Differential effects of the phosphatidylinositol 4-kinases, PI4KIIα and PI4KIIIß, on Akt activation and apoptosis.

In this study, we investigated the role of PI4P synthesis by the phosphatidylinositol 4-kinases, PI4KIIα and PI4KIIIß, in epidermal growth factor (EGF)-stimulated phosphoinositide signaling and cell survival. In COS-7 cells, knockdown of either isozyme by RNA interference reduced basal levels of PI4P and PI(4,5)P(2), without affecting receptor activation. Only knockdown of PI4KIIα inhibited EGF-stimulated Akt phosphorylation, indicating that decreased PI(4,5)P(2) synthesis observed by loss of either isoform could not account for this PI4KIIα-specific effect. Phospholipase Cγ activation was also differentially affected by knockdown of either PI4K isozyme. Overexpression of kinase-inactive PI4KIIα, which induces defective endosomal trafficking without reducing PI(4,5)P(2) levels, also reduced Akt activation. Furthermore, PI4KIIα knockdown profoundly inhibited cell proliferation and induced apoptosis as evidenced by the cleavage of caspase-3 and its substrate poly(ADP-ribose) polymerase. However, in MDA-MB-231 breast cancer cells, apoptosis was observed subsequent to knockdown of either PI4KIIα or PI4KIIIß and this correlated with enhanced proapoptotic Akt phosphorylation. The differential effects of phosphatidylinositol 4-kinase knockdown in the two cell lines lead to the conclusion that phosphoinositide turnover is inhibited through PI4P substrate depletion, whereas impaired antiapoptotic Akt signaling is an indirect consequence of dysfunctional endosomal trafficking.

EGF receptors as transcription factors: ridiculous or sublime?

The notion that a transmembrane receptor at the cell surface can somehow reappear as a transcription factor in the nucleus is bound to be controversial. However, there are two reported examples of this. If this hypothesis can withstand the inevitable and necessary battery of additional empirical tests, then our understanding of signal transduction needs to move in a new direction.

Signalling and non-caveolar rafts.

Rafts are small membrane domains containing discrete subsets of lipids and proteins. Although microscopic raft structures termed 'caveolae' were described nearly 50 years ago, the importance of rafts, particularly signalling within rafts, is only beginning to be understood. Our studies focus on receptor-dependent phosphoinositide signalling. Using their characteristic buoyancy in density gradients, we and others found that the epidermal growth factor (EGF) receptor, phosphatidylinositol 4-kinase and phosphoinositides are localized within a caveolin-rich fraction of A431 carcinoma cells. We subsequently found that membrane fragments containing the EGF receptor and most cellular phosphoinositides can be separated from caveolae. Consequently, components of EGF-dependent phosphoinositide signalling localize to one or more novel types of raft, the composition of which we are currently determining. A key component is the type II phosphatidylinositol 4-kinase, which, for many years, has proven difficult to purify and clone. We describe our recent purification from rafts and cloning of this elusive enzyme, and discuss how the structure sheds light on the rafting of this enzyme.

Cloning of a human type II phosphatidylinositol 4-kinase reveals a novel lipid kinase family.

Phosphoinositide lipids regulate numerous cellular processes in all eukaryotes. The versatility of this phospholipid is provided by combinations of phosphorylation on the 3', 4', and 5' positions of the inositol head group. Two distinct structural families of phosphoinositide (PI) kinases have so far been identified and named after their prototypic members, the PI 3-kinase and phosphatidylinositol (PtdIns) phosphate kinase families, both of which have been found to contain structural homologues possessing PI 4-kinase activity. Nevertheless, the prevalent PtdIns 4-kinase activity in many mammalian cell types is conferred by the widespread type II PtdIns 4-kinase, which has so far resisted molecular characterization. We have partially purified the human type II isoform from plasma membrane rafts of human A431 epidermoid carcinoma cells and obtained peptide mass and sequence data. The results allowed the cDNA containing the full open reading frame to be cloned. The predicted amino acid sequence revealed that the type II enzyme is the prototypic member of a novel, third family of PI kinases. We have named the purified protein type IIalpha and a second human isoform, type IIbeta. The type IIalpha mRNA appears to be expressed ubiquitously in human tissues, and homologues appear to be expressed in all eukaryotes.

