Patent Application
20060084790
This invention relates to a novel protein useful as an anti-inflammatory target, to nucleic acid encoding the protein, and to use of the protein in assays for identification of anti-inflammatory agents.
Monomeric GTPases are key regulators of intracellular signalling (Bourne et al. 1990). Rac proteins (Rac1, 2 and 3) are a subfamily of the Rho-family of monomeric GTPases involved in receptor regulation of responses such as transcriptional activation, lamellipodia formation and stimulation of reactive oxygen species (ROS) production (Tapon and Hall 1997). Rho-family monomeric GTPases are molecular switches that are ‘on’, and can activate effector proteins, when GTP-bound and ‘off’ when GDP-bound. The GTPases can be activated by guanine-nucleotide exchange factors (GEFs) that act to accelerate nucleotide exchange by prising open the binding site of specifically the GDP-bound form of the GTPases (Worthylake et al. 2000).
There is a large family of Rac-GEFs (though some can also act as GEFs for other monomeric GTPases). These include Vav (1, 2, 3), Tiam (1, 2), PIX (α, β), Ras-GRF (1, 2), and Sos (Manser et al. 1998, Scita et al. 1999, Stam and Collard 1999). Protein kinases currently seem the major direct regulators of Rac-GEF activity. For example, Vav1 can be phosphorylated on tyrosine 174 and activated by Lck (Crespo et al. 1997, Han et al. 1997). Similarly, Ras-GRF1 has to be tyrosine-phosphorylated to display Rac-GEF activity (Kiyono et al. 1999), and Tiam1 is phosphorylated and regulated possibly by Ca2+/calmodulin-dependent protein kinase II (Fleming et al. 2000). Other regulators of Rac-GEFs, for example phosphoinositide 3-kinases (PI3Ks) and Gβγs, largely work by affecting these phosphorylations (Han et al. 1998, Kiyono et al. 1999).
Type 1 PI3Ks can be activated by cell-surface receptors to synthesize the intracellular messenger phosphatidylinositol(3,4,5)P3 (PtdIns(3,4,5)P3). The signalling targets of PtdIns(3,4,5)P3 typically possess a PH domain that can bind the lipid and drive translocation of the host protein to the site of PtdIns(3,4,5)P3-accumulation in the plasma membrane (not all PH domains bind PtdIns(3,4,5)P3) (Lemmon and Ferguson 2000). In many cells, type 1 PI3Ks have been shown to be necessary for receptor-driven stimulation of Rac, and activated type 1 PI3Ks are sufficient to activate Rac (Hawkins et al. 1995, Reif et al. 1996). These pathways are widely important and underpin responses such as lamellipodia formation and associated membrane raffling and possibly ROS formation. Despite this, the mechanisms by which type 1 PI3Ks and/or PtdIns(3,4,5)P3 can activate Rac are unclear in many cellular contexts. This is partly a consequence of the fact that no Rac-GEFs have been purified and identified on the basis of their activity and, relevantly here, from a cellular context that displays PI3K-dependent activation of Rac. On the basis of studies subsequent to their original discovery and characterization, four subgroups of the currently known Rac-GEFs have been claimed to be regulated in a PI3K-dependent fashion, namely Tiam, Vav, Sos, and PIX. However, these effects of PI3K and/or PtdIns(3,4,5)P3 are, where direct, small or, where indirect, via modulation of unknown or phosphorylation-based mechanisms (Han et al. 1998, Rameh et al. 1997, Fleming et al. 2000, Buchanan et al. 2000, Yoshii et al. 1999, Scita et al. 1999, Das et al. 2000, Nimnual et al. 1998), the mechanism by which PI3Ks regulate this complex is unclear.
In neutrophil-like cells, Rac plays important roles in a variety of signalling pathways, particularly activation of PAK kinases and phospholipase D and further downstream responses such as chemotaxis, phagocytosis and ROS formation (Roberts et al. 1999, Dorseuil et al. 1992). Its roles in co-ordinating receptor-stimulated ROS formation are probably best understood. Rac (Rac2 in most species) is along with p47phox, p67phox, gp91phox and gp22phox, a component of the catalytically active oxidase complex that is assembled on the phagosomal/endosomal membrane system of appropriately stimulated cells (Babior 1999). This process has been correlated with activation of Rac (Akasaki et al. 1999, Benard et al. 1999). It has been demonstrated to be inhibited by Rac-GTPase activating proteins (GAPs) in vitro (Geiszt et al. 2001), augmented in Rac-GAP knockouts (Bcr) (Roberts et al. 1999), inhibited in some immundeficient patients that carry key mutations in Rac2 (Ambruso et al. 2000), and GTP-bound but not GDP-bound Rac can both bind p67phox and p91phox and activate PAK kinases that are claimed to phosphorylate p47 and p67phox (Babior 1999). However, some work suggests receptor-stimulated ROS formation can occur without activation of Rac (Geijsen et al. 1999), implying basal levels of GTP-Rac are sufficient, or simply not necessary, for some regulatory mechanisms.
There is evidence that PI3Ks play a key role in neutrophils in mediating signalling between activation of G-protein linked receptors and stimulation of ROS formation. Type 1B PI3K nullizygotes fail to produce, and PI3K inhibitors block, ROS formation in response to inflammatory mediators (Condliffe and Hawkins 2000). The mechanism, however, by which PI3Ks contribute to driving ROS formation is unclear. We have shown that PtdIns3P (a potential break-down product of PtdIns(3,4,5)P3) regulates ROS formation via binding to the PX domain of p40phox, an effect that can be detected in the presence of GTPγS-Rac and hence cannot involve Rac activation (Ellson et al. 2001). Some data does support the idea that in neutrophils type 1 PI3Ks may be upstream of activation of Rac. The PI3K inhibitors LY294002 and wortmannin have been shown to significantly reduce activation of Rac in response to inflammatory mediators (Akasaki et al. 1999, Benard et al. 1999). However one paper has presented convincing data showing that fMLP-stimulated activation of Rac is resistant to PI3K inhibitors, apparently contradicting the work described above (but see discussion) (Geijsen et al. 1999) and has instead, along with the precedent set by p115Rho-GEF that is activated by Gα3 (Hart et al. 1998), lead to the suggestion a Gα subunit may activate one or more neutrophil Rac-GEF activities (Geijsen et al. 1999).
The identity of the Rac-GEF(s) that is (are) involved in receptor-stimulated activation of Rac and/or ROS formation in neutrophils remain unknown. In the context of the distributions and properties of the known Rac-GEFs and the types of receptors that can drive ROS formation, it seems plausible that Vav and/or SOS proteins could be downstream of the protein-tyrosine linked receptors whilst there are no clear candidates for a similar role downstream of the G-protein linked receptors.
We have purified a PtdIns(3,4,5)P3-sensitive activator of Rac from neutrophil cytosol. It is an abundant, novel, 185 kD guanine-nucleotide exchange factor (GEF), which we cloned and named P-Rex1. The recombinant enzyme has Rac-GEF activity that is directly, substantially and synergistically activated by PtdIns(3,4,5)P3 and Gβγs both in vitro and in vivo. P-Rex1 antisense oligonucleotides reduced endogenous P-Rex1 expression and C5a-stimulated reactive oxygen species formation in a neutrophil-like cell line. P-Rex1 appears to be a novel coincidence detector in PtdIns(3,4,5)P3 and Gβγ signalling pathways that is particularly adapted to function downstream of activation of heterotrimeric G proteins in neutrophils.
According to the invention there is provided a protein in substantially isolated form comprising the amino acid sequence of P-Rex1, or a derivative thereof which has P-Rex1 activity.
Preferably the protein comprises the amino acid sequence of human P-Rex1 (SEQ ID NO:1, shown in
A derivative of P-Rex1 may be a protein which differs from wild-type P-Rex1 by one or more amino acid alterations (substitutions, additions, deletions, or modifications including post-translational modifications) but which retains at least one of the activities of wild-type P-Rex1. These activities include Rac-GEF activity, binding with PtdIns(3,4,5)P3, and binding with Gβγ-subunits. Preferably the derivative of P-Rex1 retains Rac-GEF activity. Preferably a derivative of P-Rex1 comprises up to about 40, more preferably up to about 20, amino acid alterations per each 100 amino acid residues of SEQ ID NO:1.
