Biomedical subjects

P J Cullen

Publications and source records attributed to P J Cullen.

At least 19 recordsLinked to original sources

PubMed2001

Modular phosphoinositide-binding domains--their role in signalling and membrane trafficking.

The membrane phospholipid phosphatidylinositol is the precursor of a family of lipid second-messengers, known as phosphoinositides, which differ in the phosphorylation status of their inositol group. A major advance in understanding phosphoinositide signalling has been the identification of a number of highly conserved modular protein domains whose function appears to be to bind various phosphoinositides. Such 'cut and paste' modules are found in a diverse array of multidomain proteins and recruit their host protein to specific regions in cells via interactions with phosphoinositides. Here, with particular reference to proteins involved in membrane traffic pathways, we discuss recent advances in our understanding of phosphoinositide-binding domains.

Amino Acid Sequence↗

PubMed2001

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.

Adenosine Triphosphate↗

PubMed2001

Ras effectors: buying shares in Ras plc.

The debate over whether activated Ras can regulate phosphoinositide-specific phospholipase C (PLC) has been contentious and at times heated. The argument may be resolved by the recent identification of a novel Ras-regulated PLC, but some unexpected properties of this protein are sure to stimulate further controversy.

Humans↗

PubMed2001

Casein kinase I associates with members of the centaurin-alpha family of phosphatidylinositol 3,4,5-trisphosphate-binding proteins.

Mammalian casein kinases I (CKI) belong to a family of serine/threonine protein kinases involved in diverse cellular processes including cell cycle progression, membrane trafficking, circadian rhythms, and Wnt signaling. Here we show that CKIalpha co-purifies with centaurin-alpha(1) in brain and that they interact in vitro and form a complex in cells. In addition, we show that the association is direct and occurs through the kinase domain of CKI within a loop comprising residues 217-233. These residues are well conserved in all members of the CKI family, and we show that centaurin-alpha(1) associates in vitro with all mammalian CKI isoforms. To date, CKIalpha represents the first protein partner identified for centaurin-alpha(1). However, our data suggest that centaurin-alpha(1) is not a substrate for CKIalpha and has no effect on CKIalpha activity. Centaurin-alpha(1) has been identified as a phosphatidylinositol 3,4,5-trisphosphate-binding protein. Centaurin-alpha(1) contains a cysteine-rich domain that is shared by members of a newly identified family of ADP-ribosylation factor guanosine trisphosphatase-activating proteins. These proteins are involved in membrane trafficking and actin cytoskeleton rearrangement, thus supporting a role for CKIalpha in these biological events.

Actins↗

PubMed2001

Phosphatidylinositol 3-kinase-dependent translocation of phospholipase Cgamma2 in mouse megakaryocytes is independent of Bruton tyrosine kinase translocation.

Activation of the collagen receptor glycoprotein VI (GPVI) by a collagen-related peptide (CRP) induces stimulation of platelets and megakaryocytes through the phosphatidylinositol (PI) 3-kinase-dependent pathway leading to activation of Bruton tyrosine kinase (Btk) and phospholipase Cgamma2 (PLCgamma2). Here, we present evidence that both proteins undergo PI 3-kinase-dependent translocation to the plasma membrane on CRP stimulation that is markedly inhibited by wortmannin and LY294002. Translocation of PLCgamma2 but not Btk is also seen in megakaryocytes from X-linked immunodeficiency mice, which have a mutation that reduces the affinity of the pleckstrin homology (PH) domain of Btk for PI 3,4,5-trisphosphate (PI 3,4,5-P3). Activation of PC12 cells by epidermal growth factor (EGF) results in increased PI 3-kinase activity and high PI 3,4,5-P3 levels that trigger translocation of the green fluorescent protein (GFP)-labeled PH of Btk, but not the GFP-labeled PH and tandem Src homology 2 (SH2) domains of PLCgamma2. In contrast to the results with CRP, the G protein-coupled receptor agonist thrombin stimulates PI 3-kinase-independent translocation of Btk but not PLCgamma2. In conclusion, these results demonstrate that in mouse megakaryocytes, CRP leads to PI 3-kinase-dependent translocation of PLCgamma2 and Btk that are independent of one another, whereas thrombin only induces translocation of Btk through a pathway that is independent of PI 3-kinase activity.