Cloning and characterization of a novel human phosphatidylinositol transfer protein, rdgBbeta.

The various PITP, retinal degeneration B (rdgB), and amino-terminal domain interacting receptor (Nir) phosphatidylinositol transfer proteins can be divided into two structural families. The small, soluble PITP isoforms contain only a phosphatidylinositol transfer domain and have been implicated in phosphoinositide signaling and vesicle trafficking. In contrast, the rdgB proteins, which include Nir2 and Nir3, contain an amino-terminal PITP-like domain, an acidic, Ca(2+)-binding domain, six putative transmembrane domains, and a conserved carboxyl-terminal domain. However, the biological function of rdgB proteins is unclear. Here, we report the isolation of a cDNA encoding a novel rdgB protein, mammalian rdgBbeta (MrdgBbeta). The 38-kDa MrdgBbeta protein contains an amino-terminal PITP-like domain and a short carboxyl-terminal domain. In contrast to other rdgB-like proteins, MrdgBbeta contains no transmembrane motifs or the conserved carboxyl-terminal domain. Using Northern and reverse transcription-polymerase chain reaction analysis, we demonstrate that MrdgBbeta mRNA is ubiquitously expressed. Immunofluorescence analysis of ectopic MrdgBbeta showed cytoplasmic staining, and the ability of recombinant MrdgBbeta to transfer phosphatidylinositol in vitro was similar to other PITP-like domains. Although early reports found functional degeneracy in vitro, the identification of a fifth mammalian PITP-like protein with a unique domain organization and widespread expression supports more recent results that suggest that different PITP-like domains have distinct functions in vivo.

unr, a cellular cytoplasmic RNA-binding protein with five cold-shock domains, is required for internal initiation of translation of human rhinovirus RNA.

Initiation of translation of the animal picornavirus RNAs occurs via a mechanism of direct ribosome entry, which requires a segment of the 5' UTR of the RNA, known as the internal ribosome entry site (IRES). In addition, translation of the enterovirus and rhinovirus (HRV) subgroups requires cellular trans-acting factors that are absent from, or limiting in rabbit reticulocytes, but are more abundant in HeLa cell extracts. It has been shown previously that HeLa cells contain two separable activities, each of which independently stimulates HRV IRES-dependent translation when used to supplement reticulocyte lysate; one of these activities was identified as polypyrimidine tract-binding protein (PTB). Here, the purification of the second activity is achieved by use of an RNA-affinity column based on the HRV 5' UTR. It comprises two components: a 38-kD protein (p38), which is a novel member of the GH-WD repeat protein family and has no intrinsic RNA-binding activity; and a 96- to 97-kD protein doublet, which was identified as unr, an RNA-binding protein with five cold-shock domains. Coimmunoprecipitation with antibodies against either protein shows that the two proteins interact with each other, and thus p38 is named unrip (unr-interacting protein). Recombinant unr acts synergistically with recombinant PTB to stimulate translation dependent on the rhinovirus IRES. In contrast, unr did not significantly augment the PTB-dependent stimulation of poliovirus IRES activity.

Epidermal growth factor receptor activation is localized within low-buoyant density, non-caveolar membrane domains.

