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(1/1211) Intracellular signalling: PDK1--a kinase at the hub of things.

Phosphoinositide-dependent kinase 1 (PDK1) is at the hub of many signalling pathways, activating PKB and PKC isoenzymes, as well as p70 S6 kinase and perhaps PKA. PDK1 action is determined by colocalization with substrate and by target site availability, features that may enable it to operate in both resting and stimulated cells.  (+info)

(2/1211) Rapamycin causes poorly reversible inhibition of mTOR and induces p53-independent apoptosis in human rhabdomyosarcoma cells.

The mammalian target of rapamycin (mTOR) has been shown to link growth factor signaling and posttranscriptional control of translation of proteins that are frequently involved in cell cycle progression. However, the role of this pathway in cell survival has not been demonstrated. Here, we report that rapamycin, a specific inhibitor of mTOR kinase, induces G1 cell cycle arrest and apoptosis in two rhabdomyosarcoma cell lines (Rh1 and Rh30) under conditions of autocrine cell growth. To examine the kinetics of rapamycin action, we next determined the rapamycin sensitivity of rhabdomyosarcoma cells exposed briefly (1 h) or continuously (6 days). Results demonstrate that Rh1 and Rh30 cells were equally sensitive to rapamycin-induced growth arrest and apoptosis under either condition. Apoptosis was detected between 24 and 144 h of exposure to rapamycin. Both cell lines have mutant p53; hence, rapamycin-induced apoptosis appears to be a p53-independent process. To determine whether induction of apoptosis by rapamycin was specifically due to inhibition of mTOR signaling, we engineered Rh1 and Rh30 clones to stably express a mutant form of mTOR that was resistant to rapamycin (Ser2035-->Ile; designated mTOR-rr). Rh1 and Rh30 mTOR-rr clones were highly resistant (>3000-fold) to both growth inhibition and apoptosis induced by rapamycin. These results are the first to indicate that rapamycin-induced apoptosis is mediated by inhibition of mTOR. Exogenous insulin-like growth factor (IGF)-I protected both Rh1 and Rh30 from apoptosis, without reactivating ribosomal p70 S6 kinase (p70S6K) downstream of mTOR. However, in rapamycin-treated cultures, the response to IGF-I differed between the cell lines: Rh1 cells proliferated normally, whereas Rh30 cells remained arrested in G1 phase but viable. Rapamycin is known to inhibit synthesis of specific proteins but did not inhibit synthesis or alter the levels of mTOR. To examine the rate at which the mTOR pathway recovered, the ability of IGF-I to stimulate p70S6K activity was followed in cells treated for 1 h with rapamycin and then allowed to recover in medium containing > or =100-fold excess of FK506 (to prevent rapamycin from rebinding to its cytosolic receptor FKBP-12). Our results indicate that, in Rh1 cells, rapamycin dissociates relatively slowly from FKBP-12, with a t1/2 of approximately 17.5 h. in the presence of FK506, whereas there was no recovery of p70S6K activity in the absence of this competitor. This was of interest because rapamycin was relatively unstable under conditions of cell culture having a biological t1/2 of approximately 9.9 h. These results help to explain why cells are sensitive following short exposures to rapamycin and may be useful in guiding the use of rapamycin analogues that are entering clinical trials as novel antitumor agents.  (+info)

(3/1211) Inhibition of cell cycle progression by rapamycin induces T cell clonal anergy even in the presence of costimulation.

Costimulation (signal 2) has been proposed to inhibit the induction of T cell clonal anergy by either directly antagonizing negative signals arising from TCR engagement (signal 1) or by synergizing with signal 1 to produce IL-2, which in turn leads to proliferation and dilution of negative regulatory factors. To better define the cellular events that lead to the induction of anergy, we used the immunosuppressive agent rapamycin, which blocks T cell proliferation in late G1 phase but does not affect costimulation-dependent IL-2 production. Our data demonstrate that full T cell activation (signal 1 plus 2) in the presence of rapamycin results in profound T cell anergy, despite the fact that these cells produce copious amounts of IL-2. Similar to conventional anergy (induction by signal 1 alone), the rapamycin-induced anergic cells show a decrease in mitogen-activated protein kinase activation, and these cells can be rescued by culture in IL-2. Interestingly, the rapamycin-induced anergic cells display a more profound block in IL-3 and IFN-gamma production upon rechallenge. Finally, in contrast to rapamycin, full T cell activation in the presence of hydroxyurea (which inhibits the cell cycle in early S phase) did not result in anergy. These data suggest that it is neither the direct effect of costimulation nor the subsequent T cell proliferation that prevents anergy induction, but rather the biochemical events that occur upon progression through the cell cycle from G1 into S phase.  (+info)

(4/1211) A MAP kinase docking site is required for phosphorylation and activation of p90(rsk)/MAPKAP kinase-1.