Derivatives of P-Rex1 may be made by standard mutagenesis techniques known to those of ordinary skill in the art, and may be tested for any of the activities of P-Rex1 using standard techniques.
There is also provided according to the invention a protein comprising the amino acid sequence of one or more of the different domains of human P-Rex1 (SEQ ID NOs: 2-8). These are identified in the description of
There is also provided according to the invention a protein which has PtdIns(3,4,5)P3-sensitive and/or Gβγ-subunit-sensitive Rac-GEF activity.
There is further provided according to the invention a splice variant of P-Rex1, and nucleic acid encoding the splice variant. Splice variants may be identified by standard techniques known in the art.
There is further provided according to the invention a nucleic acid in substantially isolated form comprising sequence encoding a protein of the invention. There is also provided according to the invention nucleic acid in substantially isolated form which is capable of hybridizing under stringent conditions to nucleic acid encoding a protein of the invention, or to nucleic acid which is complementary to nucleic acid encoding a protein of the invention.
The term “stringent conditions” as used herein means hybridization conditions generally understood by a person skilled in the art to correspond to stringent conditions specified in widely recognized protocols for nucleic acid hybridization. See, for example, Sambrook et al, Molecular Cloning: A laboratory Manual (2nd Edition), Cold Spring Harbor Laboratory Press (1989), pp. 1.101-1.104; 9.47-9.58 and 11.45-11.57. Typically these conditions comprise at least one wash of the hybridization membrane in 0.05× to 0.5×SSC with 0.1% SDS at 65° C., or washing conditions of equivalent stringency.
There is also provided according to the invention a P-Rex1 probe which is capable of hybridizing under stringent conditions to nucleic acid encoding human P-Rex1. The probe may be used to identify P-Rex1 genes in other animals.
According to the invention there is also provided an oligonucleotide primer for amplifying nucleic acid of the invention, for example by PCR.
There is also provided according to the invention a vector comprising nucleic acid of the invention. The vector may be an expression vector for expression of a protein of the invention.
There is also provided according to the invention a host cell comprising a vector of the invention. The host cell may be a bacterial cell, a mammalian cell, a yeast cell, a plant cell, or an insect cell
There is also provided a cell stably transfected with a nucleic acid of the invention.
According to the invention there is also provided a method for producing a protein of the invention which comprises culturing a host cell comprising a vector capable of directing expression of the protein under conditions for expression of the protein.
In some circumstances it may be desirable to up-regulate endogenous P-Rex1 expression in a cell. This may be achieved by transforming a host cell with a vector comprising a promoter capable of inserting (for example by homologous recombination) upstream of endogenous nucleic acid encoding P-Rex1 so that expression of P-Rex1 is under the control of the inserted promoter.
There is also provided according to the invention an antibody (or antibody fragment) capable of binding to a protein of the invention, preferably to an epitope which is specific to the protein thereby allowing detection of cellular P-Rex1, and/or recombinant P-Rex1. Such antibodies may be made by techniques known to those of ordinary skill in the art. Sheep polyclonal anti-P-Rex1 antibodies are described in the Experimental Procedures section below. These are anti-peptide sheep polyclonal antibodies against human P-Rex1. A pool of two sera affinity-purified together using recombinant human wild-type P-Rex1 can be used for affinity purification. These antibodies can be used for Western blots and for immunofluorescence experiments with overexpressed P-Rex1. Protocols on how to prepare samples for use with these antibodies, and use of the antibodies for Western blots are described below.
P-Rex1 has been identified as a protein involved in inflammatory pathways in white cells and is associated with superoxide formation and chemotaxis. It is also possible that P-Rex1 may have a role in metastasis, septic shock, neuro-degeneration involving inflammatory or free-radical mechanisms, and atherosclerosis. Thus, inhibitors of P-Rex1 activity, or of binding of P-Rex1 with a binding partner, or of P-Rex1 expression may reduce or inhibit any of the following: inflammation, metastasis, septic shock, neuro-degeneration, and atherosclerosis. It is also possible that stimulation of P-Rex1 activity might be of value in acute bacterial infections.
According to the invention there is further provided a fragment or derivative of P-Rex1 capable of antagonising P-Rex1 activity. Fragments or derivatives of P-Rex1 can readily be made and tested to see whether they inhibit P-Rex1 activity, or binding of P-Rex1 with a binding partner, by a person of ordinary skill in the art.
There is also provided according to the invention an antisense oligonucleotide capable of inhibiting expression of P-Rex1. The antisense oligonucleotide may be a DNA or RNA oligonucleotide capable of binding DNA of the P-Rex1 gene, or RNA expressed from the P-Rex1 gene. Thus, DNA-DNA, RNA-RNA, or DNA-RNA hybrids may be formed. There is also provided an interfering RNA (dsRNAi) capable of inhibiting expression of P-Rex1.
There is also provided according to the invention a vector comprising nucleic acid capable of undergoing homologous recombination with nucleic acid of the P-Rex1 gene to thereby inhibit P-Rex1 expression from the gene.
According to the invention there is also provided a non human animal which is heterozygous or homozygous for a disrupted P-Rex1 gene. Preferably the animal is a P-Rex1 gene knock-out mouse. There is also provided a non human animal, preferably a mouse, with a P-Rex1 transgene. There is also provided a P-Rex1 gene knock-in mouse. Such animals can be used as in vivo models in the investigation of inflammation, metastasis, septic shock, neuro-degeneration, atherosclerosis, or bacterial infection.
According to the invention there is also provided a targeting vector comprising nucleic acid capable of undergoing homologous recombination with genomic DNA encoding the P-Rex1 gene, and a selectable marker, so that when nucleic acid of the targeting vector undergoes homologous recombination with the genomic DNA, the nucleic acid encoding the selectable marker is incorporated into the genomic DNA and expression of the P-Rex1 gene is prevented or reduced.
Preferably the targeting vector comprises nucleic acid sequence of the P-Rex1 gene in which nucleic acid sequence of an exon of the gene is replaced by nucleic acid sequence encoding the selectable marker.
Preferably the targeting vector comprises at least 8-10 kb of nucleic acid sequence of the P-Rex1 gene.
Preferably nucleic acid sequence of the P-Rex1 gene is split from 20/80% to 50/50% between the 5′ and 3′ arms of the vector. Preferably from 20 to 80% of the nucleic acid sequence of the P-Rex1 gene is 5′ of the nucleic acid sequence encoding the selectable marker, with the remainder being 3′ of the nucleic acid sequence encoding the selectable marker.
Preferably exon 5 of the P-Rex1 nucleic acid sequence is replaced by the nucleic acid sequence encoding the selectable marker. Exons 1-8 of the P-Rex1 gene are all in frame. Consequently, if any of these exons are replaced with the nucleic acid sequence encoding the selectable marker, it is theoretically possible that the remaining exons could be re-spliced together and an almost full length protein could be produced missing a few amino acids. Exon 5 codes for the catalytic site of the P-Rex1 protein, so even if splicing from exon 4 to exon 6 occurs, any resulting protein would be inactive.
Preferably the nucleic acid encoding the selectable marker codes for antibiotic resistance. Preferably the antibiotic resistance is neomycin, gentomycin, hygromycin, or puromycin resistance.
There is further provided according to the invention a mouse ES cell comprising a targeting vector of the invention. There is also provided according to the invention a recombinant mouse ES cell in which expression of a P-Rex1 gene has been prevented or reduced. The mouse ES cell may be a cell of an E14, CCB, R1, or R3 ES cell line.
There is also provided according to the invention a pseudo-pregnant mouse comprising an implanted recombinant mouse ES cell in which expression of a P-Rex1 gene has been prevented or reduced.
There is further provided according to the invention a recombinant heterozygous mouse in which expression of a P-Rex1 gene has been prevented or reduced on one of the chromosome pairs.
