WIPI-dependent autophagy during neutrophil differentiation of NB4 acute promyelocytic leukemia cells.

Members of the WD-repeat protein interacting with phosphoinositides (WIPI) family are phosphatidylinositol 3-phosphate (PI3P) effectors that are essential for the formation of autophagosomes. Autophagosomes, unique double-membraned organelles, are characteristic for autophagy, a bulk degradation mechanism with cytoprotective and homeostatic function. Both, WIPI-1 and WIPI-2 are aberrantly expressed in several solid tumors, linking these genes to carcinogenesis. We now found that the expression of WIPI-1 was significantly reduced in a large cohort of 98 primary acute myeloid leukemia (AML) patient samples (complex karyotypes; t(8;21); t(15,17); inv(16)). In contrast, the expression of WIPI-2 was only reduced in acute promyelocytic leukemia (APL), a distinct subtype of AML (t(15,17)). As AML cells are blocked in their differentiation, we tested if the expression levels of WIPI-1 and WIPI-2 increase during all-trans retinoic acid (ATRA)-induced neutrophil differentiation of APL. According to the higher WIPI-1 expression in granulocytes compared with immature blast cells, WIPI-1 but not WIPI-2 expression was significantly induced during neutrophil differentiation of NB4 APL cells. Interestingly, the induction of WIPI-1 expression was dependent on the transcription factor PU.1, a master regulator of myelopoiesis, supporting our notion that WIPI-1 expression is reduced in AML patients lacking proper PU-1 activity. Further, knocking down WIPI-1 in NB4 cells markedly attenuated the autophagic flux and significantly reduced neutrophil differentiation. This result was also achieved by knocking down WIPI-2, suggesting that both WIPI-1 and WIPI-2 are functionally required and not redundant in mediating the PI3P signal at the onset of autophagy in NB4 cells. In line with these data, downregulation of PI3KC3 (hVPS34), which generates PI3P upstream of WIPIs, also inhibited neutrophil differentiation. In conclusion, we demonstrate that both WIPI-1 and WIPI-2 are required for the PI3P-dependent autophagic activity during neutrophil differentiation, and that PU.1-dependent WIPI-1 expression is significantly repressed in primary AML patient samples and that the induction of autophagic flux is associated with neutrophil differentiation of APL cells.

Mitochondrial proteolytic stress induced by loss of mortalin function is rescued by Parkin and PINK1.

The mitochondrial chaperone mortalin was implicated in Parkinson's disease (PD) because of its reduced levels in the brains of PD patients and disease-associated rare genetic variants that failed to rescue impaired mitochondrial integrity in cellular knockdown models. To uncover the molecular mechanisms underlying mortalin-related neurodegeneration, we dissected the cellular surveillance mechanisms related to mitochondrial quality control, defined the effects of reduced mortalin function at the molecular and cellular levels and investigated the functional interaction of mortalin with Parkin and PINK1, two PD-related proteins involved in mitochondrial homeostasis. We found that reduced mortalin function leads to: (1) activation of the mitochondrial unfolded protein response (UPR(mt)), (2) increased susceptibility towards intramitochondrial proteolytic stress, (3) increased autophagic degradation of fragmented mitochondria and (4) reduced mitochondrial mass in human cells in vitro and ex vivo. These alterations caused increased vulnerability toward apoptotic cell death. Proteotoxic perturbations induced by either partial loss of mortalin or chemical induction were rescued by complementation with native mortalin, but not disease-associated mortalin variants, and were independent of the integrity of autophagic pathways. However, Parkin and PINK1 rescued loss of mortalin phenotypes via increased lysosomal-mediated mitochondrial clearance and required intact autophagic machinery. Our results on loss of mortalin function reveal a direct link between impaired mitochondrial proteostasis, UPR(mt) and PD and show that effective removal of dysfunctional mitochondria via either genetic (PINK1 and Parkin overexpression) or pharmacological intervention (rapamycin) may compensate mitochondrial phenotypes.

A "no-hybrids" screen for functional antagonizers of human p53 transactivator function: dominant negativity in fission yeast.

We have developed a functional "no-hybrids" screen in the fission yeast Schizosaccharomyces pombe based on the transcription transactivator activity of human p53. The screen can be used to identify antagonizers and modulators of p53 activity. Expression of functional full-length human p53 is conditionally lethal to the screen reporter strains. Co-expression of specific inhibitory proteins promotes cell survival and growth. We have validated the "no-hybrids" system by (a) successful modeling of human wild-type p53 interaction with SV40 large T antigen, Mdm2 and a panel of tumor-derived human p53 mutants, (b) demonstrating the screening system's efficiency through identification of a dominant negative fragment of p53 itself in a library screen context and (c) using Drosophila p53 to demonstrate that the system can detect evolutionarily distant p53 homologues based on their transactivator activity. The "no-hybrids" screen will be of utility in searches for p53 function-modulators of both cellular and viral origin.

Kinase activities of c-Mos and v-Mos proteins: a single amino acid exchange is responsible for constitutive activation of the 124 v-Mos kinase.

The Mos protein kinase is a serine-/threonine-specific protein kinase with a crucial role in meiotic cell divisions in vertebrates. Several oncogenic derivatives of the c-Mos protein have been discovered in murine retroviruses. These proteins have acquired mutations and exhibit different degrees of protein kinase activity in vitro. In an attempt to understand the factors governing Mos protein kinase activity we have compared the kinase activities of the wild-type c-Mos protein and two v-Mos proteins (strain HT1 and MSV124) after expression in insect cells. Only the 124 v-Mos protein showed kinase activity in vitro as measured by autophosphorylation, vimentin phosphorylation or by phosphorylation and activation of MAP kinase kinase. By domain swapping and site-directed mutagenesis we identified a single point mutation in the 124 v-Mos protein (Arg145-->Gly) which is responsible for its constitutive activity. This residue is located in the alpha-helix C of the kinase domain close to the ATP binding fold and is conserved in all known c-Mos proteins. Introduction of the corresponding mutation into HT1 v-Mos and into murine c-Mos activated both proteins for autophosphorylation, vimentin phosphorylation and for signalling via MAP kinase kinase in vitro. We hypothesize that the Arg145-->Gly mutation found in 124 v-Mos mimicks a conformational change which might be an obligatory step in the activation of c-Mos in vivo.