Activation of the various mitogen-activated protein (MAP) kinase pathways converts many different extracellular stimuli into specific cellular responses by inducing the phosphorylation of particular groups of substrates. One important determinant for substrate specificity is likely to be the amino-acid sequence surrounding the phosphorylation site; however, these sites overlap significantly between different MAP kinase family members. The idea is now emerging that specific docking sites for protein kinases are involved in the efficient binding and phosphorylation of some substrates [1] [2] [3] [4]. The MAP kinase-activated protein (MAPKAP) kinase p90 rsk contains two kinase domains [5]: the amino-terminal domain (D1) is required for the phosphorylation of exogenous substrates whereas the carboxy-terminal domain (D2) is involved in autophosphorylation. Association between the extracellular signal-regulated kinase (Erk) MAP kinases and p90(rsk) family members has been detected in various cell types including Xenopus oocytes [6] [7] [8], where inactive p90(rsk) is bound to the inactive form of the Erk2- like MAP kinase p42(mpk1). Here, we identify a new MAP kinase docking site located at the carboxyl terminus of p90(rsk). This docking site was required for the efficient phosphorylation and activation of p90(rsk) in vitro and in vivo and was also both necessary and sufficient for the stable and specific association with p42(mpk1). The sequence of the docking site was conserved in other MAPKAP kinases, suggesting that it might represent a new class of interaction motif that facilitates efficient and specific signal transduction by MAP kinases.  (+info)

(5/1211) p70(S6K) controls selective mRNA translation during oocyte maturation and early embryogenesis in Xenopus laevis.

In mammalian cells, p70(S6K) plays a key role in translational control of cell proliferation in response to growth factors. Because of the reliance on translational control in early vertebrate development, we cloned a Xenopus homolog of p70(S6K) and investigated the activity profile of p70(S6K) during Xenopus oocyte maturation and early embryogenesis. p70(S6K) activity is high in resting oocytes and decreases to background levels upon stimulation of maturation with progesterone. During embryonic development, three peaks of activity were observed: immediately after fertilization, shortly before the midblastula transition, and during gastrulation. Rapamycin, an inhibitor of p70(S6K) activation, caused oocytes to undergo germinal vesicle breakdown earlier than control oocytes, and sensitivity to progesterone was increased. Injection of a rapamycin-insensitive, constitutively active mutant of p70(S6K) reversed the effects of rapamycin. However, increases in S6 phosphorylation were not significantly affected by rapamycin during maturation. mos mRNA, which does not contain a 5'-terminal oligopyrimidine tract (5'-TOP), was translated earlier, and a larger amount of Mos protein was produced in rapamycin-treated oocytes. In fertilized eggs rapamycin treatment increased the translation of the Cdc25A phosphatase, which lacks a 5'-TOP. Translation assays in vivo using both DNA and RNA reporter constructs with the 5'-TOP from elongation factor 2 showed decreased translational activity with rapamycin, whereas constructs without a 5'-TOP or with an internal ribosome entry site were translated more efficiently upon rapamycin treatment. These results suggest that changes in p70(S6K) activity during oocyte maturation and early embryogenesis selectively alter the translational capacity available for mRNAs lacking a 5'-TOP region.  (+info)

(6/1211) p70 S6 kinase is regulated by protein kinase Czeta and participates in a phosphoinositide 3-kinase-regulated signalling complex.

