Correspondence to: Matilda Katan, CRC Centre for Cell and Molecular Biology, Chester Beatty Laboratories, The Institute of Cancer Research, Fulham Road, London SW3 6JB, UK. Tel:44-207-352-8133 Fax:44-207-352-3299 E-mail:matilda{at}icr.ac.uk.
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Abstract |
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The translocation of fluorescently tagged PLC and requirements for this process in cells stimulated with EGF were analyzed using real time fluorescence microscopy applied for the first time to monitor growth factor receptoreffector interactions. The translocation of PLC
to the plasma membrane required the functional Src homology 2 domains and was not affected by mutations in the pleckstrin homology domain or inhibition of phosphatidylinositol (PI) 3-kinase. An array of domains specific for PLC
isoforms was sufficient for this translocation. The dynamics of translocation to the plasma membrane and redistribution of PLC
, relative to localization of the EGF receptor and PI 4,5-biphosphate (PI 4,5-P2), were shown. Colocalization with the receptor was observed in the plasma membrane and in membrane ruffles where PI 4,5-P2 substrate could also be visualized. At later times, internalization of PLC
, which could lead to separation from the substrate, was observed. The data support a direct binding of PLC
to the receptor as the main site of the plasma membrane recruitment. The presence of PLC
in membrane structures and its access to the substrate appear to be transient and are followed by a rapid incorporation into intracellular vesicles, leading to downregulation of the PLC activity.
Key Words: PLC, EGF receptor, translocation, real time imaging, SH2 and PH domains
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Introduction |
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The hydrolysis of phosphatidylinositol 4,5-bisphosphate (PI 4,5-P2)1 occurs in response to a large number of extracellular signals to generate two second messengers: inositol 1,4,5-trisphosphate (I 1,4,5-P3) and diacylglycerol. In addition, this hydrolysis may also contribute to the control of cellular PI 4,5-P2 levels and in this way, regulate function of proteins that bind this lipid directly ( (
1,
2), and PLC
(
1
4), have been extensively studied and characterized (
(
families, the type of signaling pathway that regulates enzyme activity.
PLC is mainly regulated through receptors with intrinsic tyrosine kinase activity (for example, growth factor receptors) or receptors (such as B and T cell antigen receptors) that are linked to the activation of nonreceptor tyrosine kinases through a complex signaling network (
have distinct tissue distributions. Although PLC
1 is expressed ubiquitously, the pattern of expression of PLC
2 is characterized by high levels in cells of hematopoetic origin. Transgenic studies suggested that the biological function of these isoforms is reflected in their cellular distribution. Thus, the mice deficient in PLC
1 developed normally up to embryonic day 9 when a cessation of growth occurred in all parts of the embryo (
2 allowed normal development but resulted in functional and signaling disorders in a subset of cell types, including B cells, platelets, and mast cells (
1 and PLC
2 could equally fulfill the same signaling function. This has been demonstrated in both signaling via receptor (
family, which includes the pleckstrin homology (PH) domain, two Src homology (SH)2, and one SH3 domain (see Fig 1).
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Molecular interactions that are required for the activation of PLC have been best characterized in responses via growth factor receptors (including receptors for EGF, platelet-derived growth factor [PDGF], and nerve growth factor [NGF]), where the receptor directly interacts with PLC
and phosphorylates the enzyme by the intrinsic tyrosine kinase activity of these receptors (
is involved in the regulation of the enzyme activity and that translocation to membrane structures is required for the subsequent increase in PI 4,5-P2hydrolysis. The data have also suggested that in addition to the PLC
interactions with the phosphorylated tyrosines in the cytoplasmic part of the receptors via the SH2 domains, other interactions with lipid membrane components, such as phosphatidylinositol 3,4,5-trisphosphate (PI 3,4,5-P3) and/or components of the cytoskeleton, could have an important role (
have not been clarified. In particular, questions related to the type of subcellular structures where interactions with the receptor or other targeting molecules are taking place at different points after the addition of a growth factor and the effects of these interactions on the access to its substrate and PLC activity remain unanswered.
