The Ras binary switch: an ideal processor for decoding complex Ca2+ signals?

Activation of cell-surface receptors often leads to changes in intracellular calcium concentration ([Ca(2+)](i)). Receptor-generated calcium transients are often seen as repetitive spikes of elevated intracellular calcium concentration ([Ca(2+)](i)), whose frequency varies according to the amplitude of the receptor stimuli. This suggests a requirement for molecular decoders, capable of interpreting such complex calcium signals into the correct physiological response. Ras proteins are binary molecular switches controlling a plethora of cellular responses. Whether Ras is in its active GTP-bound, or inactive GDP-bound, form is determined by the activity of guanine nucleotide exchange factors (GEFs) and GTPase-activating protein (GAPs). Calcium-regulated GEFs and GAPs have been identified, some with an exquisite sensitivity to [Ca(2+)](i), implicating a potential role of complex calcium signals in regulating Ras.

Curcumin, a molecule that inhibits the Ca2+-ATPase of sarcoplasmic reticulum but increases the rate of accumulation of Ca2+.

Curcumin, an important inhibitor of carcinogenesis, is an inhibitor of the ATPase activity of the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum (SR). Inhibition by curcumin is structurally specific, requiring the presence of a pair of -OH groups at the 4-position of the rings. Inhibition is not competitive with ATP. Unexpectedly, addition of curcumin to SR vesicles leads to an increase in the rate of accumulation of Ca(2+), unlike other inhibitors of the Ca(2+)-ATPase that result in a reduced rate of accumulation. An increase in the rate of accumulation of Ca(2+) is seen in the presence of phosphate ion, which lowers the concentration of free Ca(2+) within the lumen of the SR, showing that the effect is not passive leak across the SR membrane. Rather, simulations suggest that the effect is to reduce the rate of slippage on the ATPase, a process in which a Ca(2+)-bound, phosphorylated intermediate releases its bound Ca(2+) on the cytoplasmic rather than on the lumenal side of the membrane. The structural specificity of the effects of curcumin on ATPase activity and on Ca(2+) accumulation is the same, and the apparent dissociation constants for the two effects are similar, suggesting that the two effects of curcumin could follow from binding to a single site on the ATPase.

Tumour necrosis factor-alpha activation of protein kinase B in WEHI-164 cells is accompanied by increased phosphorylation of Ser473, but not Thr308.

Tumour necrosis factor-alpha (TNF-alpha) may activate both cell survival and cell death pathways. In the murine fibrosarcoma cell line WEHI-164, physiological concentrations (1 ng/ml) of TNF-alpha induced wortmannin-sensitive cell ruffling characteristic of the phosphoinositide 3-kinase (PI3-kinase) activation associated with cell survival. Wortmannin also enhanced cell death induced by TNF-alpha in the presence of actinomycin D, confirming that TNF-alpha activates a transcription-independent survival pathway requiring PI3-kinase activity. Both TNF-alpha and insulin-like growth factor 1 (IGF-1) caused a 6-10-fold wortmannin-sensitive increase in protein kinase B (PKB) activity within 5 min. For IGF-1, this was associated with an increase in phosphorylation of both Thr(308) and Ser(473), whereas for TNF-alpha only phosphorylation of Ser(473) was increased, even in the presence of okadaic acid to inhibit protein phosphatases 1 and 2A. TNF-alpha did not decrease the phosphorylation of Thr(308) induced by IGF-1, implying that TNF-alpha neither inhibits phosphoinositide-dependent kinase 1 (PDK1) nor activates an opposing phosphatase. In WEHI cells overexpressing a form of PKB, IGF-1 increased phosphorylation of Ser(473) on PKB, but not its kinase activity, whereas TNF-alpha failed to induce Ser(473) phosphorylation or kinase activation of either overexpressed T308A or wild-type PKB (where T308A is the mutant bearing the substitution Thr(308)-->A). IGF-1 caused translocation of green-fluorescent-protein-tagged ADP-ribosylation factor nucleotide-binding site opener (ARNO) to the plasma membrane of WEHI cells, but this was not detected with TNF-alpha. We conclude that, at physiological concentrations, TNF-alpha activates endogenous PKB by stimulating PDK2 (increase in Ser(473) phosphorylation) in a PI3-kinase-dependent (wortmannin-sensitive) manner, without causing detectable stimulation of PDK1 (no increase in Thr(308) phosphorylation) or ARNO translocation. Possible explanations of these observations are discussed.

CAPRI regulates Ca(2+)-dependent inactivation of the Ras-MAPK pathway.