According to the invention there is also provided a protein of the invention which further comprises a purification tag allowing affinity purification of the protein. There is also provided a protein of the invention further comprising an epitope tag allowing detection of the protein with an anti-epitope antibody.
There is also provided a protein of the invention comprising a label allowing detection of the protein. Preferably the label is a fluorophore or a radioactive label.
According to the invention there is also provided a fusion protein comprising a protein of the invention. The fusion protein may comprise green fluorescent protein (GFP), or a variant or derivative of GFP which has fluorescent activity to allow detection of the fusion protein. The fusion protein may comprise a purification tag allowing affinity purification of the fusion protein.
According to the invention there is also provided use of a protein of the invention, a tagged or labeled protein of the invention, or a fusion protein of the invention, as a target for drug discovery. Such use is expected to allow identification of a drug with anti-inflammatory activity. It is also possible that drugs which reduce or inhibit metastasis, septic shock, neuro-degeneration, or atherosclerosis may be identified.
The invention also provides use of a protein of the invention, or a nucleic acid of the invention, in a screening assay to identify a modulator of binding of P-Rex1 with a binding partner, a modulator of P-Rex1 activity, or a modulator of P-Rex1 expression. In vitro or cell-based assays may be used. In general such assays will be used to identify an inhibitor of P-Rex1 binding, or of P-Rex1 activity or expression because such compounds will have the potential to reduce or inhibit inflammation, metastasis, septic shock, neuro-degeneration, or atherosclerosis, or be of use in designing or identifying drugs which have such activity.
According to the invention there is provided a method for identifying a modulator of P-Rex1 activity which comprises contacting a protein of the invention with a candidate modulator and determining whether activity of the protein is modulated by the candidate modulator. In such methods, for example when assaying for Rac-GEF activity, it may be desirable to perform the method in the presence of PIP3 and/or Gβγ-subunits or derivatives thereof which can activate the Rac-GEF activity of P-Rex1.
There is also provided according to the invention a method for identifying a modulator of binding of P-Rex1 with a binding partner which comprises contacting a protein of the invention with a binding partner in the presence and absence of a candidate modulator, and determining whether binding of the protein to the binding partner is modulated by the candidate modulator.
Typically, assays for modulators will be designed to find compounds which inhibit the interaction of P-Rex-1 with either PIP3 or one or more of the proteins with which P-Rex-1 interacts. In vitro assays preferably involve use of full length P-Rex-1, a truncated sequence or fusion protein with a green fluorescent protein (GFP), all with an appropriate purification tag, expressed in an in vitro expression system such as baculovirus or E. coli and purified using an appropriate purification system. If not produced as a fusion protein with a fluorescent protein, the purified protein may either be chemically labeled with a fluorophore (typically fluorescein, rhodomine or other fluorescent dyes with an excitation maximum of>450 nm) using standard methodologies for labeling proteins, or labeled with a radioactive label. The protein may be incubated with the individual candidate modulators and the remainder of the assay reagents, and the interaction measured either by direct spectrophotometric measurement, or following separation of the bound and free P-Rex-1 constructs.
Preferred assay methods are listed below. All of these methods can be readily applied to high throughput screening of>10,000 compounds per day using commercially-available equipment.
As an adaptation of method (8) above, SPA plates could be coated with GDP-Rac and this incubated with P-Rex1 and radioisotope-labelled GTPγS. The association of the radiolabelled GTP with the immobilised Rac would be measured continuously by determination of scintillation proximity.
There is also provided according to the invention use of a cell-based assay to identify a modulator of binding of P-Rex1 with a binding partner, a modulator of P-Rex1 activity, or a modulator of P-Rex1 expression.
A cell-based assay according to the invention for identifying a modulator of binding of P-Rex1 with a binding partner, or a modulator of P-Rex1 activity comprises:
The cell may be a wild-type cell expressing P-Rex1, or comprise exogenous nucleic acid directing expression of P-Rex1 in the cell.
There is also provided according to the invention a cell-based assay for identifying a modulator of binding of P-Rex1 with a binding partner or a modulator of P-Rex1 activity which comprises:
The binding or activity of P-Rex1 may be determined by superoxide formation, chemotaxis, lamellipodia formation, or by use of reporter gene expression, fluorescence, measurement of protein movement from one cellular location or compartment to another, or by examination of lamellipodia formation. The stimulus is preferably an inflammatory mediator, for example one that stimulates superoxide formation, chemotaxis, or lamellipodia formation.
A further cell-based assay comprises over-expressing P-Rex1 in a cell in the presence and absence of a candidate modulator, and determining whether lamellipodia formation is altered by the candidate modulator.
It will be appreciated that appropriate controls will be necessary to ensure that any modulators identified are modulating P-Rex1 activity or binding of P-Rex1 with a binding partner.
Preferred cell-based assay methods are listed below. In these assays, the cells are pre-incubated for 30 minutes in medium with a candidate modulator, prior to addition of the stimulus, to allow cell penetration.
A yeast two-hybrid (or three-hybrid) system may be used for identification of a modulator of binding of P-Rex1 to a binding partner.
There is also provided according to the invention an inactive mutant of P-Rex1, a nucleic acid encoding the mutant, and an antibody capable of binding the mutant with higher affinity than wild-type P-Rex1, to thereby allow specific detection of the mutant. There is further provided use of the mutant, nucleic acid, or antibody in a screening assay.
According to the invention there is also provided a P-Rex1-negative cell, a cell comprising an inhibitor which inhibits P-Rex1 expression in the cell, and extracts from such cells. There is also provided use of such cells or extracts in a screening assay.
Such mutants, cells, and extracts may be used as controls in screening assays to identify a modulator of binding of P-Rex1 with a binding partner, or a modulator of P-Rex1 activity.
It is possible that mutations in the P-Rex1 gene or an expression product of the P-Rex1 gene, or differences in the expression level of P-Rex1, or in the pattern of expression of the P-Rex1 gene, may be associated with a disease or disorder. Such mutations, or differences in expression could be identified by standard techniques known to those of skill in the art in which disease and normal biological material (such as tissue, cells or extracts) are compared to see whether there are any mutations or differences in expression which are associated with the disease tissue, but not the normal tissue. Detection of any differences identified could then be used as the basis of a diagnostic test to identify individuals with, or susceptible to, the disease or disorder.
There is also provided according to the invention use of an in vitro or cell-based assay to identify a modulator of a P-Rex1 dependent signalling pathway. Such use will preferably also require use of a protein, nucleic acid, vector, antibody, cell, or extract of the invention.
Embodiments of the invention are further described with reference to the accompanying drawings:
A) P13K and Gβγ synergistically stimulate ROS formation. Neutrophil cytosol and low-density membranes were incubated with 45 nM recombinant p101/p110 PI3K and/or 54 nM bovine-brain Gβγ, either with wortmannin (200 nM grey bars), or without (black bars), or with dominant-negative N17-Rac (200 nM, hatched bars), and ROS formation (SPC, single photon counts in 0.1 min) was measured. Data are means (n=4)±SD from two experiments. B) PtdIns(3,4,5)P3 stimulates ROS formation. Neutrophil cytosol and low-density membranes were incubated with isomers of PtdIns(3,4,5)P3, PtdIns(3,4,)P2 or PtdIns(4,5)P2, (S/A, stearoyl-arachidonyl, P/P, dipalmitoyl) and ROS formation was measured. Data are means (n=2-6)±range. C) PI3K and Gβγ synergistically stimulate Rac. Neutrophil cytosol and low-density membranes were incubated with PI3K (50 nM), PtdIns(3,4,5)P3 (30 μM) and/or Gβγ (40 nM in left panel, 200 nM in right panel) either with (grey bars) or without (black bars) wortmannin (200 nM) and incorporation of [α32P]-GTP into EE-Rac1 (30 nM) was quantified (means (n=4-8)±SD from 4 experiments). D) Active Rac induces ROS formation. Neutrophil cytosol and low-density membranes were incubated either with Wt-Rac (black bars) or dominant-negative N17-Rac (200 nM, white bars), preloaded and incubated with the indicated guanine nucleotides and, for Wt-Rac, with wortmannin (200 nM, grey bar), and ROS formation was measured. Data are means (n=2-6)±range from three experiments.
