Flowchart: Preparation: AKTText Box:  MainPage
 
 
 
 

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Breast

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mTOR/RICTOR is the Ser473 kinase for Akt/PKB in 3T3-L1 adipocytes.

Hresko RC, Mueckler M.

Department of Cell Biology and Physiology, Washington University, School of Medicine, St. Louis, MO 63110-1093.

The insulin-signaling pathway leading to the activation of Akt/PKB has been well-characterized except for a single step, the phosphorylation of Akt at Ser473. Double-stranded DNA-dependent protein kinase (DNA-PK). ataxia telangiectasia mutated (ATM) gene product, Integrin-linked kinase (ILK), Protein Kinase Ca (PKCa), and mammalian target of Rapamycin (mTOR) when complexed to rapamycin insensitive companion of mTOR (RICTOR) have all been identified as playing a critical role in Akt-Ser473 phosphorylation. However, the apparently disparate results reported in these studies are difficult to evaluate, given that different stimuli and cell types were examined and that all of the candidate proteins have never been systematically studied in a single system. Additionally, none of these studies were performed in a classical insulin-responsive cell-type or tissue such as muscle or fat. We therefore examined each of these candidates in 3T3-L1 adipocytes. In vitro kinase assays, using different subcellular fractions of 3T3-L1 adipocytes, revealed that phosphatidylinositol 3,4,5-trisphosphate (PIP3)-stimulated Ser473 phosphorylation correlated well with the amount of DNA-PK, mTOR, and RICTOR but did not correlate with levels of ATM, ILK, and PKCa. PKCa was completely absent from compartments with Ser473 phosphorylation activity. Although purified DNA-PK could phosphorylate a peptide derived from Akt that contains amino acid Ser473, it could not phosphorylate full-length Akt2. Vesicles immunoprecipitated from low-density microsomes (LDM) using antibodies directed against mTOR or RICTOR had PIP3-stimulated Ser473 activity that was sensitive to wortmannin but not staurosporine. In contrast, immunopurified LDM vesicles containing ILK could not phosphorylate Akt on Ser473 in vitro. Small interference RNA (siRNA)-knockdown of RICTOR, but not DNA-PK, ATM, or ILK, suppressed insulin-activated Ser473 phosphorylation and to a lesser extent Thr308 phosphorylation in 3T3-L1 adipocytes. Based on our cell-free kinase and siRNA results, we conclude mTOR complexed to RICTOR is the Ser473 kinase in 3T3-L1 adipocytes.

 

Lycopene induces apoptosis in immortalized fibroblasts exposed to tobacco smoke condensate through arresting cell cycle and down-regulating cyclin D1, pAKT and pBad.

 

 

 

 

 

 

 



Palozza P, Sheriff A, Serini S, Boninsegna A, Maggiano N, Ranelletti FO, Calviello G, Cittadini A.

 

 

 



Institute of General Pathology, Catholic University, Rome.

There is a lot of interest in the health benefits of dietary carotenoids and on the relationship of these compounds with smoke. In particular, it is unknown if the enhanced cancer risk observed in smokers following beta-carotene supplementation can be also found using other carotenoids. Here, we studied the effects of the tomato carotenoid lycopene on molecular pathways involved in cell cycle progression, apoptosis and survival in immortalized RAT-1 fibroblasts exposed to cigarette smoke condensate (TAR). Lycopene (0.5-2.0 muM) inhibited cell growth in a dose-and time-dependent manner, by arresting cell cycle progression and by promoting apoptosis in cells exposed to TAR. The arrest of cell cycle was independent of p53 and of 8-OH-dG DNA damage and related to a decreased expression of cyclin D1. Moreover, the carotenoid up-regulated apoptosis and down-regulated the phosphorylation of AKT and Bad in cells exposed to TAR. Such an effect was associated to an inhibition of TAR-induced expression of Cox-2 and hsp90, which is known to maintain AKT activity. This study suggests that lycopene, differently from beta-carotene, can exert protective effects against cigarette smoke condensate.

PMID: 16215689 [PubMed - as supplied

 

 

conclude mTOR complexed to RICTOR is the Ser473 kinase in 3T3-L1 adipocytes.

PMID: 16221682 [PubMed - as supplied by publisher]

 

 

 

 

 

 

Ovarian follicle development is dependent on growth factors that stimulate cell proliferation and act as survival factors to prevent apoptosis of follicle cells. We examined the mechanism of the protective effect of IGF-I against Fas ligand (FasL)-induced apoptosis of granulosa cells and its relationship to cell proliferation. IGF-I activated both the phosphoinositide 3'-OH kinase (PI3K) and the mitogen-activated protein kinase (MAPK) pathways. Experiments using specific inhibitors of these pathways showed that protection by IGF-I was mediated by the PI3K pathway and not the MAPK pathway. Recombinant adenoviruses were used to test whether the downstream target of PI3K activation, Akt kinase, was required for protection against apoptosis. Expression of dominant negative Akt (dnAkt) prevented protection by IGF-I while expression of constitutively active Akt (myrAkt) mimicked the effect of IGF-I. Treatment with IGF-I, or expression of myrAkt, increased progression from G0/G1 to S phase of the cell cycle while expression of dnAkt inhibited G0/G1 to S phase progression and prevented the stimulatory effect of IGF-I. We tested whether cell cycle progression was required for protection from apoptosis using the cyclin dependent kinase-2 inhibitor roscovitine, which blocks cells at the G1/S transition. Roscovitine prevented the protective effect of IGF-I and myrAkt expression against apoptosis. Therefore, activation of Akt is not sufficient to protect granulosa cells from apoptosis in the absence of cell cycle progression. In summary, IGF-I protects granulosa cells from apoptosis by activation of the PI3K/Akt pathway. This protective effect can only occur when progression from G1 to S phase of the cell cycle regulated by the PI3K/Akt pathway is unperturbed