Ca(2+) is a universal second messenger that is critical for cell growth and is intimately associated with many Ras-dependent cellular processes such as proliferation and differentiation. Ras is a small GTP binding protein that operates as a molecular switch regulating the control of gene expression, cell growth, and differentiation through a pathway from receptors to mitogen-activated protein kinases (MAPKs). A role for intracellular Ca(2+) in the activation of Ras has been previously demonstrated, e.g., via the nonreceptor tyrosine kinase PYK2 and by Ca(2+)/calmodulin-dependent guanine nucleotide exchange factors (GEFs) such as Ras-GRF; however, there is no Ca(2+)-dependent mechanism for direct inactivation. An important advance toward greater understanding of the complex coordination within the Ras-signaling network is the spatio-temporal analysis of signaling events in vivo. Here, we describe the identification of CAPRI (Ca(2+)-promoted Ras inactivator), a Ca(2+)-dependent Ras GTPase-activating protein (GAP) that switches off the Ras-MAPK pathway following a stimulus that elevates intracellular Ca(2+). Analysis of the spatio-temporal dynamics of CAPRI indicates that Ca(2+) regulates the GAP by a fast C2 domain-dependent translocation mechanism.

GAP1IP4BP contains a novel group I pleckstrin homology domain that directs constitutive plasma membrane association.

The group I family of pleckstrin homology (PH) domains are characterized by their inherent ability to specifically bind phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) and its corresponding inositol head-group inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P(4)). In vivo this interaction results in the regulated plasma membrane recruitment of cytosolic group I PH domain-containing proteins following agonist-stimulated PtdIns(3,4,5)P(3) production. Among group I PH domain-containing proteins, the Ras GTPase-activating protein GAP1(IP4BP) is unique in being constitutively associated with the plasma membrane. Here we show that, although the GAP1(IP4BP) PH domain interacts with PtdIns(3,4, 5)P(3), it also binds, with a comparable affinity, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) (K(d) values of 0.5 +/- 0.2 and 0.8 +/- 0.5 microm, respectively). Intriguingly, whereas this binding site overlaps with that for Ins(1,3,4,5)P(4), consistent with the constitutive plasma membrane association of GAP1(IP4BP) resulting from its PH domain-binding PtdIns(4,5)P(2), we show that in vivo depletion of PtdIns(4,5)P(2), but not PtdIns(3,4,5)P(3), results in dissociation of GAP1(IP4BP) from this membrane. Thus, the Ins(1,3,4,5)P(4)-binding PH domain from GAP1(IP4BP) defines a novel class of group I PH domains that constitutively targets the protein to the plasma membrane and may allow GAP1(IP4BP) to be regulated in vivo by Ins(1,3,4,5)P(4) rather than PtdIns(3,4,5)P(3).

Tissue-specific expression and endogenous subcellular distribution of the inositol 1,3,4,5-tetrakisphosphate-binding proteins GAP1(IP4BP) and GAP1(m).

GAP1(IP4BP) and GAP1(m) belong to the GAP1 family of Ras GTPase-activating proteins that are candidate InsP4 receptors. Here we show they are ubiquitously expressed in human tissues and are likely to have tissue-specific splice variants. Analysis by subcellular fractionation of RBL-2H3 rat basophilic leukemia cells confirms that endogenous GAP1(IP4BP) is primarily localised to the plasma membrane, whereas GAP1(m) appears localised to the cytoplasm (cytosol and internal membranes) but not the plasma membrane. Subcellular fractionation did not indicate a specific co-localisation between membrane-bound GAP1(m) and several Ca2+ store markers, consistent with the lack of co-localisation between GAP1(m) and SERCA1 upon co-expression in COS-7 cells. This difference suggests that GAP1(m) does not reside at a site where it could regulate the ability of InsP4 to release intracellular Ca2+. As GAP1(m) is primarily localised to the cytosol of unstimulated cells it may be spatially regulated in order to interact with Ras at the plasma membrane.

Identification of the ras GTPase-activating protein GAP1(m) as a phosphatidylinositol-3,4,5-trisphosphate-binding protein in vivo.