Increasing evidence for the organization of cell-surface proteins and lipids into different detergent-insoluble rafts led us to investigate epidermal growth factor (EGF) receptor activation in the plasma membranes of A431 carcinoma cells, using a combination of cell fractionation and immunoprecipitation techniques. Density-gradient centrifugation of sodium carbonate cell extracts revealed that the vast majority of both stimulated and unstimulated EGF receptors were concentrated in a caveolin-rich light membrane (CLM) fraction, with the biochemical characteristics of detergent-insoluble glycolipid-rich domains (DIGs). However, ultrastructural analysis of the CLM fraction revealed that it contained a heterogeneous collection of vesicles, some with sizes greater than that expected for individual caveolae. Experiments with detergent-solubilized cells and isolated CLMs indicated that, in contrast with caveolin, EGF receptors were unlikely to be localized to DIG domains. Furthermore, immunoisolation of caveolin from CLMs revealed that EGF receptor activation occurs in a compartment distinct from caveolae. Similarly, using an anti-(EGF receptor) antibody, the bulk of the cellular caveolin was not co-immunoprecipitated from CLMs, thereby confirming that these two proteins reside in separate membrane domains. The deduction that caveolar signalling and EGF receptor activation occur in separable rafts argues for a multiplicity of signal transduction compartments within the plasma membrane. In addition, by demonstrating that EGF receptor activation is compartmentalized within low-density, non-caveolar regions of the plasma membrane, it is also shown that the co-localization of proteins in a CLM fraction is insufficient to prove caveolar localization.

The gp200-MR6 molecule which is functionally associated with the IL-4 receptor modulates B cell phenotype and is a novel member of the human macrophage mannose receptor family.

The human gp200-MR6 molecule has previously been shown to have either an antagonistic or agonistic effect on IL-4 function, demonstrated by inhibition of IL-4-induced proliferation of T cells or mimicking of IL-4-induced maturation of epithelium, respectively. We now show that gp200-MR6 ligation can also mimic IL-4 and have an anti-proliferative pro-maturational influence within the immune system, causing up-regulation of co-stimulatory molecules on B lymphocytes. Biochemical analysis and cDNA cloning reveal that gp200-MR6 belongs to the human macrophage mannose receptor family of multidomain molecules. It comprises 1722 amino acids in toto (mature protein, 1695 amino acids; signal sequence, 27 amino acids) organized into 12 external domains (an N-terminal cysteine-rich domain, a fibronectin type II domain and 10 C-type carbohydrate recognition domains), a transmembrane region and a small cytoplasmic C terminus (31 amino acids) containing a single tyrosine residue (Y1679), but no obvious kinase domain. Strong amino acid sequence identity (77%) suggests that gp200-MR6 is the human homologue of the murine DEC-205, indicating that this molecule has much wider functional activity than its classical endocytic role. We also show that the gp200-MR6 molecule is closely associated with tyrosine kinase activity; the link between gp200-MR6 and the IL-4 receptor may therefore be via intracellular signaling pathways, with multifunctionality residing in its extracellular multidomain structure.

Phosphatidylinositol 4-phosphate synthesis in immunoisolated caveolae-like vesicles and low buoyant density non-caveolar membranes.

This study examined phosphatidylinositol 4-phosphate (PtdIns4P) synthesis in caveolae that have been suggested to be discrete signaling microdomains of the plasma membrane and are enriched in the marker protein caveolin. Caveolin-rich light membranes (CLMs) were isolated from A431 cells by detergent-free, discontinuous density-gradient centrifugation method. The CLM fraction was separated from the bulk of the cellular protein and was greatly enriched in PtdIns, PtdIns4P, and phosphatidylinositol 4, 5-bisphosphate (PtdIns(4,5)P2) and an adenosine-sensitive type II PtdIns 4-kinase activity. Preparation of CLMs by an OptiPrep-based cell fractionation procedure confirmed the co-localization of PtdIns 4-kinase and caveolin. Electron microscopy confirmed that an anti-caveolin antiserum immunopurified vesicles from CLMs that were within the size range described for caveolae in other systems. Co-immunoprecipitated PtdIns 4-kinase activity could utilize endogenous PtdIns, present within the caveolae-like vesicles, to produce PtdIns4P. The addition of recombinant phosphatidylinositol transfer protein increased PtdIns 4-kinase activity both in immunoisolated caveolae and CLMs. However, less than 1% of the total cellular PtdIns and PtdIns 4-kinase activity was present in caveolae-like vesicles, indicating that non-caveolar light membrane rafts are the main site for cellular PtdIns4P production.