A) Chromatography profiles. The PtdIns(3,4,5)P3-dependent Rac-GEF activity was purified from 90 l of pigs' blood using this column sequence. The dotted line represents absorption at 280 nm, the dashed line shows salt concentration. Column fractions were assayed for Rac-GEF activity using liposomes either with (thick black line) or without (hatched line) PtdIns(3,4,5)P3. Grey bars represent the fractions selected for the following purification step.
B) Silver-stained SDS-page of fractions including the peak of PtdIns(3,4,5)P3-dependent Rac-GEF activity recovered from columns at the gel filtration (1% fraction vol.) and Mono S (1.67% fraction vol.) purification steps. C) Purification summary. The absolute activity of the starting material (100%) was calculated to be stimulating the loading of 1.4 p.mol of GTPγS onto Rac (above Rac alone) min−1.mg−1 protein, in the presence of PtdIns(3,4,5)P3, under the conditions described in the methods.
A) Amino acid sequence of human P-Rex1 (SEQ ID NO:1). Tryptic peptides obtained from purified P-Rex1 are residues 182(K)-198(R), 913(T)-920(R), 1463(L)-1470(K), 1501(V)-1506(R), and 1590(S)-1604(R). Protein homology domains are underlined. These are:
B) Schematic representation of the domain structure of P-Rex1.
A) Northern blots. Multiple tissue northern blots from Clontech were probed for P-Rex1 mRNA expression. B) Western blot. EE-epitope tagged P-Rex1 was transiently expressed in COS-7 cells, then extracted with 1% Triton-X100 containing buffer, and aliquots of a 10,000 g supernatant (equivalent to 5×103, 5×104, 5×105 cells/lane) were immunoblotted with anti-EE antibody. C) Recombinant human P-Rex1 GEF activity was assayed using liposomes (PtdCho, PtdS, PtdIns, 200 μM each) with (dark bars) or without (hatched bars) PtdIns(3,4,5)P3 (10 μM) and the indicated purified GTPases (100 nM). Data are duplicate means±range from one of three experiments.
A) PtdIns(3,4,5)P3 dose response. P-Rex1-dependent activation of EE-Rac1 was assayed in the presence of liposomes containing 200 μM each of PtdCho, PtdS, PtdCho and the indicated concentrations of PtdIns(3,4,5)P3 (final P-Rex1 concentration was 100 nM). Data are means (n=2-4)±range from two pooled experiments. B) Lipid specificity of P-Rex1-dependent activation of Rac was measured in the presence of liposomes (as in A) with either 10 μM (dark bars) or 0.3 μM (white inset bars) of the indicated phosphoinositides (S/A, stearoyl-arachidonyl, P/P, dipalmitoyl). Data are duplicate means±range obtained from one of two separate experiments. C) Phosphoinositide-dependent binding of P-Rex1 (100 nM) to liposomes containing PtdE, PtdS, PtdCho (330 μM each) and the indicated phosphoinositides (6 mol-%) was measured by Biacore. Data are means±SD from 4 pooled experiments. D) Gβγ dose response. P-Rex1-dependent activation of Rac was assayed using the indicated concentrations of purified bovine brain Gβγ. Final cholate concentration was 0.0072% except for 1 μM Gβγ samples (0.0104%). Data are duplicate means±range from one of three experiments. E) Controls for Gβγ effects. P-Rex1 -dependent activation of Rac was assayed using, where indicated, Gβγ (0.3 μM, bovine brain-derived except where indicated to be prenylated or non-prenylated, which were derived from Sf9 cells), mixed Gα subunits (0.23 μM), A1F (10 μM), boiled bovine-brain Gβγ (0.5 μM), recombinant prenylated Gβγ (0.5 μM), or recombinant non-prenylated Gβγ (0.5 μM. For combinations of Gβγ and Gα, these (or control buffers) were preincubated for 30 min on ice. Final cholate concentration was 0.012%. Data are duplicate means±range from one experiment. F) Synergy between PtdIns(3,4,5)P3 (0.2 μM and bovine-brain Gβγ (0.3 μM) in the regulation of P-Rex1 Rac-GEF activity. Final cholate concentration was 0.0048%. Data are duplicate means±range from one of three experiments.
A) Western blots of Rac activation by P-Rex1 in vivo. Aliquots of 5×106 Sf9 cells in 6 cm dishes were infected with combinations of viruses encoding P-Rex1, Gβ1, Gγ2, p101, p110γ or control viruses where indicated. After 42.5 h in growth medium, then 4 h serum-free, the cells were subjected to a PAK-Crib pull-down assay. Inmunoblots were probed with anti Rac (top and second panel) or anti-CDC42 (third panel) antibodies. The equivalent of 0.18 dishes of cells was loaded from PAK-Crib pull downs and 0.05 dishes for total lysates. The bottom panel shows the second panel filter after staining with coomassie. B) Synergistic PI3K and Gβγ-dependent activation of Rac by P-Rex1 in vivo. Sf9 cells were infected with the above viruses as indicated, then treated as in A). ECL-exposed films were digitized, and the data shown are means±range (n=4) from two pooled experiments. C) Gβγ and/or PI3K-induced formation of PtdIns(3,4,5)P3 in Sf9 cells was measured (data are means (n=5)±range) and plotted against P-Rex1-dependent Gβγ and/or PI3K-induced activation of Rac (data from B).
A) Immunofluorescence micrographs of serum starved normal (first and second panel) or stably V12-Rac transfected (third panel) PAE cells after stimulation with 10 ng/ml PDGF for 5 min (second panel) or without (first and third panel). Fixed cells were labelled with FITC-phalloidin to stain filamentous actin. B and C) Expression of P-Rex1 in PAE cells. Myc-tagged P-Rex1 or DAPP1 were transiently expressed in PAB cells, these were grown (10 h), serum starved (8 h), treated with wortrnannin (100 nM, 10 min) or not, and then stimulated with a range of PDGF concentrations for 5 min, as indicated. Cells were fixed and stained with anti-myc antibody followed by FFIC secondary antibody to label P-Rex1 or DAPP1 and TRITC-phalloidin to label filamentous actin. B) Immunofluorescence micrographs. C) Quantification of immunofluorescence microscopy data. Results were obtained by counting 100 P-Rex1-positive cells (dark bars) or DAPP1-positive cells (hatched bars) per coverslip. P-Rex1 data are from duplicate coverslips (means±range) from one of two independent experiments. DAPP1 data are from one coverslip per condition from one experiment.
Human promyelocytic NB4 cells were differentiated for 2 days with 1 μM all-trans retinoic acid and treated with 10 μM of either P-Rex1 antisense oligonucleotide or randomised control oligonucleotide and then subjected to the experiments below. A) Oligonucleotide-treated NB4 cells were stimulated with 0.15 nM C5a and ROS formation (SPC, single photon counts) was measured. Data are mean±stdev (n=4) from one of 3 experiments. B) Total lysates of P-Rex1-transfected or control Cos7 cells, human neutrophils, or oligonucleotide-treated NB4 cells were analysed for P-Rex1 expression level by Western blot using a polyclonal anti-P-Rex1 antibody. C) Oligonucleotide-treated NB4 cells were serum-starved and then stimulated with C5a as indicated for 3 min at RT. MapK activation was measured by Western blot using a phospho-MapK antibody and densitometric scanning of the blots. Data are mean±range (n=2) from 2 experiments.
Genomic sequence (SEQ ID NO:14) for human P-Rex1 is given in the Sequence Listing.
