Regulation of the number ofFurthermore, the effect of EPI64C was dependent upon its GTPase-activating proteins activity. Co-immunoprecipitation studies confirmed an association between KCa2.3 and both Rab35 and RME-1. In contrast to KCa2.3, KCa3.1 was rapidly endocytosed and degraded in an RME-1 and Rab35-independent manner. A series of N-terminal deletions identified a 12-amino acid region, Gly 206 -Pro 217 , as being required for the rapid recycling of KCa2.3. Deletion of Gly 206 -Pro 217 had no effect on the association of KCa2.3 with Rab35 but significantly decreased the association with RME-1. These represent the first studies elucidating the mechanisms by which KCa2.3 is maintained at the plasma membrane.

2 /M arrest and how MMR mechanistically participates in this process are unknown. Here, we show that MNNG exposure results in activation of the cell cycle checkpoint kinases ATM, Chk1, and Chk2, each of which has been implicated in the triggering of the G 2 /M checkpoint response. We document that MNNG induces a robust, dose-dependent G 2 arrest in MMR and ATM-proficient cells, whereas this response is abrogated in MMR-deficient cells and attenuated in ATM-deficient cells treated with moderate doses of MNNG. Pharmacological and RNA interference approaches indicated that Chk1 and Chk2 are both required components for normal MNNG-induced G 2 arrest. MNNG-induced nuclear exclusion of the cell cycle regulatory phosphatase Cdc25C occurred in an MMRdependent manner and was compromised in cells lacking ATM. Finally, both Chk1 and Chk2 interact with the MMR protein MSH2, and this interaction is enhanced after MNNG exposure, supporting the notion that the MMR system functions as a molecular scaffold at the sites of DNA damage that facilitates activation of these kinases. INTRODUCTIONCells are continually exposed to numerous forces and toxins capable of damaging DNA. To ensure maintenance of genome stability, cells have evolved a complex set of mechanisms to appropriately respond to genotoxic damage. Such responses include genome surveillance and DNA repair, activation of cell cycle checkpoints, and apoptosis. Tumor initiation and progression are directly linked to genomic instability and often correlate with loss of gene(s) involved in genome damage response (Hartwell and Kastan, 1994;Loeb et al., 2003). Paradoxically, many cancer treatment regimens induce DNA damage and exert their therapeutic effects through activation of growth arrest or apoptotic responses. Thus, elucidating the mechanisms and molecules that govern DNA damage response is key to understanding the molecular basis of both tumor formation and the therapeutic effects of many anticancer drugs.The nitrosourea N-methyl-NЈ-nitro-N-nitrosoguanidine (MNNG) is a well characterized monofunctional DNA alkylating agent. The cytotoxic and mutagenic potential of MNNG is chiefly attributable to its ability to alkylate (methylate) the O 6 -position of guanine, resulting in formation of O 6 -methylguanine (O 6 MeG) adducts (Goldmacher et al., 1986;Karran and Bignami, 1992). O 6 MeG forces O 6 MeG-T mispairing after DNA replication due to blocking of a hydrogen bonding position involved in complementary base pairing. This mutagenic lesion is primarily repaired via direct demethylation by the DNA repair protein methylguanine-DNA methyltransferase (MGMT) (Lindahl et al., 1982). Moreover, loss of MGMT activity renders cells extremely sensitive to MNNG and like alkylators (Kalamegham et al., 1988), underscoring the role that O 6 MeG lesions play in triggering response to this drug. O 6 MeG lesions are also recognized and repaired by the mismatch repair (MMR) system (Griffin et al., 1994;Duckett et al., 1996).In response to MNNG and other methylators, cells undergo a robust G...

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The number of intermediate-conductance, Ca2+-activated K+ channels (KCa3.1) present at the plasma membrane is deterministic in any physiological response. However, the mechanisms by which KCa3.1 channels are removed from the plasma membrane and targeted for degradation are poorly understood. Recently, we demonstrated that KCa3.1 is rapidly internalized from the plasma membrane, having a short half-life in both human embryonic kidney cells (HEK293) and human microvascular endothelial cells (HMEC-1). In this study, we investigate the molecular mechanisms controlling the degradation of KCa3.1 heterologously expressed in HEK and HMEC-1 cells. Using immunofluorescence and electron microscopy, as well as quantitative biochemical analysis, we demonstrate that membrane KCa3.1 is targeted to the lysosomes for degradation. Furthermore, we demonstrate that either overexpressing a dominant negative Rab7 or short interfering RNA-mediated knockdown of Rab7 results in a significant inhibition of channel degradation rate. Coimmunoprecipitation confirmed a close association between Rab7 and KCa3.1. On the basis of these findings, we assessed the role of the ESCRT machinery in the degradation of heterologously expressed KCa3.1, including TSG101 [endosomal sorting complex required for transport (ESCRT)-I] and CHMP4 (ESCRT-III) as well as VPS4, a protein involved in the disassembly of the ESCRT machinery. We demonstrate that TSG101 is closely associated with KCa3.1 via coimmunoprecipitation and that a dominant negative TSG101 inhibits KCa3.1 degradation. In addition, both dominant negative CHMP4 and VPS4 significantly decrease the rate of membrane KCa3.1 degradation, compared with wild-type controls. These results are the first to demonstrate that plasma membrane-associated KCa3.1 is targeted for lysosomal degradation via a Rab7 and ESCRT-dependent pathway.