p70 S6 kinase (p70S6K) is an important regulator of cell proliferation. Its activation by growth factor requires phosphorylation by various inputs on multiple sites. Data accumulated thus far support a model whereby p70S6K activation requires sequential phosphorylations at proline-directed residues in the putative autoinhibitory pseudosubstrate domain, as well as threonine 389. Threonine 229, a site in the catalytic loop is phosphorylated by phosphoinositide-dependent kinase 1 (PDK-1). Experimental evidence suggests that p70S6K activation requires a phosphoinositide 3-kinase (PI3-K)-dependent signal(s). However, the intermediates between PI3-K and p70S6K remain unclear. Here, we have identified PI3-K-regulated atypical protein kinase C (PKC) isoform PKCzeta as an upstream regulator of p70S6K. In coexpression experiments, we found that a kinase-inactive PKCzeta mutant antagonized activation of p70S6K by epidermal growth factor, PDK-1, and activated Cdc42 and PI3-K. While overexpression of a constitutively active PKCzeta mutant (myristoylated PKCzeta [myr-PKCzeta]) only modestly activated p70S6K, this mutant cooperated with PDK-1 activation of p70S6K. PDK-1-induced activation of a C-terminal truncation mutant of p70S6K was also enhanced by myr-PKCzeta. Moreover, we have found that p70S6K can associate with both PDK-1 and PKCzeta in vivo in a growth factor-independent manner, while PDK-1 and PKCzeta can also associate with each other, suggesting the existence of a multimeric PI3-K signalling complex. This work provides evidence for a link between a phorbol ester-insensitive PKC isoform and p70S6K. The existence of a PI3-K-dependent signalling complex may enable efficient activation of p70S6K in cells.  (+info)

(7/1211) Growth hormone-dependent differentiation of 3T3-F442A preadipocytes requires Janus kinase/signal transducer and activator of transcription but not mitogen-activated protein kinase or p70 S6 kinase signaling.

The signals mediating growth hormone (GH)-dependent differentiation of 3T3-F442A preadipocytes under serum-free conditions have been studied. GH priming of cells was required before the induction of terminal differentiation by a combination of epidermal growth factor, tri-iodothyronine, and insulin. Cellular depletion of Janus kinase-2 (JAK-2) using antisense oligodeoxynucleotides (ODNs) prevented GH-stimulated JAK-2 and signal transducer and activator of transcription (STAT)-5 tyrosine phosphorylation and severely attenuated the ability of GH to promote differentiation. Although p42(MAPK)/p44(MAPK) mitogen-activated protein kinases were activated during GH priming, treatment of cells with PD 098059, which prevented activation of these kinases, did not block GH priming. However, antisense ODN-mediated depletion of mitogen-activated protein kinases from the cells showed that their expression was necessary for terminal differentiation. Similarly, although p70(s6k) was activated during GH priming, pretreatment of cells with rapamycin, which prevented the activation of p70(s6k), had no effect on GH priming. However, rapamycin did partially block epidermal growth factor, tri-iodothyronine, and insulin-stimulated terminal differentiation. By contrast, cellular depletion of STAT-5 with antisense ODNs completely abolished the ability of GH to promote differentiation. These results indicate that JAK-2, acting specifically via STAT-5, is necessary for GH-dependent differentiation of 3T3-F442A preadipocytes. Activation of p42(MAPK)/p44(MAPK) and p70(s6k) is not essential for the promotion of differentiation by GH, although these signals are required for GH-independent terminal differentiation.  (+info)

(8/1211) Suboptimal cross-linking of antigen receptor induces Syk-dependent activation of p70S6 kinase through protein kinase C and phosphoinositol 3-kinase.

Ligation of the B cell antigen receptor (BCR) induces a cascade of signaling pathways that lead to clonal expansion, differentiation, or abortive activation-induced apoptosis of B lymphocytes. BCR-mediated cross-linking induces the rapid phosphorylation of protein tyrosine kinases. However, the pathways leading to the activation of downstream serine/threonine kinases such as mitogen-activated protein kinase, p90(Rsk), and p70S6 kinase (p70(S6k)) that mediate reorganization of the actin cytoskeleton, cell cycle progression, gene transcription, and protein synthesis have not been delineated. We recently demonstrated that cross-linking of BCR leads to activation of p70(S6k) in B lymphocytes. In this report, we demonstrate that multiple protein tyrosine kinase-dependent signal transduction pathways induced by BCR lead to the activation of p70(S6k). These distinct pathways exhibit different thresholds with respect to the extent of receptor cross-linking required for their activation. Activation of p70(S6k) by suboptimal doses of anti-Ig is Syk-dependent and is mediated by protein kinase C and phosphoinositol 3-kinase. Moreover, the activation of p70(S6k) results in phosphorylation of S6 protein which is important for ribosomal protein synthesis and may be coupled to BCR-induced protein and DNA synthesis in primary murine B cells.  (+info)