PI3K, Gβγ and PtdIns(3,4,5)P3-Regulation of Rac Activation and ROS Formation
Mixtures of cytosol and low-density membranes from neutrophils can be stimulated to produce ROS by the addition of amphiphiles such as SDS and arachidonic acid. We tested the idea that type 1 PI3Ks could operate upstream of ROS formation, by adding combinations of purified, recombinant p101/p110γ-PI3K and purified Gβγ (either recombinant SF9-derived Gβ1γ2 or bovine brain Gβγs; both of which can substantially activate p101/p110γ-PI3K), in the presence of MgATP and GTP. We have shown previously that under similar conditions PtdIns(3,4,5)P3, PtdIns(3,4)P2 and PtdIns3P are synthesized in these assays (Pacold et al. 2000). Although the assays contain endogenous PI3Ks and Gβγs, the added recombinant proteins independently activated ROS formation but acted synergistically when added together (
In context of the literature defining an important role for PI3Ks in activation of Rac and the important part Rac plays in the assembly of the oxidase complex, we asked the question; do these effects depend on activation of Rac? By adding small amounts of pure, recombinant, post-translationally lipid-modified EE-Rac1 and [α32P]-GTP into these assays we could show p101/p110γ-PI3K and Gβγ can independently and, in combination, synergistically activate Rac (
The above data suggest Rac can act downstream of p101/p110γ-PI3K, Gβγ and PtdIns(3,4,5)P3 in stimulation of ROS formation in these assays. We tested whether purified, recombinant, lipid-modified Rac could stimulate ROS formation. GTPγS-Rac1 stimulated ROS formation substantially more effectively than GDP-Rac1 or GTPγS-treated N17-Rac1 (
Purification of a PtdIns(3,4,5)P3-Sensitive Rac-GEF from Neutrophil Cytosol Fractions
We found the Ptd(3,4,5)P3-stimulated Rac-GEF activity was recovered in cytosol fractions and attempted to purify the enzyme(s) responsible from this source (
Cloning and Expression of Human P-Rex1
We cloned the relevant human gene using a combination of library screening from random-primed human U937 cell and spleen cDNA libraries and PCR from a human leukocyte marathon-ready cDNA library. Together these approaches yielded a novel full length ORF of 4980 bp (accession number AJ320261), with the start ATG being preceded by a passable Kozak sequence and a CG-rich region at the N-terminus, but no upstream stop codon, leaving a small possibility that we have not identified the true start ATG. Underlying genomic sequence showed that the coding sequence of P-Rex1 is arranged into 41 exons, stretched over more than 300 kb of chromosome 20 at q13.13 (AL131078, AL049541, AL445192, AL035106, AL133342). It also revealed the potential existence of a splice variant and a potential homologue on chromosome 8 (see database entry EST BAB14375).
The P-Rex1 protein sequence is 1659 amino acids long, predicting a protein of 185 kD, and harbours all five tryptic peptides obtained from the purified pig enzyme (
We have studied P-Rex1 mRNA expression by probing human multiple-tissue Northern blots from Clontech with a probe made from 673 bp of the P-Rex1 coding sequence. The northern blots revealed a major band of approximately 6 kb which is consistent with the expected size of full length P-Rex1 mRNA and a minor band just below. They show that P-Rex1 is expressed mainly in peripheral blood leukocytes and brain, less in spleen and lymph nodes and much weaker in most other tissues (
We transiently expressed P-Rex1 with an N-terminal EE-epitope tag in COS-7 cells, and anti-EE Western blots revealed a protein with an apparent MW of 197 kD in the cell lysates (
PtdIns(3,4,5)P3- and Gβγ-Dependent Activation of Rac by P-Rex-1 in Vitro
We expressed P-Rex1 with an N-terminal EE-tag in SF9 cells. The protein expressed well and could be purified to greater than 95% purity in one step using a monoclonal anti-EE antibody cross-linked to protein G-sepharose.
Recombinant P-Rex1 displayed PtdIns(3,4,5)P3-sensitive Rac-GEF activity very similar to that of the purified protein. We tested the specificity of P-Rex1 for various Rho-family GTPases and Rac proteins that were with or without post-translational lipid modifications or carried different epitope-tags. P-Rex1 displayed similar PtdIns(3,4,5)P3-sensitive activity against Rac1, Rac2 and CDC42 and low activity against RhoA (
Further analysis of the Rac-GEF activity of P-Rex1 showed that PtdIns(3,4,5)P3 had a 50% maximal effect at 0.3 μM (
We have analysed the interaction between soluble P-Rex1 and PtdIns(3,4,5)P3- or PtdIns(3,4)P2-containing phospholipid vesicles, immobilised on a dextran-coated L1 gold chip, utilising surface plasmon energy transfer technology (BiaCore). P-Rex1-binding to phospholipid vesicles was substantially augmented by the inclusion of PtdIns(3,4,5)P3, and to a lesser extent by PtdIns(3,4)P2 (
Although we had no direct assay data to support the possibility that Gβγs could activate P-Rex1 directly, the presence of the DEP domains, which commonly occur in proteins that interact with heterotrimeric G-proteins, and our earlier results with neutrophil cytosol/membrane mixtures, encouraged us to test the effects of Gβγs on P-Rex1 Rac-GEF activity. Pure Gβγs from bovine brain or prepared as recombinant G-EE-β1γ2 from co-infected SF9 cells both activated P-Rex1 Rac-GEF activity in vitro (
PtdIns(3,4,5)P3- and Gβγ-Dependent Activation of Rac by P-Rex-1 in Vivo
To address questions over the selectivity of P-Rex1 for Rac versus CDC42 in cells and the physiological significance of the effects of Gβγs and PtdIns(3,4,5)P3 we have observed in the test-tube, we prepared the relevant baculoviruses to allow us to study the activation of endogenous Rac and CDC42 in SF9 cells. We found that SF9 cells infected with baculoviruses driving P-Rex1, p101/p110γ-PI3K and Gβ1γ2 production showed substantial increases in the levels of endogenous GTP-Rac but no change in the levels of endogenous GTP-CDC42, suggesting that in vivo P-Rex1 acts as a Rac-GEF (
P-Rex1 Induces a Phenotype Like Constitutively-Active Rac in PDGF-Stimulated PAE Cells
We sought evidence that P-Rex1 can be regulated by signalling pathways downstream of cell-surface receptors and act as a Rac-GEF in mammalian cells. We used a porcine aortic endothelial (PAE) cell line that stably overexpresses the PDGF β-receptor. In these cells PDGF stimulates PtdIns(3,4,5)P3 accumulation, wortmannin-sensitive activation of Rac and Rac-dependent membrane ruffling and lamellipodia formation, and stable overexpression of constitutively active V12-Rac causes the formation of strongly exaggerated lamellipodia (‘fried eggs’) (
PAE cells were transiently transfected with N-terminally myc-tagged P-Rex1, serum-starved and the effects of PDGF-stimulation on cell shape and the distribution of myc-P-Rex1 were analyzed by indirect immunofluorescence microscopy (
Agonist-Stimulated ROS Formation in a Neutrophil-Like Cell Line is Dependant on P-Rex1.
We treated a promyelocytic cell line (NB4) with retinoic acid and either phosphorothioate antisense oligonucleotide targetted against P-Rex1 or a randomised control oligonucleotide. After 2 days, both populations of cells had differentiated normally and displayed indistinguishable MAPK activation in response to C5a (
Finally, as the C-terminal half of P-Rex1 has substantial homology with Inositol Polyphosphate 4-Phosphatase, we attempted to determine whether the protein possessed Inositol polyphosphate 4-phosphatase activity using 32P-PtdIns(3,4,5)P3 and 32P-PtdIns(3,4)P2 as substrates and Inositol Polyphosphate 4-Phosphatase and SHIP-1 as controls. We also used para-nitrophenolphosphate as a broad-spectrum substrate in a protein phosphatase assay, using calf intestinal alkaline phosphatase and MEG-2 tyrosine phosphatase as controls. At P-Rex1 concentrations of up to 1.45 μM and 192 nM, respectively, in these assays, the enzyme exhibited no lipid or protein phosphatase activity.
