Activation of extracellular signal-regulated protein kinase 5 downregulates FasL upon osmotic stress.

Extracellular signal-regulated protein kinase (ERK) 5 is a mitogen-activated protein kinase (MAPK) that is activated by dual phosphorylation via a unique MAPK/ERK kinase 5, MEK5. The physiological importance of this signaling cascade is underscored by the early embryonic death caused by the targeted deletion of the erk5 or the mek5 genes in mice. Here, we have found that ERK5 is required for mediating the survival of fibroblasts under basal conditions and in response to sorbitol treatment. Increased Fas ligand (FasL) expression acts as a positive feedback loop to enhance apoptosis of ERK5- or MEK5-deficient cells under conditions of osmotic stress. Compared to wild-type cells, erk5-/- and mek5-/- fibroblasts treated with sorbitol display a reduced protein kinase B (PKB) activity associated with increased Forkhead box O3a (Foxo3a) activity. Based on these results, we conclude that the ERK5 signaling pathway promotes cell survival by downregulating FasL expression via a mechanism that implicates PKB-dependent inhibition of Foxo3a downstream of phosphoinositide 3 kinase.

Phosphatidylinositol phosphate kinases.

Phosphatidylinositol 4,5-bisphosphate is a multi-functional lipid, whose proposed functions now number more than twenty, covering many aspects of cell biology in several different subcellular compartments. The enzymes primarily responsible for synthesizing this lipid, the Type I phosphatidylinositol 4-phosphate 5-kinases, are therefore a tightly regulated and diverse family. Here we review our current knowledge about these enzymes and how they may be regulated.

Cellular signalling: stressing the importance of PIP3.

The lipid second messenger PIP3 was previously thought to be generated exclusively by type I PI 3-kinases. Now, a novel route of PIP3 synthesis, controlled by an unrelated enzyme family, has been discovered, increasing our understanding of the versatility of PIP3 in cellular signalling.

Thrombin stimulation of platelets causes an increase in phosphatidylinositol 5-phosphate revealed by mass assay.

Phosphatidylinositol 5-phosphate (PtdIns5P), a novel inositol lipid, has been shown to be the major substrate for the type II PtdInsP kinases (PIPkins) ¿Rameh et al. (1997) Nature 390, 192-196. A PtdInsP fraction was prepared from cell extracts by neomycin chromatography, using a protocol devised to eliminate the interaction of acidic solvents with plasticware, since this was found to inhibit the enzyme. The PtdIns5P in this fraction was measured by incubating with ¿gamma-(32)PATP and recombinant PIPkin IIalpha, and quantifying the radiolabelled PtdInsP(2) formed. This assay was used on platelets to show that during 10 min stimulation with thrombin, the mass level of PtdIns5P increases, implying the existence of an agonist-stimulated synthetic mechanism.

Nuclear targeting of the beta isoform of type II phosphatidylinositol phosphate kinase (phosphatidylinositol 5-phosphate 4-kinase) by its alpha-helix 7.

Type II phosphatidylinositol phosphate kinases (PIPkins) have recently been found to be primarily phosphatidylinositol 5-phosphate 4-kinases, and their physiological role remains unclear. We have previously shown that a Type II PIPkin [isoform(s) unknown], is localized partly in the nucleus [Divecha, Rhee, Letcher and Irvine (1993) Biochem. J. 289, 617-620], and here we show, by transfection of HeLa cells with green-fluorescent-protein-tagged Type II PIPkins, that this is likely to be the Type IIbeta isoform. Type IIbeta PIPkin has no obvious nuclear localization sequence, and a detailed analysis of the localization of chimaeras and mutants of the alpha (cytosolic) and beta PIPkins shows that the nuclear localization requires the presence of a 17-amino-acid length of alpha-helix (alpha-helix 7) that is specific to the beta isoform, and that this helix must be present in its entirety, with a precise orientation. This resembles the nuclear targeting of the HIV protein Vpr, and Type IIbeta PIPkin is apparently therefore the first example of a eukaryotic protein that uses the same mechanism.

Regulation of type IIalpha phosphatidylinositol phosphate kinase localisation by the protein kinase CK2.

Inositol lipid synthesis is regulated by several distinct families of enzymes [1]. Members of one of these families, the type II phosphatidylinositol phosphate kinases (PIP kinases), are 4-kinases and are thought to catalyse a minor route of synthesis of the multifunctional phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) from the inositide PI(5)P [2]. Here, we demonstrate the partial purification of a protein kinase that phosphorylates the type IIalpha PIP kinase at a single site unique to that isoform - Ser304. This kinase was identified as protein kinase CK2 (formerly casein kinase 2). Mutation of Ser304 to aspartate to mimic its phosphorylation had no effect on PIP kinase activity, but promoted both redistribution of the green fluorescent protein (GFP)-tagged enzyme in HeLa cells from the cytosol to the plasma membrane, and membrane ruffling. This effect was mimicked by mutation of Ser304 to alanine, although not to threonine, suggesting a mechanism involving the unmasking of a latent membrane localisation sequence in response to phosphorylation.

