Active nuclear import of protein is controlled by nuclear localization signals (NLSs), but nuclear export is not understood well. Nuclear trafficking of the catalytic (C) subunit of cAMP-dependent protein kinase (cAPK) is critical for regulation of gene expression. The heat-stable inhibitor (PKl) of cAPK contains a nuclear export signal (NES) that triggers rapid, active net extrusion of the C-PKl complex from the nucleus. This NES (residues 35-49), fused or conjugated to heterologous proteins, was sufficient for rapid nuclear export. Hydrophobic residues were critical. The NES is a slightly weaker signal than the SV40 NLS. A sequence containing only residues 37-46, LALKLAGLDI, is also sufficient for nuclear export. This is an example of a protein-based NES having no obvious association with RNA. A similar sequence, LQLPPLERLTL, from Rev, an RNA-binding protein of HIV-1, also is an NES.

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Phosphoinositide 3 kinase/Akt pathway plays an essential role in neuronal survival. However, the cellular mechanisms by which Akt suppresses cell death and protects neurons from apoptosis remain unclear. We previously showed that transient expression of constitutively active Akt inhibits ceramide-induced death of hybrid motor neuron 1 cells. Here we show that stable expression of either constitutively active Akt or Bcl-2 inhibits apoptosis, but only Bcl-2 prevents the release of cytochrome c from mitochondria, suggesting that Akt regulates apoptosis at a postmitochondrial level. Consistent with this, overexpressing active Akt rescues cells from apoptosis without altering expression levels of endogenous Bcl-2, Bcl-x, or Bax. Akt inhibits apoptosis induced by microinjection of cytochrome c and lysates from cells expressing active Akt inhibit cytochrome c induced caspase activation in a cell-free assay while lysates from Bcl-2–expressing cells have no effect. Addition of cytochrome c and dATP to lysates from cells expressing active Akt do not activate caspase-9 or -3 and immunoprecipitated Akt added to control lysates blocks cytochrome c–induced activation of the caspase cascade. Taken together, these data suggest that Akt inhibits activation of caspase-9 and -3 by posttranslational modification of a cytosolic factor downstream of cytochrome c and before activation of caspase-9.

Valproic acid (VPA), a well-established therapy for seizures and bipolar disorder, has recently been shown to inhibit histone deacetylases (HDACs). Similar to more widely studied HDAC inhibitors, VPA can cause growth arrest and induce differentiation of transformed cells in culture. Whether this effect of VPA is through inhibition of HDACs or modulation of another target of VPA has not been tested. We have used a series of VPA analogs to establish a pharmacological profile for HDAC inhibition. We find that VPA and its analogs inhibit multiple HDACs from class I and class II (but not HDAC6 or HDAC10) with a characteristic order of potency in vitro. These analogs also induce hyperacetylation of core histones H3 and H4 in intact cells with an order of potency that parallels in vitro inhibition. VPA and VPA analogs induce differentiation in hematopoietic cell lines in a p21-dependent manner, and the order of potency for induction of differentiation parallels the potencies for inhibition in vitro, as well as for acetylation of histones associated with the p21 promoter, supporting the argument that differentiation caused by VPA is mediated through inhibition of HDACs. These findings provide additional evidence that VPA, a well-tolerated, orally administered drug with extensive clinical experience, may serve as an effective chemotherapeutic agent through targeting of HDACs.

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Abstract. Rabl is a small GTPase regulating vesicular traffic between early compartments of the secretory pathway. To explore the role of rabl we have analyzed the function of a mutant (rabla[S25N]) containing a substitution which perturbs Mg 2+ coordination and reduces the affinity for GTE resulting in a form which is likely to be restricted to the GDP-bound state. The rabla(S25N) mutant led to a marked reduction in protein export from the ER in vivo and in vitro, indicating that a guanine nucleotide exchange protein (GEP) is critical for the recruitment of rabl during vesicle budding. The mutant protein required posttranslational isoprenylation for inhibition and behaved as a competitive inhibitor of wild-type rabl function. Both rabla and rablb (92% identity) were able to antagonize the inhibitory activity of the rabla(S25N) mutant, suggesting that these two isoforms are functionally interchangeable. The rabl mutant also inhibited transport between Golgi compartments and resulted in an apparent loss of the Golgi apparatus, suggesting that Golgi integrity is coupled to rabl function in vesicular traffic.