Synaptojanin is the major constitutively active phosphatidylinositol-3,4,5-trisphosphate 5-phosphatase in rodent brain.

The major constitutive phosphatidylinositol-3,4,5-P3 (PtdIns) 5-phosphatase activity was purified and subjected to peptide sequence analysis providing extensive amino acid sequence which was subsequently used for cloning the cDNA. Peptide and cDNA sequences revealed that the purified PtdIns(3,4,5)P3 5-phosphatase was identical to a splice variant of a recently cloned inositol polyphosphate 5-phosphatase termed synaptojanin. Since synaptojanin is not known to possess PtdIns(3,4,5)P3 5-phosphatase activity, we verified that the purified PtdIns(3,4,5)P3 5-phosphatase activity and synaptojanin are identical by Western blot using specific antibodies raised against synaptojanin sequences. Immunoprecipitation from crude lysates of rat brain tissue showed that synaptojanin accounts for the major part of the active PtdIns(3, 4,5)P3 5-phosphatase activity. It is also shown that the protein is localized to the soluble fraction. Expression of a truncated recombinant protein demonstrates that the conserved 5-phosphatase region of the synaptojanin gene expresses PtdIns(3,4,5)P3 5-phosphatase activity. However, immunological analysis demonstrates that the PtdIns(3,4,5)P3 5-phosphatase activity expressed from the synaptojanin gene in brain is due to a particular splice variant which contains a 16-amino acid insert as shown by immunoprecipitation using a specific antibody raised against this particular splice variant.

Growth factor-dependent phosphoinositide signalling.

A wide variety of messages, in the form of diffusible growth factors, hormones and cytokines, are carried throughout multicellular organisms to coordinate important physiological properties of target cells, such as proliferation, differentiation, migration, apoptosis and metabolism. Most messengers bind to cognate receptors on target cells, which initiate a characteristic cascade of reactions within the cell, ultimately leading to the desired response. The cellular response is defined by the combination of signalling components whose individual activity depends upon the number and type of surface receptors. Consequently the responses of different cell types to one or more stimuli can be quite disparate. A molecular understanding of the signalling pathways employed by each type of receptor therefore underlies the ability to rationalize many cellular functions and to correct disfunctions. As a well studied example of the primary signalling events that take place on the cytoplasmic leaflet of the plasma membrane following receptor activation, we will discuss how the widely expressed receptor for epidermal growth factor (EGF) causes the phosphorylation and hydrolysis of a signalling precursor, the membrane lipid phosphatidylinositol. This paradigm will be used to illustrate certain general principles of signalling, including formation of multienzyme complexes, compartmentation of second messengers and intermediates, and cross-talk between different signalling pathways.

Cytosolic phox proteins interact with and regulate the assembly of coronin in neutrophils.

The NADPH oxidase generates microbicidal superoxide in phagocytes, and when defective it leads to chronic granulomatous disease (CGD). Oxidase specific proteins in the cytosol, p47phox and p67phox, as well as the small GTP binding protein p21rac are important for activation of superoxide production. Because the activity of this oxidase is normally tightly restricted to the phagocytic vacuole, and its temporal and spatial organisation might be regulated by cytoskeletal proteins, we examined the cytosolic phox proteins for interactions with cytoskeletal elements. p67phox copurified with a 57 kDa protein, identified as coronin, an actin binding protein that is important for movement and phagocytosis in Dictyostelium. Binding studies revealed that coronin attaches to the C-terminal half of p40phox, a binding partner of p67phox. The phox proteins and coronin had a similar distribution in the cell, and both accumulated around the phagocytic vacuole. PMA activation of adherent neutrophils resulted in a major rearrangement of these proteins, and of actin, which were lost from the periphery of the cell and condensed around the nucleus. The rearrangement of F-actin and coronin in adherent cells, were absent, or markedly diminished, in cells from patients lacking p47phox or p67phox in which an abnormally large proportion of the coronin was present as part of a large complex. The cytosolic phox proteins might play a regulatory role in the reorganisation of the cytoskeleton accompanying superoxide generation.