PIPkins1, their substrates and their products: new functions for old enzymes.

The phosphatidylinositolphosphate kinases (PIPkins) are a unique family of enzymes that catalyse the production of phosphorylated inositol lipids. Recent advances have revealed that, due to their ability to utilise a number of different lipid substrates (at least in vitro), this family is potentially able to generate several distinct, physiologically important inositol lipids. Despite their importance, however, our understanding of the regulation of the PIPkins and of their physiological role in cellular signalling and regulation is still poor. Here we describe in turn the diverse physiological functions of the known substrates and major products of the PIPkins. We then examine what is known about the members of the PIPkin family themselves, and their characteristics and regulation.

Regulation of PtdIns4P 5-kinase C by thrombin-stimulated changes in its phosphorylation state in human platelets.

PtdIns(4,5)P2 production by the enzyme PtdIns4P 5-kinase C (PIPkin C) was examined in thrombin-stimulated human platelets. Thrombin caused a rapid, transient 2-3-fold increase in PIPkin activity and a transient net dephosphorylation of the enzyme. PIPkin C was phosphorylated on serine and threonine residues in unstimulated platelets; no evidence for tyrosine phosphorylation was found. The phosphatase inhibitor okadaic acid promoted PIPkin C hyperphosphorylation and a concomitant marked inhibition of its activity in immunoprecipitates. Activity was restored by treatment with alkaline phosphatase, suggesting the existence of an inhibitory phosphorylation site. In support of this idea, alkaline phosphatase treatment of PIPkin C immunoprecipitated from unstimulated platelets caused a modest (1.6-fold) but significant activation of the enzyme. However, alkaline phosphatase treatment of PIPkin C immunoprecipitated from thrombin-stimulated platelets caused a decrease in activity to approximately the same levels, suggesting that the phosphorylation of PIPkin C also contributes to the observed stimulation. Two-dimensional phosphopeptide mapping of immunoprecipitated PIPkin C revealed that the enzyme is multiply phosphorylated and that, whereas some phosphopeptides are indeed lost on stimulation, consistent with the net dephosphorylation of the enzyme, at least two novel sites become phosphorylated. This suggests that thrombin causes complex changes in the phosphorylation state of PIPkin C, one consequence of which is its activation.

Aggregation-dependent, integrin-mediated increases in cytoskeletally associated PtdInsP2 (4,5) levels in human platelets are controlled by translocation of PtdIns 4-P 5-kinase C to the cytoskeleton.

Thrombin-stimulated aggregation of human platelets promotes an increase in the phosphatidylinositol 4-phosphate (PtdIns 4-P) 5-kinase (PIPkin) activity in the cytoskeleton. This phenomenon is associated with translocation of PIPkin isoform C to the cytoskeleton and with an increase in the amount of phosphatidylinositol bisphosphate (PtdInsP2) bound to the cytoskeletal pellet. All three of these effects are prevented if the platelets are not stirred or if RGD-containing peptides are present, demonstrating that they require integrin activation. All three are also abolished by pretreatment with okadaic acid, which also prevents the aggregation-dependent translocation of pp60(c-src) to the cytoskeleton. The results point to the existence of a cytoskeletally associated PtdInsP2 pool under the control of integrin-mediated signals that act via PIPkin C and suggest that a common, okadaic acid-sensitive mechanism may underlie the aggregation-dependent translocation of certain signalling molecules to the platelet cytoskeleton.

The cloning and sequence of the C isoform of PtdIns4P 5-kinase.

In this study we describe the purification and sequencing of the C isoform of platelet PtdIns4P 5-kinase. Subsequently a cDNA was isolated from a human circulating-leucocyte library, which when sequenced was shown to contain all of the peptides identified in the purified protein. In addition, expression of this cDNA in bacteria led to the production of a protein which was recognized by specific monoclonal antibodies raised to the bovine brain enzyme [Brooksbank, Hutchings, Butcher, Irvine and Divecha (1993) Biochem. J. 291, 77-82] and also led to the appearance of PtdIns4P 5-kinase activity in the bacterial lysates. Interestingly, the cDNA showed no similarity to any of the previously cloned inositide kinases. A search of the DNA databases showed that two proteins from Saccharomyces cerevisiae shared close similarity to this enzyme, one of which, the mss4 gene product, has been implicated in the yeast inositol lipid pathway. These data suggest that the PtdIns4P 5-kinases are a new family of inositide kinases unrelated to the previously cloned phosphoinositide 3/4-kinases.