Rho-dependence of Schizosaccharomyces pombe Pck2.

BACKGROUND: In metazoans, the HR1 domain, a motif found in a number of proteins including the protein kinase C-related PRKs, is responsible for an interaction with Rho-GTPases. The structural similarity between the Schizosaccaromyces pombe Pck proteins and the mammalian Rho-dependent protein kinase C-related family, has led us to investigate the relationship between the function of Rho and that of Pck1/2. RESULTS: Rho1 is shown to interact with the conserved N-terminal HR1 domain of Pck1/2 in vitro and in vivo. Lethal overproduction of Rho1 is neutralized by co-expression of the Pck2 HR1 domain, which by itself compromises growth when overproduced. The Pck2-Rho1 interaction has a profound effect on the steady state expression of Pck2 and this is shown to parallel the immunoprecipitated activity and phosphorylation of Pck2 at its activation loop site. It is further shown that Pck2 becomes localized at the septum, where Rho1 is also located. CONCLUSIONS: The results demonstrate that the Pck proteins are Rho1 effectors in fission yeast and that the HR1 domain is a universal motif for the Rho-GTPase interaction. Furthermore, the evidence supports the contention that the yeast Pck1 and Pck2 proteins are primitive protein kinases, which in vertebrates have evolved into the two distinct PKC and PRK families.

Osmotic stress activates phosphatidylinositol-3,5-bisphosphate synthesis.

Inositol phospholipids play multiple roles in cell signalling systems. Two widespread eukaryotic phosphoinositide-based signal transduction mechanisms, phosphoinositidase C-catalysed phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) hydrolysis and 3-OH kinase-catalysed PtdIns(4,5)P2 phosphorylation, make the second messengers inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) sn-1,2-diacylglycerol and PtdIns(3,4,5)P3. In addition, PtdIns(4,5)P2 and PtdIns3P have been implicated in exocytosis and membrane trafficking. We now show that when the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe are hyperosmotically stressed, they rapidly synthesize phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P2) by a process that involves activation of a PtdIns3P 5-OH kinase. This PtdIns(3,5)P2 accumulation only occurs in yeasts that have an active vps34-encoded PtdIns 3-OH kinase, showing that this latter kinase makes the PtdIns3P needed for PtdIns(3,5)P2 synthesis and indicating that PtdIns(3,5)P2 may have a role in sorting vesicular proteins. PtdIns(3,5)P2 is also present in mammalian and plant cells: in monkey Cos-7 cells, its labelling is inversely related to the external osmotic pressure. The stimulation of a PtdIns3P 5-OH kinase-catalysed synthesis of PtdIns(3,5)P2, a molecule that might be a new type of phosphoinositide 'second messenger, thus appears to be central to a widespread and previously uncharacterized regulatory pathway.

Intracellular targeting and homotetramer formation of a truncated inositol 1,4,5-trisphosphate receptor-green fluorescent protein chimera in Xenopus laevis oocytes: evidence for the involvement of the transmembrane spanning domain in endoplasmic reticulum targeting and homotetramer complex formation.

In an attempt to define structural regions of the type I inositol 1, 4,5-trisphosphate [Ins(1,4,5)P3] receptor [Ins(1,4,5)P3R] involved in its intracellular targeting to the endoplasmic reticulum (ER), we have employed the use of green fluorescent protein (GFP) to monitor the localization of a truncated Ins(1,4,5)P3R mutant containing just the putative transmembrane spanning domain and the C-terminal cytoplasmic domain [amino acids 2216-2749; termed inositol trisphosphate receptor(ES)]. We expressed a chimeric GFP-Ins(1,4, 5)P3R(ES) fusion protein in Xenopus laevis oocytes, and used fluorescence confocal microscopy to monitor its intracellular localization. Fluorescence confocal microscopy data showed an intense fluorescence in the perinuclear region and in a reticular-network under the animal pole of the oocyte, consistent with the targeting of expressed GFP-Ins(1,4,5)P3R(ES) to perinuclear ER and ER under the animal pole. These findings are consistent with the intracellular localization of the endogenous Xenopus Ins(1,4, 5)P3R shown previously. Furthermore, electron microscopy data indicate that expressed GFP-Ins(1,4,5)P3R(ES) is in fact targeted to the ER. Sodium carbonate extraction of microsomal membranes and cross-linking experiments indicate that the expressed chimeric protein is in fact membrane anchored and able to form a homotetrameric complex. Our data provides evidence that Ins(1,4, 5)P3R(ES) constitutes the membrane spanning domain of the Ins(1,4, 5)P3R and is able to mediate homotetramer formation, without the need for the large N-terminal cytoplasmic domain. Furthermore, the localization of GFP-Ins(1,4,5)P3R(ES) on the ER indicates that an ER retention/targeting signal is contained within the transmembrane spanning domain of the inositol trisphosphate receptor.

Pharmacological modulators of the inositol 1,4,5-trisphosphate receptor.

Elevation of cytosolic calcium concentrations, induced by many neurotransmitters, plays a crucial role in neuronal function. Some neurotransmitters produce the second messenger InsP3 which activates an intracellular calcium channel (InsP3 receptor) usually located in the endoplasmic reticulum. This article undertakes a comprehensive survey of most pharmacological modulators of the InsP3 receptor so far reported. This review discusses in detail competitive antagonists, non-competitive antagonists and thiol reactive reagents, highlighting their modes of action and in some cases indicating drawbacks in their use.