Agammaglobulinaemia Tyrosine Kinase↗

PubMed2000

Effects of elevated expression of inositol 1,4,5-trisphosphate 3-kinase B on Ca2+ homoeostasis in HeLa cells.

Ins(1,4,5)P(3) 3-kinase (IP3K) phosphorylates the Ca(2+)-mobilizing second messenger Ins(1,4,5)P(3) to yield the putative second messenger Ins(1,3,4,5)P(4). A HeLa cell line was established expressing the rat B isoform of IP3K under the control of an inducible promoter. The IP3KB-transfected cell line possessed 23-fold greater IP3K activity than untransfected cells after induction of IP3KB expression, but only 0.23-fold greater activity when IP3KB expression was not induced. Elevating IP3KB expression significantly reduced levels of Ins(1,4,5)P(3) and increased levels of Ins(1,3,4,5)P(4) after stimulation of cells with histamine, but had no effect on basal levels. Histamine- and ATP-evoked cytosolic Ca(2+) responses were dramatically reduced upon elevation of IP3KB expression. On stimulation with a supramaximal dose of histamine, 67% of cells induced to express IP3KB gave no detectable elevation in cytosolic Ca(2+), compared with 3% of uninduced cells. The quantity of Ca(2+) within thapsigargin-sensitive and -insensitive stores was unaffected by elevation of IP3KB expression, as was capacitative Ca(2+) entry. These data suggest that IP3KB may play a significant role in the regulation of Ins(1,4,5)P(3) levels, and consequently in Ca(2+) responses following stimulation of cells with Ins(1,4,5)P(3)-elevating agonists.

Adenosine Triphosphate↗

PubMed2000

Glucose depletion causes haploid invasive growth in yeast.

Haploid yeast invades solid agar in response to nutrient limitation. To decipher the cues that underlie invasion, we have developed a single cell invasive growth assay. Using this assay, as well as the traditional plate-washing assay, we show that invasive growth occurs in response to glucose depletion. In the absence of glucose (or other fermentable sugar), individual cells adopted a nonaxial budding pattern and elongated morphology within the first cell divisions, and invasion into the agar was observed in microcolonies containing as few as 10 cells. In support of this observation, we found that glucose suppressed the hyperinvasive growth morphology of STE11-4, pbs2, hsl7, and RAS2V19 mutations. In addition, removal of glucose from YPD medium caused constitutive invasion in wild-type cells. We tested glucose control proteins for a role in invasion and found that Snf1, a protein required for derepression of glucose-repressed genes, was required for invasive growth. The transcription factor Sip4, which interacts with Snf1 and is induced during the diauxic shift, had an inhibitory role on invasive growth, suggesting that multiple mechanisms are required for glucose depletion-dependent invasion.

Glucose↗

PubMed2000

Membrane targeting: what a difference a G makes.

Pleckstrin homology domains are modular domains that direct membrane targeting of their host proteins by binding to polyphosphoinositides; recent results have increased our appreciation of how some of these domains actually bind 3-phosphoinositides, and along the way thrown up some unexpected observations.

Adaptor Proteins, Signal Transducing↗

PubMed2000

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).

Amino Acid Substitution↗

PubMed2000

Molecular modelling and site-directed mutagenesis of the inositol 1,3,4,5-tetrakisphosphate-binding pleckstrin homology domain from the Ras GTPase-activating protein GAP1IP4BP.

GAP1(IP4BP) is a Ras GTPase-activating protein (GAP) that in vitro is regulated by the cytosolic second messenger inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P(4)]. We have studied Ins(1,3,4,5)P(4) binding to GAP1(IP4BP), and shown that the inositol phosphate specificity and binding affinity are similar to Ins(1,3,4,5)P(4) binding to Bruton's tyrosine kinase (Btk), evidence which suggests a similar mechanism for Ins(1,3,4,5)P(4) binding. The crystal structure of the Btk pleckstrin homology (PH) domain in complex with Ins(1,3,4,5)P(4) has shown that the binding site is located in a partially buried pocket between the beta 1/beta 2- and beta 3/beta 4-loops. Many of the residues involved in the binding are conserved in GAP1(IP4BP). Therefore we generated a model of the PH domain of GAP1(IP4BP) in complex with Ins(1,3,4,5)P(4) based on the Btk-Ins(1,3,4,5)P(4) complex crystal structure. This model had the typical PH domain fold, with the proposed binding site modelling well on the Btk structure. The model has been verified by site-directed mutagenesis of various residues in and around the proposed binding site. These mutations have markedly reduced affinity for Ins(1,3,4,5)P(4), indicating a specific and tight fit for the substrate. The model can also be used to explain the specificity of inositol phosphate binding.