GAP1(m) is a member of the GAP1 family of Ras GTPase-activating proteins (GAPs) [1]. In vitro, it has been shown to bind inositol 1, 3,4,5-tetrakisphosphate (IP4), the water-soluble inositol head group of the lipid second messenger phosphatidylinositol 3,4, 5-trisphosphate (PIP3) [2] [3]. This has led to the suggestion that GAP1(m) might function as a PIP3 receptor in vivo [4]. Here, using rat pheochromocytoma PC12 cells transiently transfected with a plasmid expressing a chimera of green fluorescent protein fused to GAP1(m) (GFP-GAP1(m)), we show that epidermal growth factor (EGF) induces a rapid (less than 60 seconds) recruitment of GFP-GAP1(m) from the cytosol to the plasma membrane. This recruitment required a functional GAP1(m) pleckstrin homology (PH) domain, because a specific point mutation (R629C) in the PH domain that inhibits IP4 binding in vitro [5] totally blocked EGF-induced GAP1(m) translocation. Furthermore, the membrane translocation was dependent on PI 3-kinase, and the time course of translocation paralleled the rate by which EGF stimulates the generation of plasma membrane PIP3 [6]. Significantly, the PIP3-induced recruitment of GAP1(m) did not appear to result in any detectable enhancement in its basal Ras GAP activity. From these results, we conclude that GAP1(m) binds PIP3 in vivo, and it is recruited to the plasma membrane, but does not appear to be activated, following agonist stimulation of PI 3-kinase.

Structural and functional analysis of the putative inositol 1,3,4, 5-tetrakisphosphate receptors GAP1(IP4BP) and GAP1(m).

Previously we have purified and cloned a high affinity isomerically specific inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4)-binding protein which, because it is clearly a member of the GAP1 family of Ras GTPase-activating proteins (GAP), we have termed GAP1(IP4BP). Here we show that expressed full-length GAP1(IP4BP) binds Ins(1,3,4, 5)P4 with an affinity and specificity similar to that of the originally purified protein, a binding activity which is dependent on a functional PH/Btk domain. Furthermore, we highlight a fundamental distinction between GAP1(IP4BP) and its homologue GAP1(m), namely that both proteins function as Ras GAPs but only GAP1(IP4BP) displays Rap GAP activity.

Cloning and expression of an insect Ca(2+)-ATPase from Heliothis virescens.

A complementary DNA for the Tobacco Budworm, Heliothis virescens, sarco(endo)plasmic reticulum-type Ca(2+)-ATPase (HVSERCA) has been cloned and sequenced. cDNA fragments of adult rabbit fast-twitch muscle Ca(2+)-ATPase (SERCA1a) were used as heterologous probes to isolate a partial cDNA clone coding for a protein with high homology to the Ca(2+)-ATPase from Drosophila melanogaster (DRSERCA) and vertebrate ER/SR Ca2+ pumps. The entire cDNA clone contains an ORF encoding a protein of 1000 amino acids which shares the characteristic motifs of a P-type ATPase. HVSERCA shares 89% identity with DRSERCA, 80% identity with the Artemia Ca(2+)-ATPase and 72% identity with avian and mammalian SERCAs. An insect Ca(2+)-ATPase-specific polyclonal antiserum has been raised against a fusion protein containing sequence from the cytoplasmic domain of HVSERCA. Heterologous expression of the insect pump in COS-7 cells has been demonstrated by immunocytochemistry and the reticular pattern of staining is consistent with an ER localisation. However, the expressed enzyme from COS-7 cells does not appear to be active.

Distinct subcellular localisations of the putative inositol 1,3,4,5-tetrakisphosphate receptors GAP1IP4BP and GAP1m result from the GAP1IP4BP PH domain directing plasma membrane targeting.

Inositol 1,3,4,5-tetrakisphosphate (IP4), is a ubiquitous inositol phosphate that has been suggested to function as a second messenger. Recently, we purified and cloned a putative IP4 receptor, termed GAP1(IP4BP)[1], which is also a member of the GAP1 family of GTPase-activating proteins for the Ras family of GTPases. A homologue of GAP1(IP4BP), called GAP1(m), has been identified [2] and here we describe the cloning of a GAP1(m) cDNA from a human circulating-blood cDNA library. We found that a deletion mutant of GAP1(m), in which the putative phospholipid-binding domains (C2A and C2B) have been removed, binds to IP4 with a similar affinity and specificity to that of the corresponding GAP1(IP4BP) mutant. Expression studies of the proteins in either COS-7 or HeLa cells showed that, whereas GAP1(IP4BP) is located solely at the plasma membrane, GAP1(m) seems to have a distinct perinuclear localisation. By mutational analysis, we have shown that the contrast in subcellular distribution of these two closely related proteins may be a function of their respective pleckstrin homology (PH) domains. This difference in localisation has fundamental significance for our understanding of the second messenger functions of IP4.