Membrane trafficking is regulated in part by small GTP-binding proteins of the ADP-ribosylation factor (Arf) family. Arf function depends on the controlled exchange and hydrolysis of GTP. We have purified and cloned two variants of a 130-kDa phosphatidylinositol 4,5-biphosphate (PIP 2 )-dependent Arf1 GTPase-activating protein (GAP), which we call ASAP1a and ASAP1b. Both contain a pleckstrin homology (PH) domain, a zinc finger similar to that found in another Arf GAP, three ankyrin (ANK) repeats, a proline-rich region with alternative splicing and SH3 binding motifs, eight repeats of the sequence E/DLPPKP, and an SH3 domain. Together, the PH, zinc finger, and ANK repeat regions possess PIP 2 -dependent GAP activity on Arf1 and Arf5, less activity on Arf6, and no detectable activity on Arl2 in vitro. The cDNA for ASAP1 was independently identified in a screen for proteins that interact with the SH3 domain of the tyrosine kinase Src. ASAP1 associates in vitro with the SH3 domains of Src family members and with the Crk adapter protein. ASAP1 coprecipitates with Src from cell lysates and is phosphorylated on tyrosine residues in cells expressing activated Src. Both coimmunoprecipitation and tyrosine phosphorylation depend on the same proline-rich class II Src SH3 binding site required for in vitro association. By directly interacting with both Arfs and tyrosine kinases involved in regulating cell growth and cytoskeletal organization, ASAP1 could coordinate membrane remodeling events with these processes.

Arf family GTP-binding proteins are best characterized as regulators of membrane traffic, but recent studies indicate an additional role in cytoskeletal organization. An Arf GTPase-activating protein of the centaurin ␤ family, ASAP1 (also known as centaurin ␤4), binds Arf and two other known regulators of the actin cytoskeleton, the tyrosine kinase Src and phosphatidylinositol 4,5-bisphosphate. In this paper, we show that ASAP1 localizes to focal adhesions and cycles with focal adhesion proteins when cells are stimulated to move. Overexpression of ASAP1 altered the morphology of focal adhesions and blocked both cell spreading and formation of dorsal ruffles induced by platelet-derived growth factor (PDGF). On the other hand, ASAP1, with a mutation that disrupted GTPase-activating protein activity, had a reduced effect on cell spreading and increased the number of cells forming dorsal ruffles in response to PDGF. These data support a role for an Arf GTPase-activating protein, ASAP1, as a regulator of cytoskeletal remodeling and raise the possibility that the Arf pathway is a target for PDGF signaling.

Gonadotropin-releasing hormone (GnRH) neurons are critical to controlling fertility. In vivo, estradiol can inhibit or stimulate GnRH release depending on concentration and physiological state. We examined rapid, nongenomic effects of estradiol. Whole-cell recordings were made of GnRH neurons in brain slices from ovariectomized mice with ionotropic GABA and glutamate receptors blocked. Estradiol was bath applied and measurements completed within 15 min. Estradiol from high physiological (preovulatory) concentrations (100 pM) to 100 nM enhanced action potential firing, reduced afterhyperpolarizing potential (AHP) and increased slow afterdepolarization amplitudes (ADP), and reduced I AHP and enhanced I ADP . The reduction of I AHP was occluded by previous blockade of calcium-activated potassium channels. These effects were mimicked by an estrogen receptor (ER) ␤-specific agonist and were blocked by the classical receptor antagonist ICI182780. ER␣ or GPR30 agonists had no effect. The acute stimulatory effect of high physiological estradiol on firing rate was dependent on signaling via protein kinase A. In contrast, low physiological levels of estradiol (10 pM) did not affect intrinsic properties. Without blockade of ionotropic GABA and glutamate receptors, however, 10 pM estradiol reduced firing of GnRH neurons; this was mimicked by an ER␣ agonist. ER␣ agonists reduced the frequency of GABA transmission to GnRH neurons; GABA can excite to these cells. In contrast, ER␤ agonists increased GABA transmission and postsynaptic response. These data suggest rapid intrinsic and network modulation of GnRH neurons by estradiol is dependent on both dose and receptor subtype. In cooperation with genomic actions, nongenomic effects may play a role in feedback regulation of GnRH secretion.

Summary Stress granules aid cell survival in response to environmental stressors by acting as sites of translational repression. We report an unanticipated link between stress granules and the Ser/Thr kinase, RSK2. In stressed breast cells endogenous RSK2 co-localizes in granules with TIA-1 and poly-(A) binding protein 1, and the sequestration of RSK2 and TIA-1 exhibits co-dependency. The RSK2 N-terminal kinase domain controls the direct interaction with the prion-related domain of TIA-1. Silencing RSK2 decreases cell survival in response to stress. Mitogen releases RSK2 from the stress granules and permits its nuclear import via a nucleo-cytoplasmic shuttling sequence in the C-terminal domain. Nuclear accumulation is dependent on TIA-1. Surprisingly, nuclear localization of RSK2 is sufficient to enhance proliferation through induction of cyclin D1, in the absence of other active signaling pathways. Hence, RSK2 is a pivotal factor linking the stress response to survival and proliferation.

