-Mobilisierung beinhaltet sowohl die Freisetzung aus intrazellulären Ca2+-Speichern als auch den Einstrom über die Plasmamembran. Das Ausmaß der Ca2+-Mobilisierung aus diesen beiden Speichern hängt von dem Entwicklungsstadium des B-Lymphozyten ab. Die räumliche und zeitliche Veränderung von Ca2+-Profilen kann zu verschiedenen B-Zell-Antworten führen, d.h. Proliferation, Differenzierung, Anergie oder Apoptose. Gegenwärtig sind zentrale Fragen in der BCR-induzierten Ca2+-Mobilisierung: (1) wie wird der Ca2+-Initiationskomplex an die Membran rekrutiert, (2) was ist der exakte Mechanismus der BCR-vermittelten PLC-[gamma]2 Aktivierung, (3) wie ist die intrazelluläre Ca2+-Freisetzung mit dem Ca2+-Einstrom über die Plasmamembran verbunden, und (4) wie erfolgt die differentielle Regulierung der Ca2+-Mobilisierung während der B-Zell-Lymphopoese? In dieser Studie wurden diese Fragen mittels zielgerichteter Gen-Inaktivierung und nachfolgenden Rekonstitutionen analysiert. Es konnte gezeigt werden, dass der SH2/SH3-Adapter Grb2 die Ca2+-Mobilisierung aus intra- und extrazellulären Quellen negativ reguliert. Diese Funktion ist von der zentralen SH2- und der C-terminalen SH3-Domäne abhängig. Tyrosin-phosphoryliertes NTAL wirkt dieser Funktion durch die Grb2-Rekrutierung in lipid rafts entgegen. Es konnte des weiteren gezeigt werden, dass das NTAL/Grb2-Modul nicht den Phosphorylierungsgrad der zentralen Komponenten des Ca2+-Initiationskomplexes, d.h. SLP-65 oder PLC-[gamma]2, beeinflusst. Allerdings scheinen NTAL/Grb2 die PLC-[gamma]2-Aktivierung und/oder dessen Retention an der Plasmamembran zu kontrollieren. Es konnte im Rahmen dieser Arbeit herausgefunden werden, dass das Hauptphosphoprotein in DT40 das Grb2-Effektorprotein ist. Nachfolgend wurde dieses als das cytosolische Adapterprotein Dok-3 identifiziert. Die Phosphorylierung von Dok-3 durch Lyn ist abhängig von Grb2 und benötigt dessen zentrale SH2- und die C-terminale SH3-Domäne. Zusammenfassend deuten diese Studien darauf hin, dass Grb2 ein molekularer Schalter in der BCR-induzierten Ca2+-Mobilisierung ist. Die SH2-Domänen-abhängige Bindung von Grb2 an entweder Tyrosin-phosphoryliertes NTAL oder Dok-3 führt letztendlich zur positiven bzw. negativen Modulation der Antigenrezeptor-induzierten Ca2+-Mobilisierung in B-Zellen. In dieser Studie wurde ein neuer Ca2+-Regulationsmechanismus identifiziert. Die Adapter-vermittelte Modulation der Ca2+-Mobilisierung könnte durch die Unterstützung von entweder Aktivierung oder Toleranzinduktion die B-Zell-Signaltransduktion kritisch beeinflussen.The generation of antigen-specific B lymphocytes is essential for the humoral immune response. B cell activation depends on signals generated upon engagement of the B cell antigen receptor (BCR). Proximal BCR effector proteins are cytoplasmic protein tyrosine kinases (PTKs) which phosphorylate cytoplasmic and transmembrane adaptor proteins such as SLP-65 or NTAL, respectively. Phosphorylated SLP-65 forms the scaffold for the assembly and activation of a protein complex which initiates Ca2+ release from intracellular stores. This Ca2+ initiation complex is composed of at least Bruton's tyrosine kinase (Btk) and phospholipase C-[gamma]2 (PLC-[gamma]2). BCR-induced Ca2+ mobilization arises by both release from intracellular Ca2+ stores, and Ca2+ influx through plasma membrane channels. The extent of Ca2+ mobilization from these two sources varies depending on the developmental stage of the B lymphocyte. Shaping Ca2+ profiles in the dimensions of space and time can determine different B cell responses, i.e. proliferation, differentiation, anergy or apoptosis. Currently, central questions of BCR-induced Ca2+ mobilization are: (1) how the Ca2+ initiation complex is targeted to the plasma membrane, (2) what is the exact mechanism of BCR-triggered PLC-[gamma]2 activation, (3) how the intracellular Ca2+ release is connected to Ca2+ influx across the plasma membrane, and (4) how Ca2+ mobilization is differentially regulated during B cell lymphopoiesis. In this study, these questions were addressed by gene targeting experiments and subsequent reconstitutions in the chicken B cell line DT40. It was demonstrated that the SH2/SH3 adaptor Grb2 negatively regulates Ca2+ mobilization from intra- and extracellular sources. This function depends on the central SH2 and the C-terminal SH3 domain. Tyrosine-phosphorylated NTAL counter-acts Grb2 by recruiting it into lipid rafts. It was further shown that the NTAL/Grb2 module does not affect the phosphorylation state of the central components of the Ca2+ initiation complex, i.e. SLP-65 or PLC-[gamma]2. However, it appeared that PLC-[gamma]2 activation and/or retention at the plasma membrane are controlled by NTAL/Grb2. The downstream effector of Grb2 was determined to be the major tyrosine phosphorylated protein in DT40 B cells, and was subsequently identified as the cytosolic adaptor Dok-3. Phosphorylation of Dok-3 by Lyn depends on Grb2 and requires its central SH2 and C-terminal SH3 domain. Collectively, Grb2 seems to be a molecular switch in BCR-induced Ca2+ mobilization. The SH2 domain-dependent binding of Grb2 to either tyrosine phosphorylated NTAL or Dok-3 ultimately leads to positive or negative modulation of antigen receptor-induced Ca2+ mobilization in B cells. Thus, in this study a novel Ca2+ regulation pathway in B lymphocytes was identified. The adaptor-mediated modulation of Ca2+ mobilization may critically influence B cell signalling by supporting either activation or tolerance induction