The effects of cyclic AMP (cAMP) on cell proliferation are cell type specific. Although the growth-inhibitory effects of cAMP have been well studied, much less is known regarding how cAMP stimulates proliferation. We report that cAMP stimulates proliferation through both protein kinase A (PKA)-dependent and PKA-independent signaling pathways and that phosphatidylinositol 3-kinase (PI3K) is required for cAMP-stimulated mitogenesis. In cells where cAMP is a mitogen, cAMP-elevating agents stimulate membrane ruffling, Akt phosphorylation, and p70 ribosomal S6 protein kinase (p70s6k) activity. cAMP effects on ruffle formation and Akt were PKA independent but sensitive to wortmannin. In contrast, cAMP-stimulated p70s6k activity was repressed by PKA inhibitors but not by wortmannin or microinjection of the N-terminal SH2 domain of the p85 regulatory subunit of PI3K, indicating that p70s6k and Akt can be regulated independently. Microinjection of highly specific inhibitors of PI3K or Rac1, or treatment with the p70s6k inhibitor rapamycin, impaired cAMP-stimulated DNA synthesis, demonstrating that PKA-dependent and -independent pathways contribute to cAMP-mediated mitogenesis. Direct elevation of PI3K activity through microinjection of an antibody that stimulates PI3K activity or stable expression of membrane-localized p110 was sufficient to confer hormoneindependent DNA synthesis when accompanied by elevations in p70s6k activity. These findings indicate that multiple pathways contribute to cAMP-stimulated mitogenesis, only some of which are PKA dependent. Furthermore, they demonstrate that the ability of cAMP to stimulate both p70s6k-and PI3K-dependent pathways is an important facet of cAMP-regulated cell cycle progression.Cyclic AMP (cAMP) exerts differential effects on cell proliferation. In many cells, including CHO cells, aortic smooth muscle cells, and Rat-1 fibroblasts, cAMP inhibits the mitogenic response to growth factors (8). Growth-inhibitory effects of cAMP are mediated partly through activation of cAMPdependent protein kinase A (PKA), which interferes with Raf activation and signaling (23). Less is known regarding how cAMP stimulates growth, although accumulating evidence has dissociated the mitogenic effects of cAMP from effects on mitogen-activated protein kinase (MAPK) (37,42,76). In contrast, the effects of cAMP on p70s6k correlate with effects on proliferation, and inhibition of p70s6k activation abolishes cAMP-stimulated DNA synthesis (10). These results prompted us to examine the role of phosphatidylinositol 3-kinase (PI3K)-dependent signaling pathways in cAMP-stimulated proliferation.Multiple isoforms of PI3K that vary in lipid substrate specificity and subunit structure have been identified (reviewed in reference 71). Typically, mitogens that activate receptor tyrosine kinases stimulate PI3K␣/␤ whereas those that activate G-protein-coupled receptors stimulate PI3K␥, although exceptions have been noted (36,52,66,67). PI3K is required for the mitogenic activity of many growth factors, including platel...

Summary The specific molecular events that characterize the intrinsic apoptosis pathway have been the subject of intense research due to its fundamental role in development, homeostasis, and cancer. This pathway is defined by the release of cytochrome c from mitochondria into the cytosol, and subsequent binding of cytochrome c to the caspase activator Apaf-1. Here we report that both mitochondrial and cytosolic transfer RNA (tRNA) bind to cytochrome c. This binding prevents cytochrome c interaction with Apaf-1, blocking Apaf-1 oligomerization and caspase activation. tRNA hydrolysis in living cells and cell lysates enhances apoptosis and caspase activation, while microinjection of tRNA into living cells blocks apoptosis. These findings suggest that tRNA, in addition to its well-established role in gene expression, may determine cellular responsiveness to apoptotic stimuli.