No abstract

Akt is involved in the regulation of diverse cellular functions such as cell proliferation, energy metabolism, and apoptosis. Although three Akt isoforms are known, the function of each isoform is poorly understood. To gain a better understanding of each Akt isoform, we examined the subcellular localization and expression of each isoform in transformed and nontransformed cells. Akt1 was localized in the cytoplasm, which is in agreement with the currently accepted model that cytoplasmic Akt is translocated and activated at the inner leaflet of the plasma membrane. Interestingly, HEK-293 and HEK-293T cells contained Akt1 in the nucleus and cytoplasm, respectively, suggesting that SV40 T-antigen plays a crucial role in the cytoplasmic localization and activation of Akt1 in HEK-293T. Akt2 was colocalized with the mitochondria, while Akt3 was localized in both the nucleus and nuclear membrane. The subcellular localization of the Akt isoforms was not substantially altered in response to ionizing radiation or EGF. Furthermore, the ablation of one Akt isoform by small interfering RNA (siRNA) did not alter the subcellular location of the remaining isoforms, suggesting that the major function of one isoform is not compensated for by other isoforms. Together, our data support the notion that Akt2 and Akt3 are regulated at the mitochondrial and nuclear membranes, respectively. The mitochondrial localization of Akt2 raises the possibility that this isoform may be involved in both glucose-based energy metabolism and suppression of apoptosis, two Akt functions previously identified with anti-pan-Akt antibodies.

No abstract

The diverse function of proliferating cell nuclear antigen (PCNA) may be regulated by interactions with different protein partners. Interestingly, the binding sites for all known PCNA-associating proteins are on the outer surface or the C termini ("front") sides of the PCNA trimer. Using cell extracts and purified human PCNA protein, we show here that two PCNA homotrimers form a back-to-back doublet. Mutation analysis suggests that the Arg-5 and Lys-110 residues on the PCNA back side are the contact points of the two homotrimers in the doublet. Furthermore, short synthetic peptides encompassing either Arg-5 or Lys-110 inhibit double trimer formation. We also found that a PCNA double trimer, but not a homotrimer alone, can simultaneously accommodate chromatin assembly factor-1 and polymerase ␦. Together, our data supports a model that chromatin remodeling by chromatin assembly factor-1 (and, possibly, many other cellular activities) are tightly coupled with DNA replication (and repair) through a PCNA double trimer complex.

MINT‐7995351: G3P (uniprotkb:P04406) and PCNA (uniprotkb:P12004) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT‐7995334: ENOA (uniprotkb:P06733) and PCNA (uniprotkb:P12004) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT‐7995368: ALDOA (uniprotkb:P04075) and PCNA (uniprotkb:P12004) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT‐7995141: G3P (uniprotkb:P04406) binds (MI:0407) to PCNA (uniprotkb:P12004) by far western blotting (MI:0047)MINT‐7995182: ENOA (uniprotkb:P06733) binds (MI:0407) to PCNA (uniprotkb:P12004) by far western blotting (MI:0047)MINT‐7995132: G3P (uniprotkb:P04406) physically interacts (MI:0915) with PCNA (uniprotkb:P12004) by far western blotting (MI:0047)MINT‐7995228: PRDX6 (uniprotkb:P30041) physically interacts (MI:0915) with PCNA (uniprotkb:P12004) by far western blotting (MI:0047)MINT‐7995220: CAH2 (uniprotkb:P00918) physically interacts (MI:0915) with PCNA (uniprotkb:P12004) by far western blotting (MI:0047)MINT‐7995114: Triosephosphate isomerase (uniprotkb:P60174) binds (MI:0407) to PCNA (uniprotkb:P12004) by far western blotting (MI:0047)MINT‐7995244: K2C7 (uniprotkb:P08729) physically interacts (MI:0915) with PCNA (uniprotkb:P12004) by far western blotting (MI:0047)MINT‐7995252: ANXA2 (uniprotkb:P07355) physically interacts (MI:0915) with PCNA (uniprotkb:P12004) by far western blotting (MI:0047)MINT‐7995122: Triosephosphate isomerase (uniprotkb:P60174) physically interacts (MI:0915) with PCNA (uniprotkb:P12004) by far western blotting (MI:0047)MINT‐7995093: ALDOA (uniprotkb:P04075) physically interacts (MI:0915) with PCNA (uniprotkb:P12004) by far western blotting (MI:0047)MINT‐7995148: PGK1 (uniprotkb:P00558) physically interacts (MI:0915) with PCNA (uniprotkb:P12004) by far western blotting (MI:0047)MINT‐7995158: PGAM1 (uniprotkb:P18669) physically interacts (MI:0915) with PCNA (uniprotkb:P12004) by far western blotting (MI:0047)MINT‐7995166: PGAM1 (uniprotkb:P18669) binds (MI:0407) to PCNA (uniprotkb:P12004) by far western blotting (MI:0047)MINT‐7995105: ALDOA (uniprotkb:P04075) binds (MI:0407) to PCNA (uniprotkb:P12004) by far western blotting (MI:0047)MINT‐7995260: PPIA (uniprotkb:P62937) physically interacts (MI:0915) with PCNA (uniprotkb:P12004) by far western blotting (MI:0047)MINT‐7995173: ENOA (uniprotkb:P06733) physically interacts (MI:0915) with PCNA (uniprotkb:P12004) by far western blotting (MI:0047)MINT‐7995268: EF1A (uniprotkb:P68104) physically interacts (MI:0915) with PCNA (uniprotkb:P12004) by far western blotting (MI:0047)MINT‐7995236: MDHM (uniprotkb:P40926) physically interacts (MI:0915) with PCNA (uniprotkb:P12004) by far western blotting (MI:0047)MINT‐7995189: RSSA (uniprotkb:P08865) physically interacts (MI:0915) with PCNA (uniprotkb:P12004) by far western blotting (MI:0047)MINT‐7995282: PCNA (uniprotkb:P12004) physically interacts (MI:0915) with ALDOA (uniprotkb:P00883) and G3P (uniprotkb:P46406) by anti bait coimmunoprecipitation (MI:0006).

