PI3K regulation of the SKP-2/p27 axis through mTORC2.

The cyclin-dependent kinase inhibitor p27 is a key regulator of cell-cycle progression. Its expression and localization are altered in several types of malignancies, which has prognostic significance in cancers such as renal cell carcinoma (RCC). S-phase kinase-associated protein 2 (SKP-2) is an F-box protein that is part of the SKP-1/Cul1/F-box ubiquitin ligase complex that targets nuclear p27 among many other cell-cycle proteins for proteosomal degradation. Its overexpression has been observed in several tumor types. Signaling by phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) has previously been shown to regulate the SKP-2/p27 axis. Recent evidence suggests that PI3K signaling may activate mammalian target of rapamycin complex 2 (mTORC2) activity. As PI3K signaling is known to regulate SKP-2 and p27, we sought to determine whether these effects were mediated by mTORC2. Here we provide additional genetic evidence that PI3K signaling activates mTORC2 kinase activity. We also demonstrate a novel role for mTORC2 in the modulation of nuclear p27 levels. In particular, mTORC2 signaling promotes the reduction of nuclear p27 protein levels through the increased protein expression of SKP-2. These are the first data to demonstrate a role for mTOR in the regulation of SKP-2. In concordance with these findings, mTORC2 activity promotes cell proliferation of RCC cells at the G1-S interphase of the cell cycle. Collectively, these data implicate mTORC2 signaling in the regulation of the SKP-2/p27 axis, a signaling node commonly altered in cancer.

Recovery of developmentally defined gene sets from high-density cDNA macroarrays.

New technologies for isolating differentially expressed genes from large arrayed cDNA libraries are reported. These methods can be used to identify genes that lie downstream of developmentally important transcription factors and genes that are expressed in specific tissues, processes, or stages of embryonic development. Though developed for the study of gene expression during the early embryogenesis of the sea urchin Strongylocentrotus purpuratus, these technologies can be applied generally. Hybridization parameters were determined for the reaction of complex cDNA probes to cDNA libraries carried on six nylon filters, each containing duplicate spots from 18,432 bacterial clones (macroarrays). These libraries are of sufficient size to include nearly all genes expressed in the embryo. The screening strategy we have devised is designed to overcome inherent sensitivity limitations of macroarray hybridization and thus to isolate differentially expressed genes that are represented only by low-prevalence mRNAs. To this end, we have developed improved methods for the amplification of cDNA from small amounts of tissue (as little as approximately 300 sea urchin embryos, or 2 x 10(5) cells, or about 10 ng of mRNA) and for the differential enhancement of probe sequence concentration by subtractive hybridization. Quantitative analysis of macroarray hybridization shows that these probes now suffice for detection of differentially expressed mRNAs down to a level below five molecules per average embryo cell.

Effect of guanine nucleotides on [3H]glutamate binding and on adenylate cyclase activity in rat brain membranes.

GMP-PNP, a non-hydrolyzable analog of GTP binds tightly to G-protein in the presence of Mg2+, so that the binding is stable even after exhaustive washings. This property was exploited to prepare membrane samples of rat brain where G-protein GTP-binding sites were saturated with GMP-PNP. Experiments carried out with these membranes showed that GTP, GMP-PNP, GDP-S and GMP (1 mM) inhibit the sodium-independent [3H]glutamate binding by 30-40% [F(4,40) = 5.9; p < .001], whereas only GMP-PNP activates adenylate cyclase activity [F(6,42) = 3.56; p < .01]. The inhibition of sodium-independent [3H]glutamate binding occurred in the absence of Mg2+. These findings suggest that guanine nucleotides may inhibit glutamate binding and activate adenylate cyclase through distinct mechanisms by acting on different sites.