To our knowledge, no Rho-family or Ras-family GEFs have been successfully purified and identified on the basis of their GEF activity. In the case of the Rac-GEFs, this means that activities in lysates or those involved in specific signalling events have only rarely been attributed to a specific GEF, and further the contributions any GEF makes towards total cellular GEF activities are unclear. We have resolved neutrophil cytosol by chromatography on Q-Sepharose and found a major peak of PtdIns(3,4,5)P3-sensitive Rac-GEF activity (in the presence of PtdIns(3,4,5)P3, it represented about 65% of total Rac-GEF activity) which we have purified, cloned and named P-Rex1. P-Rex1 is a surprisingly abundant protein, about 0.1% of cytosolic protein (cf. 0.001% for the type 1B PI3K purified from similar fractions).
Our results show that PtdIns(3,4,5)P3 can substantially activate P-Rex1 Rac-GEF activity in vitro, and that cell-surface receptors can activate P-Rex1 in a PI3K-dependent fashion in vivo. Further, we demonstrate P-Rex1 can selectively bind PtdIns(3,4,5)P3-containing phospholipid vesicles. However, P-Rex1 does not substantially translocate from the cytosol to the sites of PtdIns(3,4,5)P3-accumulation in vivo, rather the enzyme is partially localised to the membrane in serum-starved cells. The implication of these results is that PtdIns(3,4,5)P3 is able to activate the enzyme by inducing a catalytically significant conformational shift or by re-orientating P-Rex1 at the membrane surface rather than by targetting it to the membrane. This is totally compatible with the emerging view of the role of the PH domain in the tandem DH/PH domains of Rho-family GEFs as a phosphoinositide-inhibited repressor of DH domain GEF activity (Worthylake et al. 2000, see Introduction). This is quite distinct to the generally accepted view of the role of the PH domain in proteins such as PLCδ where lipid binding acts purely as a membrane-targetting device.
Some data has suggested activation of heterotrimeric G-proteins in neutrophil-like cells can stimulate Rac-GEF activities (Geijsen et al. 1999), and furthermore a small (about 40% above control) effect of Gβγ on Rac-GEF activity in neutrophil lysates has been reported (Arcaro 1998). P-Rex1 is the first example of a Rac-GEF that can be activated directly by Gβγ. The ability of Gβγs and PtdIns(3,4,5)P3 to synergistically activate P-Rex1 suggests these regulators can bind simultaneously to independent sites although we have not identified these sites. P-Rex1 hence becomes one of a growing list of effector proteins that are regulated by Gβγ subunits in neutrophil, including p101/110γ-PI3K and PLCs (Stemweis and Smrcka 1992) (see above).
G protein-mediated signalling pathways in neutrophils respond rapidly, eg. maximal activation of Rac can occur within 10 sec. The fact that both p101/p110γ-PI3K and P-Rex1 appear to be partially membrane-localized in serum-starved cells and are activated at the level of the membrane without any requirement for translocation from the cytosol (Krugmann et al. 2002) probably contributes to this rapidity.
Our data are consistent with the existence of a signalling pathway in neutrophils from G-protein linked receptors and via Gβγs, type 1B PI3K and PtdIns(3,4,5)P3 and activation of Rac to enhanced ROS formation. There is significant work that has also suggested these links, however, this appeared weak in the absence of an appropriate Rac-GEF. The literature also contains high-quality work that is apparently inconsistent with this model: activation of Rac by ligands such as fMLP has been shown to be resistant to PI3K inhibitors (Geijsen et al. 1999, see Introduction). Our results offer a possible explanation; they suggest that, at the earliest times of stimulation with ligands like fMLP, Gβγ activation of P-Rex1 may be more important than activation via PtdIns(3,4,5)P3, as the levels of PtdIns(3,4,5)P3 rise due to Gβγ stimulation of p101/p110γ-PI3K. Moreover, this phenomenon would be exaggerated in unprimed neutrophils which produce up to 20 times less PtdIns(3,4,5)P3 in response to fMLP (Condliffe and Hawkins, 2000). This is exactly what is observed in the literature, workers with demonstratedly unprimed neutrophils who also stimulate for the shortest times (10 s) found Rac activation by fMLP to be largely resistant to PI3K inhibitors (Geijsen et al. 1999). Those workers who tested PI3K inhibitors at later times of stimulation (1 min) find that PI3K inhibitors substantially, but not completely, inhibit activation of Rac (Akasaki et al. 1999, Benard et al. 1999).
P-Rex1 is a coincidence detector apparently designed to respond to the combined versus isolated appearance of PtdIns(3,4,5)P3 and Gβγ, probably in the same membrane. In neutrophils, this set of signals is naturally delivered by activation of G-protein linked receptors in the context of a large population of Gi proteins in the plasma membrane and Gβγ-sensitive p101/110γ-PI3K (that is particularly enriched in hematopoietically-derived cells) which drives accumulation of PtdIns(3,4,5)P3 in the membranes actually harbouring Gβγs (Stephens et al. 1997). The importance of this synergy is possibly reflected in the fact that ligands like GM-CSF that activate type 1A PI3Ks primarily via protein-tyrosine kinase-based mechanisms do not cause detectable activation of Rac (Geijsen et al. 1999), despite the fact they cause significant accumulation of PtdIns(3,4,5)P3 (Corey et al. 1993). In other cellular contexts, perhaps in the brain, it is easy to imagine that P-Rex1 could be relevant in the detection of specific patterns of signalling that deliver coincident activation of type 1A PI3Ks and activation of Gi/Go proteins. This type of regulation could be particularly significant in forming or strengthening particular cell contacts in view of the key role Rac plays in, for example, neurite outgrowth (Luo et al. 1997).
Experimental Procedures
Materials
Monomeric GTPases (EE-Rac1, GST-Rac1, EE-N17 Rac1, EE-Rac2, GST-CDC42, GST-RhoA) were purified from Sf9 cells in the GDP-bound state to>95% purity, and stored in 1% (w/v) cholate, 5 μM GDP, 5 mM MgCl2, 1 mM EGTA, 1 mM DTT, 0.15 M NaCl, 40 mM Hepes/NaOH pH 7.4 (4° C.). Non-lipid-modified GST-Rac was derived from bacteria Gβγ were purified from bovine brain (Sternweis and Robishaw 1984), or from Sf9 cells (both wild-type EE-β1,γ2 and non-prenylated mutants EE-β1,C186S-γ2), and stored in 1% cholate, 1 mM DTT, 20 mM Hepes/NaOH pH 8.0 (4° C.), 5 μM GDP (for bovine-brain Gβγs) and 1 mM EDTA. Gα subunits (a mixture of αi1, αi2 and αo) were purified from bovine brain (Sternweis and Pang 1990) and stored in 50 mM Hepes/NaOH pH 8.0 (4° C.), 1 mM EDTA, 1 mM DTT, 100 mM NaCl, 1% cholate and 10 μM GDP. Phosphorothioate antisense oligonucleotides and controls directed to P-Rex1 have been designed and manufactured by BIOGNOSTIK, Göttingen, Germany (antisense: TCA TTG ATG GAG TAG ATC (SEQ ID NO:9), randomised control: ACT ACT ACA CTA GAC TAC (SEQ ID NO:10)). Recombinant EE-p101/hexa-His-p110PI3K was produced from Sf9 cells (Stephens et al. 1997). Recombinant NH2-terminally EE-tagged P-Rex1 was purified from Sf9 cells utilizing the EE-tag and stored in PBS, 1 mM EGTA, 1 mM DTT, 0.01% Na azide. Stearyl-arachidonyl (S/A)-PtdIns(3,4,5)P3 stereoisomers were synthesised by P. Gaffney (Gaffney and Reese 1997). All dipalmitoyl (P/P)-phosphoinositides were made by G. Painter (Painter et al. 1999). In this manuscript, the term PtdIns(3,4,5)P3 refers to D/D-(S/A)-PtdIns(3,4,5)P3 unless otherwise stated.
Two independent affinity-purified sheep polyclonal anti-P-Rex1 antibodies (raised against conjugated peptides based on P-Rex1 sequence: CLHPEPQSQHE (SEQ ID NO:11) and CAAARESERQLRLR (SEQ ID NO:12)) were pooled and used to detect endogenous and heterologous P-Rex1 by immunoblotting.
