Inhibition of protein phosphatase 2A (PP2A) activity has been identified as a prerequisite for the transformation of human cells. However, the molecular mechanisms by which PP2A activity is inhibited in human cancers are currently unclear. In this study, we describe a cellular inhibitor of PP2A with oncogenic activity. The protein, designated Cancerous Inhibitor of PP2A (CIP2A), interacts directly with the oncogenic transcription factor c-Myc, inhibits PP2A activity toward c-Myc serine 62 (S62), and thereby prevents c-Myc proteolytic degradation. In addition to its function in c-Myc stabilization, CIP2A promotes anchorage-independent cell growth and in vivo tumor formation. The oncogenic activity of CIP2A is demonstrated by transformation of human cells by overexpression of CIP2A. Importantly, CIP2A is overexpressed in two common human malignancies, head and neck squamous cell carcinoma (HNSCC) and colon cancer. Thus, our data show that CIP2A is a human oncoprotein that inhibits PP2A and stabilizes c-Myc in human malignancies.

A σ-2 receptor ligand siramesine induces lysosomal leakage and cathepsin-dependent death of cancer cells in vitro and displays potent anti-cancer activity in vivo. The mechanism by which siramesine destabilizes lysosomes is, however, unknown. Here, we show that siramesine induces a rapid rise in the lysosomal pH that is followed by lysosomal leakage and dysfunction. The rapid accumulation of siramesine into cancer cell lysosomes, its ability to destabilize isolated lysosomes, and its chemical structure as an amphiphilic amine indicate that it is a lysosomotropic detergent. Notably, siramesine triggers also a substantial Atg6-and Atg7-dependent accumulation of autophagosomes that is associated with a rapid and sustained inhibition of mammalian target of rapamycin complex 1 (mTORC1; an inhibitor of autophagy). Siramesine fails, however, to increase the degradation rate of long-lived proteins. Thus, the massive accumulation of autophagosomes is likely to be due to a combined effect of activation of autophagy signaling and decreased autophagosome turnover. Importantly, pharmacological and RNA interference-based inhibition of autophagosome formation further sensitizes cancer cells to siramesine-induced cytotoxicity. These data identify siramesine as a lysosomotropic detergent that triggers cell death via a direct destabilization of lysosomes and cytoprotection by inducing the accumulation of autophagosomes. Threrefore, the combination of siramesine with inhibitors of autophagosome formation appears as a promising approach for future cancer therapy.

Extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase pathway activity is regulated by the antagonist function of activating kinases and inactivating protein phosphatases. Sustained ERK pathway activity is commonly observed in human malignancies; however, the mechanisms by which the pathway is protected from phosphatasemediated inactivation in the tumor tissue remain obscure. Here, we show that methylesterase PME-1-mediated inhibition of the protein phosphatase 2A promotes basal ERK pathway activity and is required for efficient growth factor response. Mechanistically, PME-1 is shown to support ERK pathway signaling upstream of Raf, but downstream of growth factor receptors and protein kinase C. In malignant gliomas, PME-1 expression levels correlate with both ERK activity and cell proliferation in vivo. Moreover, PME-1 expression significantly correlates with disease progression in human astrocytic gliomas (n = 222). Together, these observations identify PME-1 expression as one mechanism by which ERK pathway activity is maintained in cancer cells and suggest an important functional role for PME-1 in the disease progression of human astrocytic gliomas.