Mammalian actin-related protein 2/3 complex localizes to regions of lamellipodial protrusion and is composed of evolutionarily conserved proteins.

Human neutrophils contain a complex of proteins similar to the actin-related protein 2/3 (Arp2/3) complex of Acanthamoeba. We have obtained peptide sequence information for each member of the putative seven-protein complex previously described for Acanthamoeba and human platelets. From the peptide sequences we have identified cDNA species encoding three novel proteins in this complex. We find that in addition to Arp2 and Arp3, this complex contains a relative of the human (Suppressor of Profilin) SOP2Hs protein and four previously unknown proteins. These proteins localize in the cytoplasm of fibroblasts that lack lamellipodia, but are enriched in lamellipodia on stimulation with serum or platelet-derived growth factor. We propose a conserved and dynamic role for this complex in the organization of the actin cytoskeleton.

Identification of regions of the Wiskott-Aldrich syndrome protein responsible for association with selected Src homology 3 domains.

Src homology 3 (SH3) domains have been shown to mediate selected interactions between signaling molecules and are essential for the activation of a number of receptor-driven pathways. The Wiskott-Aldrich syndrome protein was identified as a protein that associated selectively with the SH3 domains derived from c-Src, p85alpha, phospholipase Cgamma1, and c-Fgr. Significantly reduced association was detected to the N-terminal SH3 domain and the tandem SH3 domains of p47(phox), and no binding was detected to the SH3 domain of n-Src, the C-terminal SH3 domain of p47(phox), or either of the SH3 domains of p67(phox). Three peptides corresponding to potential Wiskott-Aldrich syndrome protein SH3 domain binding motifs were found to inhibit its association with c-Src, Fgr, and phospholipase Cgamma1 SH3 domains, but not the p85alpha SH3 domain. These peptides have the sequences MRRQEPLPPPPPPSRG, TGRSGPLPPPPPGA, and KGRSGPLPPVPLGI and show homology with other SH3 domain binding motifs. It is possible that the intracellular association of Wiskott-Aldrich syndrome protein with other signaling proteins is mediated by its SH3 domain-binding regions, and this may play a role in its putative function as a regulatory molecule in immune cells.

The Friedreich's ataxia gene encodes a novel phosphatidylinositol-4- phosphate 5-kinase.

The STM7 gene on chromosome 9 was recently 'excluded' as a candidate for Friedreich's ataxia following the identification of an expanded intronic GAA triplet repeat in the adjacent gene, X25, in patients with the disease. Using RT-PCR, northern and sequence analyses, we now demonstrate that X25 comprises part of the STM7 gene, contributing to at least four splice variants, and report the identification of new coding sequences. Functional analysis of the STM7 recombinant protein corresponding to the reported 2.7-kilobase transcript has demonstrated PtdlnsP 5-kinase activity, supporting the idea that the disease is caused by a defect in the phosphoinositide pathway, possibly affecting vesicular trafficking or synaptic transmission.

A family of AP-2 proteins regulates c-erbB-2 expression in mammary carcinoma.