Amino Acid Sequence↗

PubMed2000

Signalling via ADP-ribosylation factor 6 lies downstream of phosphatidylinositide 3-kinase.

ADP-ribosylation factor (ARF) 6 regulates plasma membrane trafficking and cortical actin formation by cycling between inactive GDP and active GTP-bound conformations. Here we show that agonist stimulation of phosphatidylinositide 3-kinase (PI 3-kinase) activates a pathway that leads to ARF6 activation. We also describe experiments that propose a central role in this pathway for the PI 3-kinase-dependent plasma membrane recruitment of the cytohesin-1 family of PtdIns(3,4,5)P(3)-binding ARF-exchange factors.

ADP-Ribosylation Factor 6↗

PubMed2000

Defects in protein glycosylation cause SHO1-dependent activation of a STE12 signaling pathway in yeast.

In haploid Saccharomyces cerevisiae, mating occurs by activation of the pheromone response pathway. A genetic selection for mutants that activate this pathway uncovered a class of mutants defective in cell wall integrity. Partial loss-of-function alleles of PGI1, PMI40, PSA1, DPM1, ALG1, MNN10, SPT14, and OCH1, genes required for mannose utilization and protein glycosylation, activated a pheromone-response-pathway-dependent reporter (FUS1) in cells lacking a basal signal (ste4). Pathway activation was suppressed by the addition of mannose to hexose isomerase mutants pgi1-101 and pmi40-101, which bypassed the requirement for mannose biosynthesis in these mutants. Pathway activation was also suppressed in dpm1-101 mutants by plasmids that contained RER2 or PSA1, which produce the substrates for Dpm1. Activation of FUS1 transcription in the mannose utilization/protein glycosylation mutants required some but not all proteins from three different signaling pathways: the pheromone response, invasive growth, and HOG pathways. We specifically suggest that a Sho1 --> Ste20/Ste50 --> Ste11 --> Ste7 --> Kss1 --> Ste12 pathway is responsible for activation of FUS1 transcription in these mutants. Because loss of pheromone response pathway components leads to a synthetic growth defect in mannose utilization/protein glycosylation mutants, we suggest that the Sho1 --> Ste12 pathway contributes to maintenance of cell wall integrity in vegetative cells.

Cell Wall↗

PubMed1999

Confocal imaging of the subcellular distribution of phosphatidylinositol 3,4,5-trisphosphate in insulin- and PDGF-stimulated 3T3-L1 adipocytes.

The activation of phosphatidylinositol 3-kinase (PI 3-kinase) and production of PtdIns(3,4,5)P(3) is crucial in the actions of numerous extracellular stimuli, including insulin-stimulated glucose uptake. Platelet-derived growth factor (PDGF) also stimulates PI 3-kinase, but only weakly promotes glucose uptake when compared with insulin. Insulin and PDGF have thus been proposed to have differential effects on the subcellular targeting of PI 3-kinase. However, owing to a lack of suitable methodologies, the subcellular localization of the PtdIns(3,4,5)P(3) generated has not been examined. The pleckstrin-homology (PH) domains of the nucleotide exchange factors, ADP-ribosylation factor nucleotide-binding-site opener (ARNO) and general receptor for 3-phosphoinositides (GRP1), which have a high affinity and specificity for PtdIns(3,4,5)P(3), were fused to green fluorescent protein and used to examine the subcellular localization of PtdIns(3,4,5)P(3) generation in living 3T3-L1 adipocytes. PtdIns(3,4,5)P(3) was produced almost exclusively in the plasma membrane in response to both agonists, although the response to insulin was greater in magnitude and occurred in considerably more cells. The results suggest that the greater ability of insulin to stimulate glucose uptake may be the result of its ability to generate significantly more plasma-membrane PtdIns(3, 4,5)P(3) than PDGF. ARNO and GRP1 are nucleotide exchange factors for the small GTP-binding protein ADP-ribosylation factor 6 (ARF6). The inability of a constitutively active GTPase-deficient mutant of ARF6 (ARF6-Q67L; Gln(67)-->Leu) to cause glucose transporter GLUT4 translocation suggests that activation of this pathway is not sufficient to cause GLUT4 translocation.