Plant viruses encode movement proteins (MPsA delicate balance between protein phosphorylation and dephosphorylation regulates the function of a vast variety of proteins in the cell. Recently, several lines of evidence have suggested that phosphorylation of plant virus-encoded movement proteins (MPs) 1 by host plant protein kinases may be involved in the process of virus movement (1-3). The functional role of MPs is to assist the spread of viral progeny from cell to cell and over long distances (reviewed in Refs. 4 -7). There is evidence that the 30-kDa MP of tobacco mosaic virus (TMV; genus Tobamovirus) is phosphorylated when expressed in insect cells from a baculovirus vector (8), in TMV-infected protoplasts (9, 10), and in the cell wall-enriched fractions of transgenic plants expressing the wild-type MP and its mutants (3, 11). The 17-kDa MP of potato leafroll virus (genus Luteovirus) was shown to be phosphorylated in a reconstituted system containing bacterially expressed protein and membrane preparations from potato leaves (12). In another report, phosphorylation of the 69-kDa MP of turnip yellow mosaic virus (genus Tymovirus) was demonstrated when the MP gene was expressed in insect cells using a baculovirus vector (13).It is not yet clear whether MP phosphorylation is essential for the general process of virus movement; however, there is growing evidence suggesting that phosphorylation can affect several MP functions. Originally, it was proposed that phosphorylation represents a mechanism for MP inactivation and sequestration in the cell walls of mature plants (11). Proteolytic processing was found to be an alternative mechanism to phosphorylation for inactivation of TMV MP in Arabidopsis thaliana (14). Recently, new evidence has accumulated that suggests that phosphorylation of TMV MP may directly affect its function. Either phosphorylation or the presence of serine 37 in MP of tomato mosaic virus (genus Tobamovirus) was shown to be essential for the protein intracellular localization and stability and, therefore, required for the efficient spread of the virus (1). These results indicated that phosphorylation of MPs by cellular protein kinase(s) may represent an active process required by the plant viruses to execute their movement function. Second line of evidence in support of the possible involvement of MP phosphorylation in the cell-to-cell movement came from in vitro studies showing that the phosphorylation of TMV MP abolishes its ability to repress RNA translation (2). This finding suggested a possible mechanism for how MP phosphorylation may regulate the function of movement ribonucleoprotein intermediates in the course of their cell-to-cell translocation. According to this hypothesis, MP phosphorylation converts the translation-incompetent movement intermediates into the translation-ready state, thus allowing the virus to replicate in the newly infected cell. In another recent study (3), TMV MP mutant mimicking phosphorylation was reported to be deficient in plasmodesmal transport, suggesting th...

We reported previously that the capsid protein (CP) of Potato virus A (PVA) is phosphorylated both in virus-infected plants and in vitro. In this study, an enzyme that phosphorylates PVA CP was identified as the protein kinase CK2. The ␣ -catalytic subunit of CK2 (CK2 ␣ ) was purified from tobacco and characterized using in-gel kinase assays and liquid chromatographytandem mass spectrometry. The tobacco CK2 ␣ gene was cloned and expressed in bacterial cells. Specific antibodies were raised against the recombinant enzyme and used to demonstrate the colocalization of PVA CP and CK2 ␣ in infected tobacco protoplasts. A major site of CK2 phosphorylation in PVA CP was identified by a combination of mass spectrometric analysis, radioactive phosphopeptide sequencing, and mutagenesis as Thr-242 within a CK2 consensus sequence. Amino acid substitutions that affect the CK2 consensus sequence in CP were introduced into a full-length infectious cDNA clone of PVA tagged with green fluorescent protein. Analysis of the mutant viruses showed that they were defective in cell-to-cell and longdistance movement. Using in vitro assays, we demonstrated that CK2 phosphorylation inhibited the binding of PVA CP to RNA, suggesting a molecular mechanism of CK2 action. These results suggest that the phosphorylation of PVA CP by CK2 plays an important regulatory role in virus infection.

Macroautophagy (hereafter referred to as autophagy) is a tightly regulated lysosome‐dependent catabolic pathway. During this process, cytosolic constituents are sequestered into autophagosomes, which subsequently fuse with lysosomes to become autolysosomes, where their contents are degraded. Autophagy contributes to the maintenance of the cellular energy homeostasis, to the clearance of damaged organelles and to adaptation to environmental stresses. Accordingly, autophagy defects have been linked to a wide range of human pathologies, including cancer. The recent discovery of several evolutionarily conserved genes involved in autophagosome formation has greatly stimulated the autophagy research, and the complex signalling networks regulating mammalian autophagy have begun to emerge. Here, we draw the current picture of signalling pathways connecting mitogenic and stress‐induced signals to the initiation and maturation of autophagosomes and discuss the possibilities of their targeting as therapeutic adjuvants in anticancer therapy.

No abstract

As part of a regulatory loop linking cell metabolism, growth, and proliferation, CIP2A promotes mTORC1-mediated cell growth and autophagy inhibition but is itself down-regulated by autophagy.