The inhibition of the inositol 1,4,5-trisphosphate receptor from rat cerebellum by spermine and other polyamines.

Inositol 1,4,5-trisphosphate-induced Ca2+ release from rat cerebellar microsomes can be inhibited by polyamines at mM concentrations. Spermine, one of the most abundant naturally occurring polyamines, inhibits InsP3-induced Ca2+ release with an IC50 of 1 mM. However, the antibiotic neomycin proved most efficacious at inhibiting InsP3-induced Ca2+ release (IC50 0.4mM). The order of potency being neomycin > spermine > spermidine > putrescine. Although binding of [3H]InsP3 to cerebellar microsomes is also inhibited by polyamines, this may be due to InsP3 complexing with the polyamines under the binding conditions used. Under Ca2+ release conditions InsP3 binds weakly to spermine and therefore inhibition of InsP3-induced Ca2+ release is consistent with polyamines interacting with the InsP3 receptor.

The effects of tetrahexyl ammonium cations (THA+) on inositol 1,4,5-trisphosphate-induced calcium release from porcine cerebellar microsomes: THA+ can induce calcium release selectively from the InsP3-sensitive calcium stores.

In this study we show that the potassium-channel blocker tetrahexyl ammonium chloride (THA+) is able to inhibit inositol 1,4,5-trisphosphate (InsP3)-induced calcium release in an apparently biphasic fashion with a IC50 of 3 microM. This inhibition was not alleviated by valinomycin and, therefore, is not consistent with the blocking of K+ counter-ion movement, an observation initially made by Palade et al. (Palade, P., Dettbarn, C., Volpe, P., Alderson, B. and Otero, A.S (1989) Mol. Pharmacol. 36, 664-672). THA+ affected quantal calcium release by reducing the amount of calcium released by InsP3, but did not greatly affect the concentration of InsP3 required to cause half-maximal calcium release. THA+ did not affect the metabolism of InsP3 or its binding to porcine cerebellar microsomes. THA+ could also itself induce calcium release. At concentrations below 100 microM, THA+ appears to release Ca2+ selectively from the InsP3-sensitive calcium stores, since prior depletion of these stores with supramaximal doses of InsP3 abolishes this response. At higher THA+ concentrations (above 100 microM) Ca2+ is released non-selectively from all stores. THA+ has no effect on the Ca(2+)-ATPase activity at concentrations below 100 microM, indicating that selective THA(+)-induced Ca2+ release is not due to non-specific inhibition of the microsomal Ca2+ pumps and does not affect Ca2+ leakage. A number of pharmacological modulators of intracellular calcium channels were also tested on THA(+)-induced calcium release with little effect, except for spermidine which reduced this release by up to 50%. Our observations are consistent with the view that THA+, at concentrations below 100 microM, selectively releases calcium from the InsP3-sensitive calcium stores.

The effects of thimerosal on calcium uptake and inositol 1,4,5-trisphosphate-induced calcium release in cerebellar microsomes.

Thimerosal inhibits calcium uptake in skeletal muscle sarcoplasmic reticulum and rat cerebellar microsomes by inhibiting the Ca(2+)-ATPase. In the presence of 5 mM dithiothreitol (DTT), Ca2+ uptake and ATPase activity were not inhibited by thimerosal, indicating that thimerosal modifies cysteine residues of the Ca(2+)-ATPase. Low thimerosal concentrations (2 microM) sensitize the inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2+ channel, making it open at lower InsP3 concentrations. Higher concentrations of thimerosal, however, cause inhibition of InsP3-induced Ca2+ release. Both sensitization and inhibition of the InsP3 receptor by thimerosal can be prevented by DTT. The binding and metabolism of InsP3 by cerebellar microsomes is not affected by thimerosal. The amount of InsP3-induced Ca2+ release is co-operatively linked to the InsP3 concentration with a Hill coefficient of 2.0 +/- 0.3. This is decreased to 1.0 +/- 0.2 at inhibitory concentrations of thimerosal. Under our experimental conditions, we observed no dependence of quantal Ca2+ release on intraluminal Ca2+ concentration.

The opening of the inositol 1,4,5-trisphosphate-sensitive Ca2+ channel in rat cerebellum is inhibited by caffeine.

Ins(1,4,5)P3(InsP3)-induced Ca2+ release and [3H]InsP3 binding were measured in rat cerebellar microsomes in the presence or absence of caffeine. The quantal Ca2+ release was shown to occur in an apparently co-operative fashion with a Hill coefficient (h) of 2.2. Half-maximal Ca2+ release was observed at 900 nM-InsP3. Addition of caffeine caused changes both to the concentration of InsP3 required to cause half-maximal Ca2+ release (3.9 microM at 50 mM-caffeine) and to the apparent co-operativity (h = 1.0 at 50 mM-caffeine). Under standard conditions for [3H]InsP3 binding, caffeine had no effect, and it had no effect on InsP3 metabolism. Cyclic AMP also had no effect on the quantal release induced by InsP3. These results are consistent with the view that caffeine affects the opening (Ca2+ release) events rather than the ligand-binding events in the operation of the InsP3-sensitive Ca2+ channel.