Growth factors and mitogens use the Ras/Raf/MEK/ERK signaling cascade to transmit signals from their receptors to regulate gene expression and prevent apoptosis. Some components of these pathways are mutated or aberrantly expressed in human cancer (e.g., Ras, B-Raf). Mutations also occur at genes encoding upstream receptors (e.g., EGFR and Flt-3) and chimeric chromosomal translocations (e.g., BCR-ABL) which transmit their signals through these cascades. Even in the absence of obvious genetic mutations, this pathway has been reported to be activated in over 50% of acute myelogenous leukemia and acute lymphocytic leukemia and is also frequently activated in other cancer types (e.g., breast and prostate cancers). Importantly, this increased expression is associated with a poor prognosis. The Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt pathways interact with each other to regulate growth and in some cases tumorigenesis. For example, in some cells, PTEN mutation may contribute to suppression of the Raf/MEK/ERK cascade due to the ability of activated Akt to phosphorylate and inactivate different Rafs. Although both of these pathways are commonly thought to have anti-apoptotic and drug resistance effects on cells, they display different cell lineage specific effects. For example, Raf/MEK/ERK is usually associated with proliferation and drug resistance of hematopoietic cells, while activation of the Raf/MEK/ERK cascade is suppressed in some prostate cancer cell lines which have mutations at PTEN and express high levels of activated Akt. Furthermore the Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt pathways also interact with the p53 pathway. Some of these interactions can result in controlling the activity and subcellular localization of Bim, Bak, Bax, Puma and Noxa. Raf/MEK/ERK may promote cell cycle arrest in prostate cells and this may be regulated by p53 as restoration of wild-type p53 in p53 deficient prostate cancer cells results in their enhanced sensitivity to chemotherapeutic drugs and increased expression of Raf/MEK/ERK pathway. Thus in advanced prostate cancer, it may be advantageous to induce Raf/MEK/ERK expression to promote cell cycle arrest, while in hematopoietic cancers it may be beneficial to inhibit Raf/MEK/ERK induced proliferation and drug resistance. Thus the Raf/MEK/ERK pathway has different effects on growth, prevention of apoptosis, cell cycle arrest and induction of drug resistance in cells of various lineages which may be due to the presence of functional p53 and PTEN and the expression of lineage specific factors.

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Males of advanced age represent a rapidly growing population at risk for prostate cancer. In the contemporary setting of earlier detection, a majority of prostate carcinomas are still clinically localized and often treated using radiation therapy. Our recent studies have shown that premature cellular senescence, rather than apoptosis, accounts for most of the clonogenic death induced by clinically-relevant doses of irradiation in prostate cancer cells. We show here that this treatment-induced senescence was associated with a significantly increased release of exosome-like microvesicles. In premature senescence, this novel secretory phenotype was dependent on the activation of p53. In addition, the release of exosome-like microvesicles also increased during proliferative senescence in normal human diploid fibroblasts. These data support the hypothesis that senescence, initiated either by telomere attrition (e.g., aging) or DNA damage (e.g., radiotherapy), may induce a p53-dependent increase in the biogenesis of exosome-like vesicles. Ultrastructural analysis and RNAi-mediated knockdown of Tsg101 provided significant evidence that the additional exosomes released by prematurely senescent prostate cancer cells were principally derived from multivesicular endosomes (MVEs). Moreover, these exosomes were enriched in B7-H3 protein, a recently identified diagnostic marker for prostate cancer, and an abundance of what has recently been termed “exosomal shuttle RNA”. Our findings are consistent with the proposal that exosomes can transfer cargos, with both immunoregulatory potential and genetic information, between cells through a novel mechanism that may be recruited to increase exosome release during accelerated and replicative cellular senescence.

Brain myosin V is a member of a widely distributed class of unconventional myosins that may be of central importance to organelle trafficking in all eukaryotic cells. Molecular constituents that target this molecular motor to organelles have not been previously identified. Using a combination of immunopurification, extraction, cross-linking, and coprecipitation assays, we demonstrate that the tail domain of brain myosin V forms a stable complex with the synaptic vesicle membrane proteins, synaptobrevin II and synaptophysin. While myosin V was principally bound to synaptic vesicles during rest, this putative transport complex was promptly disassembled upon the depolarization-induced entry of Ca2+ into intact nerve endings. Coimmunoprecipitation assays further indicate that Ca2+ disrupts the in vitro binding of synaptobrevin II to synaptophysin in the presence but not in the absence of Mg2+. We conclude that hydrophilic forces reversibly couple the myosin V tail to a biochemically defined class of organelles in brain nerve terminals.