Both steroids and growth factors stimulate proliferation of steroid-dependent tumor cells, and interaction between these signaling pathways occurs at several levels. Steroid receptors are classified as ligand-activated transcription factors, and steps by which they activate target gene transcription are well understood. Several steroid responses have now been functionally linked to other intracellular signaling pathways, including c-Src or tyrosine kinase receptors. Steroids such as 17β-estradiol (E2), via binding to cytoplasmic or membrane-associated receptors, were also shown to rapidly activate intracellular signaling cascades such as ERK, PI3K and STATs. These E2-stimulated phosphorylations can then contribute to altered tumor cell function. ER-positive breast cancer cells, in which proliferation is stimulated by E2 and suppressed by antiestrogens, have been of particular interest in dissecting nuclear and cytoplasmic roles of estrogen receptors (ER). In some cell contexts, ER interacts directly with the intracellular tyrosine kinase c-Src and other cytoplasmic signaling and adaptor molecules, such as Shc, PI3K, MNAR, and p130 Cas. Although the hierarchy among these associations is not known, it is clear that c-Src plays a fundamental role in both growth factor and E2-stimulated cell growth, and this may also require other growth factor receptors such as those for EGF or IGF-1. STAT transcription factors represent one pathway to integrate E2 cytoplasmic and nuclear signaling. STAT5 is phosphorylated in the cytoplasm at an activating tyrosine in response to E2 or EGF, and then is translocated to the nucleus to stimulate target gene transcription. E2 stimulates recruitment of STAT5 and ER to the promoter of several proliferative genes, and STAT5 knockdown prevents recruitment of either protein to these promoters. STAT5 activation by E2 in breast cancer cells requires c-Src and EGF receptor, and inhibition of c-Src or EGFR, or knock-down of STAT5, prevents E2 stimulation of several genes and breast cancer cell proliferation. Hyperactivation of the growth factor receptor-c-Src pathway can in some contexts decrease growth responses to E2, or render cells and tumors resistant to suppressive actions of endocrine therapies. Crosstalk between growth factors and steroids in both the cytoplasm and nucleus may thus have a profound impact on complex biological processes such as cell growth, and may play a significant role in the treatment of steroid-dependent breast cancers.

The N-terminal BAR domain of ASAP1 mediates membrane bending and is necessary for ASAP1 function. The Arf dependence of the bending activity is consistent with ASAP1 functioning as an Arf effector.

The Ser/Thr kinase family, RSK, has been implicated in numerous types of hormone-dependent and -independent cancers. However, there has been little consideration of RSKs as downstream mediators of steroid hormone non-genomic effects or of their ability to facilitate steroid receptor-mediated gene expression. Steroid hormone signaling can directly stimulate the MEK/ERK/RSK pathway to regulate cellular proliferation and survival in transformed cells. To date, multiple mechanisms of RSK and steroid hormone receptor-mediated proliferation/survival have been elucidated. For example, RSK enhances proliferation of breast and prostate cancer cells via its ability to control the levels of the estrogen receptor co-activator, cyclin D1. While in lung and other tumors RSK may control apoptosis via estrogen-mediated regulation of mitochondrial integrity. Thus the RSKs could be important anti-cancer therapeutic targets in many different transformed tissues. The recent discovery of RSK-specific inhibitors will advance our current understanding of RSK in transformation and drive these studies into animal and clinical models. In this review we explore the mechanisms associated with RSK in tumorigenesis and their relationship to steroid hormone signaling.

We have reported that p22, an N-myristoylated EF-hand Ca(2+)-binding protein, associates with microtubules and plays a role in membrane trafficking. Here, we show that p22 also associates with membranes of the early secretory pathway membranes, in particular endoplasmic reticulum (ER). On binding of Ca(2+), p22's ability to associate with membranes increases in an N-myristoylation-dependent manner, which is suggestive of a nonclassical Ca(2+)-myristoyl switch mechanism. To address the intracellular functions of p22, a digitonin-based "bulk microinjection" assay was developed to load cells with anti-p22, wild-type, or mutant p22 proteins. Antibodies against a p22 peptide induce microtubule depolymerization and ER fragmentation; this antibody-mediated effect is overcome by preincubation with the respective p22 peptide. In contrast, N-myristoylated p22 induces the formation of microtubule bundles, the accumulation of ER structures along the bundles as well as an increase in ER network formation. An N-myristoylated Ca(2+)-binding p22 mutant, which is unable to undergo Ca(2+)-mediated conformational changes, induces microtubule bundling and accumulation of ER structures along the bundles but does not increase ER network formation. Together, these data strongly suggest that p22 modulates the organization and dynamics of microtubule cytoskeleton in a Ca(2+)-independent manner and affects ER network assembly in a Ca(2+)-dependent manner.