ROS Formation Assay with Neutrophil Cytosol and Membrane Fractions.
Neutrophil-enriched leukocytes were isolated from pigs' blood, sonicated in 0.25 M sucrose, 0.1 M KCl, 50 mM Hepes/NaOH pH 7.2 (4° C.), 1 mM DTT, 2 mM EGTA, 0.1 mM PMSF and 1× antiproteases (20 μg/ml of each antipain, aprotinin, pepstatin and leupetin) and centrifuged (100,000×g, 1 h, 4° C.) to yield cytosol and light membrane fractions (4.5 mg/ml protein, collected between 0.60 and 1.35 M sucrose and washed in sonication buffer). Light membranes (3 μl) were pre-mixed with Gβγ subunits, p101/p110γ-PI3K and/or N17-Rac (or boiled controls) in 6 μl containing 5 mM ATP, 8 mM MgCl2, 20 mM Hepes/NaOH pH 7.5 (4° C.), 2 mM EGTA, 10 mM β-glycerophosphate, 0.1 mM ortho-vanadate, 0.1 M KCl, 1 mM DTT, 0.01% (w/v) Na azide. After 25 min on ice, 20 μl was added containing 15 μl cytosol and 1 mM MgGTP, 20 μM FAD, 400 μM NADPH and 200 μM luminol. After 8 min at RT, single photon counts (SPC) per 0.1 min were quantitated in a scintillation counter at intervals of 3-5 min for up to 20 min (blanks without cytosol were subtracted). When lipids were added, they were preincubated with the membranes, and 10 μM GTPγS replaced MgGTP. Where wortmannin was added, it was preincubated with both cytosol and membrane fractions for 15 min on ice. Where EE-Rac1 was added, it was preloaded with different guanine nucleotides at a 5-fold excess over bound GDP.
Rac-GEF Assay with Neutrophil Cytosol and Membrane Fractions.
This assay was essentially as for ROS production, except pure, lipid-modified EE-Rac1 (30-50 nM final concentration) was added to the cytosol, and GTP, GTPγS, NADPH, FAD and luminol omitted. Three min after mixing membrane and cytosol fractions, [α32P]-GTP (20 μCi per sample) was added. Four min later, the reaction was stopped and EE-Rac1 pulled down using anti-EE antibody coupled to protein G sepharose. The total dpm on EE-Rac1 were quantified by scintillation counting (blanks without EE-Rac1 were subtracted).
Rac-GEF Assay for P-Rex1 Purification and Recombinant P-Rex1.
Liposomes (phosphatidylcholine (PtdCho), phosphatidylserine (PtdS), phosphatidylinositol (PtdIns), final assay concentration 200 μM each) were sonicated in lipid buffer (20 mM Hepes/NaOH pH 7.5 (4° C.), 100 mM NaCl, 1 mM EGTA) with or without PtdIns(3,4,5)P3 (final assay concentration 10 μM) and incubated for 10 min on ice with 2 μl of purified, GDP-loaded, recombinant, lipid-modified EE-Rac1 in 5 mM MgCl2, 50 mg/ml BSA, 5 mM DTT, 20 mM Hepes/NaOH pH 7.5 (4° C.), 100 mM NaCl, 1 mM EGTA (final assay concentration 100 nM EE-Rac1, 0.0024% cholate). Then 4 μl of Rac-GEF activity (cytosol, column fractions, or recombinant P-Rex1) was added, followed by 2 μl of GTPγS (in lipid buffer, final assay concentration 5 μM, including 1 μCi [35S]-GTPγS). After 10 min at 30° C., the reaction was stopped and EE-Rac1 pulled down using anti-EE antibodies coupled to protein G-sepharose, and [35S]-GTPγS-loading of Rac was detected by scintillation β-counting. Recombinant P-Rex1 was diluted in ‘buffer A’ (20 mM Hepes/NaOH pH 7.5 (4° C.), 1% betaine, 0.01% Na azide, 0.5 mM EGTA, 200 mM KCl, 10% ethylene glycol) to a final assay concentration of 50 nM. In assays with Gβγ, 2 μl of Gβγ in ‘buffer A’ were added to the liposome/Rac mix before the 10 min on ice, and P-Rex1 was added as 5×.
Purification of PtdIns(3,4,5)P3-Dependent Rac-GEF.
Neutrophil-enriched leukocytes prepared from 90 l of pigs' blood were sonicated in 30 mM Tris/HCl pH 7.8 (4° C.), 0.1 M NaCl, 4 mM EGTA, 1 mM DTT, 0.1 mM PMSF and 0.5× antiproteases. The cytosol (100,000×g supernatant) was diluted to 16.7 mM NaCl in ‘buffer B’ (0.5 mM EGTA, 10% ethylene glycol, 1% betaine, 0.01% Na azide, 1 mM DTT, 50 μM PMSF, and 0.1× antiproteases) containing 10 mM Tris/HCl pH 7.8 (4° C.), applied to a 400 ml Q-sepharose fast flow column equilibrated in ‘buffer B’ containing 30 mM Tris/HCl pH 7.8 (4° C.) and 0.1 mM EDTA, and eluted with a 0.1-0.6 M NaCl gradient over 31. The peak of PtdIns(3,4,5)P3-dependent Rac-GEF activity eluted between 0.43 and 0.52 M NaCl, was desalted on a 1.4 l G25-fine column equilibrated in ‘buffer B’ containing 20 mM Hepes/NaOH pH 6.8 (4° C.), then applied to a 50 ml SP-sepharose-HP column equilibrated in the same buffer, and eluted with a 0.25-0.75 M KCl gradient over 500 ml. The activity was recovered between 0.31 and 0.375 M KCl, desalted on a 300 ml G25-fine column equilibrated in ‘buffer B’ containing 20 mM Hepes/NaOH pH 7.2 (4° C.), applied onto a 12 ml Heparin sepharose column equilibrated the same buffer, and eluted with a 0.1-0.7 M KCl gradient over 150 ml. The activity was recovered between 0.55 and 0.69 M KCl. A fraction corresponding to 0.60-0.65 M salt, selected for good fold purification, was concentrated, pH adjusted, and applied to a 200 ml HPLC size exclusion column equilibrated in ‘buffer B’ containing 20 mM Hepes/NaOH pH 6.9 (4° C.) and 120 mM NaCl. The activity was recovered after 104 ml, corresponding to an apparent size of 203 kD, loaded onto a 1 ml Mono S FPLC column equilibrated in ‘buffer B’ containing 20 mM Hepes/NaOH pH 7.0 (4° C.), and eluted with a 0.1-0.7 M KCl gradient over 54 ml. The pure, PtdIns(3,4,5)P3-dependent Rac-GEF activity eluted between 0.375 and 0.425 M KCl.
Cloning of Human P-Rex1.
A tryptic digest of purified pig PtdIns(3,4,5)P3-dependent Rac-GEF yielded 5 peptides, T14, T30, T44, T69, T72, that were analysed by MALDI-TOF and N-terminal sequencing. T72 was identical to mouse Est AA796530 (homologous to Tiam). T14 and T69 were near identical to mouse Est A1466041 (homologous to Inositol Polyphosphate 4-Phosphatase). T30 and T44 were novel. Underlying human genomic sequence placed T44 into the Inositol Polyphosphate 4-Phosphatase homology region and T72 near the N-terminus of a predicted protein. A predicted partial sequence encompassing these regions has been published (Nagase et al. 2000). Est AA796530 was cut with Bg12, labelled with [α32P]-dCTP using the prime-a-gene system (Promega) to make a 673 bp probe for screening a human U937 cell random prime λ-Zap2 cDNA library and a human spleen random prime λGT11 cDNA library, yielding 24 and 36 clones of varying lengths, respectively. In parallel, PCR primers based on underlying genomic sequence were used to screen a marathon-ready human leukocyte cDNA library (Clontech).