The proto-oncogene c-erbB-2 is overexpressed in 25-30% of breast cancers through increased transcription and amplification of the gene. We have previously described a factor, OB2-1 which upregulates c-erbB-2 transcription and which is closely related to the developmentally regulated transcription factor, AP-2. Further analysis of affinity purified OB2-1 has now shown that it is in fact a combination of proteins from three AP-2-related genes, the previously described AP-2alpha gene and two new human family members, AP-2beta and AP-2gamma whose cloning and characterisation are described here. All three AP-2 proteins show a high degree of homology and are capable of binding to the c-erbB-2 promoter as homo- or heterodimers. The three proteins can also activate a c-erbB-2 reporter construct, but AP-2alpha and AP-2gamma are 3-4 times more active in this regard than AP-2beta. In addition both AP-2alpha and AP-2gamma are expressed at elevated levels in the majority of c-erbB-2 overexpressing mammary tumour lines examined. Mechanisms which may have led to the increased AP-2 levels in these cells are discussed.

Hepatocyte nuclear factor 6, a transcription factor that contains a novel type of homeodomain and a single cut domain.

Tissue-specific transcription is regulated in part by cell type-restricted proteins that bind to defined sequences in target genes. The DNA-binding domain of these proteins is often evolutionarily conserved. On this basis, liver-enriched transcription factors were classified into five families. We describe here the mammalian prototype of a sixth family, which we therefore call hepatocyte nuclear factor 6 (HNF-6). It activates the promoter of a gene involved in the control of glucose metabolism. HNF-6 contains two different DNA-binding domains. One of these corresponds to a novel type of homeodomain. The other is homologous to the Drosophila cut domain. A similar bipartite sequence is coded by the genome of Caenorhabditis elegans.

Hereditary hepatic and systemic amyloidosis caused by a new deletion/insertion mutation in the apolipoprotein AI gene.

We report a Spanish family with autosomal-dominant non-neuropathic hereditary amyloidosis with a unique hepatic presentation and death from liver failure, usually by the sixth decade. The disease is caused by a previously unreported deletion/insertion mutation in exon 4 of the apolipoprotein AI (apoAI) gene encoding loss of residues 60-71 of normal mature apoAI and insertion at that position of two new residues, ValThr. Affected individuals are heterozygous for this mutation and have both normal apoAI and variant molecules bearing one extra positive charge, as predicted from the DNA sequence. The amyloid fibrils are composed exclusively of NH2-terminal fragments of the variant, ending mainly at positions corresponding to residues 83 and 92 in the mature wild-type sequence. Amyloid fibrils derived from the other three known amyloidogenic apoAI variants are also composed of similar NH2-terminal fragments. All known amyloidogenic apoAI variants carry one extra positive charge in this region, suggesting that it may be responsible for their enhanced amyloidogenicity. In addition to causing a new phenotype, this is the first deletion mutation to be described in association with hereditary amyloidosis and it significantly extends the value of the apoAI model for investigation of molecular mechanisms of amyloid fibrillogenesis.

Cloning and expression of human G/T mismatch-specific thymine-DNA glycosylase.

Hydrolytic deamination of 5-methylcytosine leads to the formation of G/T mismatches. We have shown previously that these G/T mispairs are corrected to G/C pairs by a mismatch-specific thymine-DNA glycosylase, TDG, which we subsequently purified from human cells. Here we describe the cloning of the human cDNA encoding TDG. We have identified two distinct cDNA species that differ by 100 nucleotides at the 3'-untranslated region. These cDNAs contain a 410-amino acid open reading frame that encodes a 46-kDa polypeptide. The G/T glycosylase, expressed both in vitro and in Escherichia coli, migrated in denaturing polyacrylamide gels with an apparent size of 60 kDa. The substrate specificity of the recombinant protein corresponded to that of the cellular enzyme, and polyclonal antisera raised against the recombinant protein neutralized both activities. We therefore conclude that the cDNA described below encodes human TDG. Data base searches identified a serendipitously cloned mouse cDNA sequence that could be shown to encode the murine TDG homologue. No common amino acid sequence motifs between the G/T-specific enzyme and other DNA glycosylases could be found, suggesting that TDG belongs to a new class of base-excision repair enzymes.