In this study, PLC containing a fluorescent tag was analyzed in real time after stimulation of the cell with EGF. This approach, applied for the first time in studies of PLC
, circumvented many problems encountered when using other methods. Here, we show dynamic changes in the subcellular localization of PLC
relative to the presence of EGF receptor (EGFR) and the substrate and describe possible effects of cellular localization on stimulation and downregulation of the PLC activity.
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Materials and Methods |
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Construction of Expression Plasmids
The construct of the PLC1 PH domain and catalytically inactive PLC
1 with green fluorescent protein (GFP) were described previously (
1 and human PLC
2 in the pMT2 vector have been described previously (
1 and PLC
2 corresponding to the NH2-terminal PH domains (encoding residues 14150 for PLC
1 and 8144 for PLC
2 and extended constructs encoding residues 1309 for PLC
1 and 1301 for PLC
2) and
SA regions (encoding residues 484936 for PLC
1 and 468919 for PLC
2) were amplified by PCR. Each fragment was subcloned into the GFP fusion protein expression vector pEGFP-C1 (CLONTECH Laboratories, Inc.) using the BglII and EcoRI restriction sites. Specific domain array (SA) region of PLC
2 was also subcloned into pDsRed-C1 vector (CLONTECH Laboratories, Inc.), which produces a red fluorescent protein (RFP) using the BglII and EcoRI sites. Mutated
SA regions with the Arg 564 to Ala (R564A) replacement within the N-SH2 domain or Arg 672 to Ala (R672A) replacement within the C-SH2 domain in PLC
2 were generated using two-stage PCR-based overlap extension method. Forward mutagenesis primers were: 5'-CTGGTTGCGGAGAGCGAGAC for R564A and 5'-TCCTGATCGCGAAGCGAGAGG for R672A. The PCR products were subcloned into pEGFP-C1 vector using the BglII and EcoRI sites and the sequences verified. The full-length cDNAs of PLC
1 and PLC
2 were subcloned into pDsRed-N1 vector (CLONTECH Laboratories, Inc.) using Eco47III and BamHI sites (pDsRed vector), NotI (filled in by mungbean nuclease [New England Biolabs, Inc.]), and BamHI. The constructs of R564A replacement in the context of the full-length was made by replacing a part of the wild-type
SA region with mutated fragments of the same region using XmnI and SacII restriction sites. For the expression in DT40 cells, the full-length cDNA of PLC
2 was also inserted into pApuro vector (
2pDsRed and pApuro constructs were made by replacing the wild-type PH domain region with the mutated fragments generated by the PCR-based mutagenesis described above, using EcoRI sites. The forward mutagenesis primers were: 5'-TGGAGCAAGGACGACGACGAGATCGAG for acidic substitutions and 5'-TGGAGCGCGGCCGCCGCCGCGATCGAG for alanine substitutions. The deletion mutant of the PH domain in the full-length PLC
2 construct was made by exchanging the PH and EF hand regions with only the EF hand region, which was amplified by PCR and subcloned using EcoRI sites, thus generating construct encoding amino acid residues 1341256 of PLC
2. The EGFRGFP construct was described previously (
Cell Culture and Transfection
A431 and COS-7 cells were cultured in DME supplemented with 10% FBS. The transfection of GFP or RFP fusion protein constructs were made using cells in a 60-mm dish (Nunc), a glass-bottomed 50-mm dish (MatTek Corporation), or on 22-mm-diameter coverslips (British Drug House) using 2.0, 0.4, or 0.8 mg of plasmid DNA, respectively, and LipofectAMINE Plus reagent (GIBCO BRL) according to the manufacturer's instructions. 0.2 mg of each DNA was used in cotransfection of EGFR and PLC constructs on a 60-mm dish with a coverslip at the bottom. After 24 h, cells were serum starved overnight in DME with 1% FBS before EGF stimulation. PC-12 cells were cultured and stimulated with NGF as described previously (
DT40 cells (chicken Blymphoma cell lines), the wild-type and PLC2-deficient (
2 were introduced into DT40 PLC
-deficient cells by electroporation (950 V, 25 µF,
, Gene Pulser; Bio-Rad Laboratories). After 1 d, puromycin (0.35 µg/ml) (Sigma-Aldrich) was added to the medium. 1012 d after the selection, colonies were picked, and the puromycin (0.2 µg/ml) selection was repeated for 58 d. Subsequently, the puromycin-resistant colonies were grown in normal medium for a further 5 d or until cells were growing normally.