The full length sequence was obtained from three fragments and cloned into pBluescript (Stratagene) as follows: Clone 1 was cut Sal1/Sph1 to yield pBluescript with the N-terminus of P-Rex1 up to the first Sph1 site. Clone 2 was cut Sph1 /Bcl1 to give the middle of P-Rex1, and clone 3, the PCR-derived C-terminus, was cut Bcl1/Sal1 out of the T-tail vector. The fragments were three-way ligated. The resulting polylinker of pBluescript had additional Spe1, Not1 and Pst1 sites 5′ of Sal1. The 5′ overhang was replaced by PCR, creating an in-frame EcoRI/startATG. A 60 bp 3′ overhang was kept. Full length P-Rex1 was subcloned into pCMV3 mammalian expression vectors with N-terminal myc- or EE-epitope tags (Welch et al. 1998) or pAc0G1 Sf9 cell expression vector with N-terminal EE-tag by ligating P-Rex1 from EcoR1/Spe1-cut pBluescript-P-Rex1 into EcoR1/Xba1 cut vectors.
Northern Blots.
The same probe as described above for library screening was used to hybridize Clontech multiple tissue northern blots as specified by the manufacturers.
Surface Plasmon Resonance.
Assays were conducted as described (Ellson et al. 2001), using mixed phospholipid vesicles (PtdCho, PtdS, phosphatidylethanolamine (PtdE), 330 μM each final concentration) with or without added phosphoinositides (6 mol-% final concentration) to load the L1 vesicle capture chip (Biacore) prior to the injection of 100 nM purified recombinant Sf9-cell derived P-Rex1.
Rac and CDC42-GEF Assays and Measurement of PtdIns(3,4,5)P3 Formation in SE9 Cells.
Rac and CDC42-GEF in vivo assays were performed as PAK-Crib pull down assays (based on the fact that only activated GTP-bound but not GDP-bound Rac and CDC42 bind to the Crib domain of PAK) as described (Sander et al. 1998), with endogenous Rac and CDC42 from Sf9 cells that were infected to produce combinations of P-Rex1, Gβγ and PI3K. Measurement of PtdIns(3,4,5)P3 formation in Sf9 cells was done by radioligand displacement assay essentially as described (Van der Kaay et al. 1996).
Immunofluorescence Microscopy.
Pig aortic endothelial (PAE) cells were transiently transfected with pCMV3-myc-P-Rex1 or pCMV3-myc-DAPP1 by electroporation, grown on coverslips for 10 h and then serum starved for 8 h. They were then treated or not with 100 nM wortmannin for 10 min followed by stimulation with varying doses of PDGF for 5 min. Cells were fixed and prepared for immunofluorescence microscopy by staining of P-Rex1 and DAPP1 with anti-myc epitope-tag primary and FITC-goat anti-mouse secondary antibodies and filamentuous actin with TRITC-phalloidin as previously described (Welch et al. 1998).
NB4 Cell Culture and MAPK and ROS Formation Assays.
NB4 cells (from M. Lanotte, Paris) were cultured and differentiated in the presence of 1 μM all-trans retinoic acid as described (Lanotte et al., 1991) in the presence of either control or P-Rex1 antisense oligonucleotides for 2-3 days. MAPK activation in response to C5a was monitored by immunoblotting with an anti-phospho-MAPK antibody (from Cell Signalling Technology; used as recommended) (5×104 cells per sample). ROS formation was monitored using a luminol-based detection in a scintillation counter in single photon count mode (3×104 cells per sample).
Generation of P-Rex1 Knockout Mouse (P-Rex1−/− Mouse)
1) Expected Phenotype:
Based on the restricted tissue distribution of P-Rex1, its abundance in neutrophils, and our study with antisense oligonucleotides (Welch et al, 2002), we expect a significant impact of P-Rex1 deficiency on neutrophil function. Mouse models for two other enzymes involved in P-Rex1 signalling, Rac2 and class 1B PI3K (catalytic and regulatory subunits), show that these enzymes are essential for neutrophil function (Roberts et al; Hirsch et al; Li et al; Sasaki et al; and our unpublished results). Rac2−/− mice are characterised by neutrophilia, reduced inflammatory peritoneal exudate formation and increased mortality from Aspergillus fumigatus infections (Roberts et al). Leukocytes from these mice show defects in their actin cytoskeleton structure, ROS formation and chemotaxis (Roberts et al). Human patients with a congenital Rac2 deficiency caused by a D57N point mutation suffer from recurrent, life-threatening infections and their neutrophils show similar defects as those of Rac2−/− mice (Williams et al; Ambruso et al). Mice lacking class 1B PI3K have similar phenotypes both on the cellular and the organism level Hirsch et al; Li et al; Sasaki et al; and our unpublished results). We expect also a similar phenotype in P-Rex1−/− mice. However, as P-Rex1 can potentially integrate signals from two different classes of PI3Ks and from Gβγs, and as it should use not only Rac2 as substrate but also the other Rac isoforms (Rac1 and Rac3), we expect the P-Rex1−/− mouse to show some distinctive and pronounced traits. Hence, we will focus the characterisation of the P-Rex1−/− mouse on defects in neutrophil function, both on the molecular and cellular levels, and on defects in neutrophil recruitment and the ability of the mice to clear infections.
2) Generation of the P-Rex1−/− mouse:
This is done as a total knock-out by standard homologous recombination, replacing exon 5 (coding for critical residues of the catalytic DH domain (see
2a) Targeting Vector:
The genomic DNA coding for the targeted region was obtained by screening of the mouse PAK library RPCI21 (UK HGMP Resource Centre) with a probe corresponding to exons 2-7 of human P-Rex1. Clones obtained were checked by Southern blotting with a probe corresponding to exons 4 and 5 of human P-Rex1, which yielded 6 identical positive clones. A region spanning 8 kb 5′ of exon 5 to 3 kb 3′ of exon 5 (white area in
2b) Generation of P-Rex1-Targeted ES Cells:
The purified and linearised targeting vector was electroporated into 2 different ES cell lines, E14 and CCB. ES cell clones were grown using neomycin as the selection medium.
2c) Southern Blot Screening:
Neomycin-resistant ES cells were screened for correct insertion of the P-Rex1 targeting vector by Southern blotting using a 3′ external probe (
Positive clones from the 3′ probe/XmnI screen (6 from E14 cells and one from CCB cells) were then verified by further Southern blotting using a 5′ external probe combined with a PsiI/SpeI double-digest of the genomic DNA and also using an internal probe together with XbaI-digested DNA. All of the clones that were positive in the 3′ probe/XmnI screen were also positive in both other screens.
2d) Generation of Mice:
Three positive ES cell clones (two from the E14 cell line and one from CCB cell line) were independently injected into isolated Black 6 mouse blastocytes and these were implanted into pseudo-pregnant Black 6 female mice (C57BI/6 J). From this, more than 50 chimeric pups were born. These were scored according to the degree of targeted-ES cell derived body sections (mainly by coat colour) and the most promising chimeric males have been paired with Black 6 females (C57BI/6 J) in order to breed heterozygous offspring. Pups with the agouti coat colour (for mice derived from CCB cell line) or cream coat colour (for mice derived from E14 cell line) are expected when germline transmission is achieved. Potentially heterozygous offspring can then be “tailed” and the tail DNA screened for P-Rex1-targeting by Southern blot using the 3′ probe/XmnI strategy as described above. It is expected that positive heterozygote mice can then be bred together to give P-Rex1−/− mice.
Sample Preparation, Human Neutrophil Total Lysate for Anti-P-Rex1 Western Blots
Storage of Affinity Purified Sheep Anti-Human P-Rex1 Polyclonal:
at −20° C. ! (as got 50% glycerol in, so it won't freeze), has got azide in, do not freeze at −80° C.
Positive Western Blot Control:
P-Rex1-transfected COS7 cell lysate. Use between 1 and 10 μl per lane.
| Number | Date | Country | Kind |
|---|---|---|---|
| 0206684.3 | Mar 2002 | GB | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/GB03/01238 | 3/21/2003 | WO | 5/13/2005 |