No abstract

BackgroundAkt/PKB is a promising anticancer therapeutic target, since abnormally elevated Akt activity is directly correlated to tumor development, progression, poor prognosis and resistance to cancer therapies. Currently, the unique role of each Akt isoform and their relevance to human breast cancer are poorly understood.Methodology/Principal FindingsWe previously found that Akt1, 2 and 3 are localized at specific subcellular compartments (the cytoplasm, mitochondria and nucleus, respectively), raising the possibility that each isoform may have unique functions and employ different regulation mechanisms. By systematically studying Akt-ablated MDA-MB231 breast cancer cells with isoform-specific siRNA, we here show that Akt2 is the most relevant isoform to cell proliferation and survival in our cancer model. Prolonged ablation of Akt2 with siRNA resulted in cell-cycle arrest in G0/G1 by downregulating Cdk2 and cyclin D, and upregulating p27. The analysis of the Akt downstream signaling pathways suggested that Akt2 specifically targets and activates the p70S6K signaling pathway. We also found that Akt2 ablation initially resulted in an increase in the mitochondrial volume concomitantly with the upregulation of PGC-1α, a regulator of mitochondrial biogenesis. Prolonged ablation of Akt2, but not Akt1 or Akt3, eventually led to cell death by autophagy of the mitochondria (i.e., mitophagy).Conclusions/SignificanceCollectively, our data demonstrates that Akt2 augments cell proliferation by facilitating cell cycle progression through the upregulation of the cell cycle engine, and protects a cell from pathological autophagy by modulating mitochondrial homeostasis. Our data, thus, raises the possibility that Akt2 can be an effective anticancer target for the control of (breast) cancer.

It has recently been shown that the tumor suppressor p53 mediates a signal transduction pathway that responds to DNA damage by arresting cells in the late G1 period of the cell cycle. However, the operation of this pathway alone cannot explain the 50%o reduction in the rate of DNA synthesis that occurs within 30 min of irradiation of an asynchronous cell population. We are using the amplified dihydrofolate reductase (DHFR) domain in the methotrexate-resistant CHO cell line, CHOC 400, as a model replicon in which to study this acute radiation effect. We first show that the CHOC 400 cell line retains the classical acute-phase response but does not display the late G1 arrest that characterizes the p53-mediated checkpoint. Using a two-dimensional gel replicon-mapping method, we then show that when asynchronous cultures are irradiated with 900 cGy, initiation in the DHFR locus is completely inhibited within 30 min and does not resume for 3 to 4 h. Since initiation in this locus occurs throughout the first 2 h of the S period, this result implies the existence of a p53-independent S-phase damage-sensing pathway that functions at the level of individual origins. Results obtained with the replication inhibitor mimosine define a position near the GJ1S boundary beyond which cells are unable to prevent initiation at early-firing origins in response to irradiation. This is the first direct demonstration at a defined chromosomal origin that radiation quantitatively down-regulates initiation.The effects of ionizing radiation on DNA have been studied intensively for decades. An understanding of these effects and the cellular response to them is required to comprehend the genetic basis of radiation-induced mutation and to develop novel approaches to enhance the effectiveness of radiotherapy for the treatment of cancer and other diseases.Mammalian cells have elaborated complex mechanisms to deal with assaults on the integrity of genomic DNA. It is well known, for example, that cells down-regulate DNA replication ([3H]thymidine incorporation) by 50 to 55% within 30 min of a challenge with high-dose ionizing radiation (25,31,38); replication rates return to normal within a few hours (38). Presumably, this transient arrest of DNA synthesis allows the removal of adducts or the repair of strand breaks, or both, before these lesions can be permanently fixed by DNA replication into catastrophic chromosome breaks or mutations. The radiation dose-response curve has a steep initial component at lower doses, which is thought to result from inhibition of new initiations at origins of replication; a much smaller, refractile component is observed at high doses, which is thought to reflect direct effects on chain elongation (25,31,40). Because of the consequent high residual rate of [3H]thymidine incorporation by unaffected replication forks, it has been difficult to assess the extent to which initiation is specifically inhibited at any given dose of radiation.Various models have been proposed to explain how damage to the template itself, or c...