Abstract. Individual isoforms of the protein kinase C (PKC) family of kinases may have assumed distinct responsibilities for the control of complex and diverse cellular functions. In this study, we show that an isoform specific interaction between PKCe and filamentous actin may serve as a necessary prelude to the enhancement of glutamate exocytosis from nerve terminals. Using a combination of cosedimentation, overlay, and direct binding assays, we demonstrate that filamentous actin is a principal anchoring protein for PKCe within intact nerve endings. The unusual stability and direct nature of this physical interaction indicate that actin filaments represent a new class of PKC-binding protein. The binding of PKCe to actin required that the kinase be activated, presumably to expose a cryptic binding site that we have identified and shown to be located between the first and second cysteine-rich regions within the regulatory domain of only this individual isoform of PKC. Arachidonic acid (AA) synergistically interacted with diacylglycerol to stimulate actin binding to PKCe. Once established, this protein-protein interaction securely anchored PKCe to the cytoskeletal matrix while also serving as a chaperone that maintained the kinase in a catalytically active conformation. Thus, actin appears to be a bifunctional anchoring protein that is specific for the PKCe isoform. The assembly of this isoform-specific signaling complex appears to play a primary role in the PKC-dependent facilitation of glutamate exocytosis.VOLUTION has conserved a fundamental mechanism that ensures the sorting and fusion of secretory vesicles upon their delivery to appropriate destinations along the secretory pathway. In many eukaryotes, however, alternative pathways have emerged in which vesicle delivery and fusion have become rigorously regulated events that are capable of manifesting use-dependent variations in the probability of their occurrence. Glutamate exocytosis is a particularly striking example of a secretory event that, in presynaptic nerve terminals, has acquired a highly evolved mechanism for regulating the efficiency of excitatory synaptic transmission. Moreover, it has been established that use-dependent changes in the reliability (Stevens and Wang, 1994) and extent (Schulz et al., 1994) of glutamate exocytosis directly participate in certain forms of synaptic plasticity.Studies from many laboratories suggest that a conditional relationship may exist between the efficiency of glutamate release and the activity of presynaptic protein

Ectopic expression of mutant forms of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) lacking lipid (G129E) or lipid and protein (C124S) phosphatase activity decreased sensitivity of MCF-7 breast cancer cells, which have wild-type PTEN, to doxorubicin and increased sensitivity to the mammalian target of rapamycin (mTOR) inhibitor rapamycin. Cells transfected with a mutant PTEN gene lacking both lipid and protein phosphatase activities were more resistant to doxorubicin than cells transfected with the PTEN mutant lacking lipid phosphatase activity indicating that the protein phosphatase activity of PTEN was also important in controlling the sensitivity to doxorubicin, while no difference was observed between the lipid (G129E) and lipid and protein (C124S) phosphatase PTEN mutants in terms of sensitivity to rapamycin. A synergistic inhibitory interaction was observed when doxorubicin was combined with rapamycin in the phosphatase-deficient PTENtransfected cells. Interference with the lipid phosphatase activity of PTEN was sufficient to activate Akt/mTOR/ p70S6K signaling. These studies indicate that disruption of the normal activity of the PTEN phosphatase can have dramatic effects on the therapeutic sensitivity of breast cancer cells. Mutations in the key residues which control PTEN lipid and protein phosphatase may act as dominant-negative mutants to suppress endogenous PTEN and alter the sensitivity of breast cancer patients to chemo-and targeted therapies.

Protein kinase C-epsilon (PKC-⑀) contains a putative actin binding motif that is unique to this individual member of the PKC gene family. We have used deletion mutagenesis to determine whether this hexapeptide motif is required for the physical association of PKC-⑀ and actin. Full-length recombinant PKC-⑀, but not PKC-␤II, -␦, -, or -, bound to filamentous actin in a phorbol ester-dependent manner. Deletion of PKC-⑀ amino acids 222-230, encompassing a putative actin binding motif, completely abrogated this binding activity. When NIH 3T3 cells overexpressing either PKC-⑀ or the deletion mutant of this isozyme were treated with phorbol ester only wild-type PKC-⑀ colocalized with actin in zones of cell adhesion. In binary reactions, it was possible to demonstrate that purified filamentous actin is capable of directly stimulating PKC-⑀ phosphotransferase activity. These and other findings support the hypothesis that a conformationally hidden actin binding motif in the PKC-⑀ sequence becomes exposed upon activation of this isozyme and functions as a dominant localization signal in NIH 3T3 fibroblasts. This protein-protein interaction is sufficient to maintain PKC-⑀ in a catalytically active conformation.

ACKnoWLeDgeMenTSSupported in part by grants from the NIH (R01098195 to J.A.M.) and the Brody Brothers Foundation Endowment (#997729 to J.A.M. and D.M.T.). ReportA Dominant Role for p53-Dependent Cellular Senescence in Radiosensitization of Human Prostate Cancer Cells ABSTrACT Because p53 inactivation may limit the effectiveness of radiation therapy for localized prostate cancer, it is important to understand how this gene regulates clonogenic survival after an exposure to ionizing radiation. Here, we show that premature cellular senescence is the principal mode of cell death accounting for the radiosensitivity of human prostate cancer cell lines retaining p53 function. Alternative stress response pathways controlled by this tumor suppressor, including cell cycle arrest, DNA damage repair, mitotic catastrophe and apoptosis, contributed significantly less to radiation-induced clonogenic death. Using a dominant negative C-terminal fragment of p53, we present the first evidence that a complete loss of endogenous p53 function is sufficient to limit the irradiation-induced senescence and clonogenic death of prostate cancer cells. Conversely, inheritance of wild-type p53 by prostate cancer cells lacking a functional allele of this gene (i.e., DU145) significantly increases clonogenic death through p53-dependent cellular senescence and apoptotic pathways. Our data provide evidence that mutations of even one p53 allele may be sufficient to alter their clonogenic fate. In addition, they support the idea that the p53 pathway can be used as a specific target for enhancing the radiosensitivity of prostate cancer cells. Activation of p53 by the drug nutlin-3 is shown to be an effective radiosensitizer of prostate cancer cells retaining functional alleles of p53 and this effect was entirely attributable to an increased induction of p53-dependent cellular senescence.