3T3 Cells↗

PubMed1999

Molecular cloning and functional characterization of a human homologue of centaurin-alpha.

We report here the molecular cloning, expression, and characterisation of a human homologue of rat centaurin-alpha, which we have termed centaurin-alpha(1). The cDNA contains a single open reading frame, which encodes a 373-amino-acid protein with a calculated molecular weight of 43,429 Daltons. Centaurin-alpha(1) shows high identity at the amino acid level with the other centaurin-alpha homologues, p42(IP4) and PIP(3)BP. Northern analysis revealed that centaurin-alpha(1) expresses as a single 2.5-kb transcript, mainly in the brain. Recombinant centaurin-alpha(1) binds the inositol head group of PtdIns(3,4,5)P(3) and Ins(1,3,4, 5)P(4), with high affinity (K(d) 139.7 +/- 10.5 nM) and inositol phosphate specificity, consistent with it functioning as a putative PtdIns(3,4,5)P(3) receptor. In keeping with this conclusion, we have shown that GFP-tagged centaurin-alpha(1) recruits to the plasma membrane in a PI 3-kinase-dependent manner and the recruitment is inhibited by the PI 3-kinase inhibitor wortmannin. These results suggest that centaurin-alpha(1) can function as an in vivo PtdIns(3, 4,5)P(3) receptor.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Identification of centaurin-alpha1 as a potential in vivo phosphatidylinositol 3,4,5-trisphosphate-binding protein that is functionally homologous to the yeast ADP-ribosylation factor (ARF) GTPase-activating protein, Gcs1.

Centaurin-alpha is a 46 kDa in vitro binding protein for the lipid second messenger PtdIns(3,4,5)P3. In this report we have addressed whether centaurin-alpha1, a human homologue of centaurin-alpha, binds PtdIns(3,4,5)P3 in vivo and furthermore, identified a potential physiological function for centaurin-alpha1. Using confocal microscopy of live PC12 cells, transiently transfected with a chimera of green fluorescent protein (GFP) fused to the N-terminus of centaurin-alpha1 (GFP-centaurin-alpha1), we demonstrated the rapid plasma membrane recruitment of cytosolic GFP-centaurin-alpha1 following stimulation with either nerve growth factor or epidermal growth factor. This recruitment was dependent on the centaurin-alpha1 pleckstrin homology domains and was blocked by the PtdIns(4,5)P2 3-kinase (PI 3-kinase) inhibitors wortmannin (100 nM) and LY294002 (50 microM), and also by co-expression with a dominant negative p85. Functionally, we demonstrated that centaurin-alpha1 could complement a yeast strain deficient in the ADP-ribosylation factor (ARF) GTPase-activating protein Gcs1; a complementation that was blocked by mutagenesis of conserved cysteine residues within the ARF GTPase-activating protein analogous domain of centaurin-alpha1. Taken together, our data demonstrated that centaurin-alpha1 could potentially function as an ARF GTPase-activating protein that, on agonist stimulation, was recruited to the plasma membrane possibly through an ability to interact with PtdIns(3,4,5)P3.

ADP-Ribosylation Factors↗

PubMed1999

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.

Animals↗

PubMed1999

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.

Animals↗

PubMed1999

Potential regulation of ADP-ribosylation factor 6 signalling by phosphatidylinositol 3,4,5-trisphosphate.

In this review we have described data supporting the conclusion that PtdIns(3,4,5)P3 may regulate the activation state of ARF6 through an ability to recruit the ARF exchange factors ARNO, GRP1 and cytohesin-1 to the plasma membrane. The downstream consequences of such a PtdIns(3,4,5)P3-dependent activation of ARF6 are presently unclear. However, given the role of ARF6 in fusion events at the plasma membrane, we have proposed that PtdIns(3,4,5)P3 may regulate vesicle trafficking at this membrane through its ability to activate ARF6. This is an attractive possibility given the number of PtdIns(3,4,5)P3-dependent pathways which involve some aspect of vesicle trafficking at the plasma membrane, for instance glucose transport, membrane ruffling and cell movement.

ADP-Ribosylation Factor 6↗