Confocal Microscopy
To visualize responses to EGF, the transfected cells were serum-starved overnight in the presence of 1% FBS. The medium of cells cultured in glass-bottomed 50-mm dishes (MatTek Corporation) was subsequently changed to HBSS without phenol-red (GIBCO BRL). EGF (Becton Dickinson) was added at a concentration of 100 ng/ml. For the treatment with LY294002, the cells were incubated with 250 µM LY294002 (Calbiochem) for 40 min at 37°C before the addition of EGF.
Intracellular localization of fluorescent fusion proteins in living cells was performed on a Bio-Rad Laboratories MRC1024 confocal imaging system in conjunction with a Nikon Eclipse 600 microscope and LazerSharp software. Detection of GFP and RFP was carried out using a krypton-argon laser together with standard FITC and Texas red filter sets. The cells were imaged using a 60x plan-apo oil immersion objective. The images were recorded in 5-s intervals. The cells were analyzed at room temperature immediately after being taken out from the 37°C incubator. The white bars in the figures indicate 10 µm.
For further analysis of images, we used several programs in the LaserPix (version 4.0) package (Bio-Rad Laboratories) including intensity/optical density analysis, relative quantitation, and timecourse analysis. For single cells, cross section areas have been selected; whereas, for the fields of cells all areas of the cytoplasm and plasma membrane (corrected for the fluorescence contributed by the cytoplasm) have been used to calculate average intensities at different timepoints.
The cell staining using antibody to PI 4,5-P2 (1020-fold lower binding to much less abundant PI P3) was as follows: after stimulation with EGF, the cells were fixed in 0.4% formaldehyde for 10 min; the cells were permeabilized for 10 min with 100 mM glycine, 0.1% Triton X-100 after which nonspecific binding was blocked by incubating for 5 min in 0.1% BSA in PBS; antiPI 4,5-P2 antibody (1:100) was added for 1 h at room temperature; after washing, the Texas red or FITC-conjugated antibody was added for 30 min at room temperature; and the cells were then washed, mounted, and viewed by confocal microscopy.
Measurement of Intracellular Calcium Concentration
The method used to measure intracellular calcium concentrations was according to
For measurements of intracellular calcium concentrations in DT40 cells, a cell suspension containing 5 x 106 cells was loaded with 2 µM Fluo-3AM (Molecular Probes) for 1 h at room temperature. After the cells were washed with PBS, their calcium mobilization was measured at 40°C after stimulation with 10 µg/ml goat antichicken IgM, using a stirred suspension of cells in a LS-50B fluorimeter (PerkinElmer). The excitation was at 490 nm, and the emission was monitored at 535 nm.
Analysis of the Protein Expression and Phosphorylation by Western Blotting and/or Immunoprecipitation
Transfected COS-7 or A431 cells were harvested and resuspended in PBS buffer containing a protease inhibitor cocktail (Boehringer), sonicated, and subjected to centrifugation at 35,000 g. Aliquots of the supernatants were separated by SDS-PAGE, transferred to polyvinyldifluoride membranes, and the membrane incubated with the appropriate primary and secondary antibodies. The anti-PLC1 antibody (1:3,000; Upstate Biotechnology) and anti-PLC
2 antibody (1:4,000; Santa Cruz Biotechnology, Inc.) were used to analyze the expression of all full-length PLC
constructs. The anti-GFP antibody (1:2,500; CLONTECH Laboratories, Inc.) was used to analyze the expression of the GFPN-PH or
SA fusion proteins. The detection of PLC
1 phosphorylation was performed with anti-PLC
1(pY783) phosphospecific antibody (1:2,000; BioSource International), whereas phosphorylation of PLC
2 was monitored after immunoprecipitation (incubation with protein Gagarose-conjugated anti-PLC
2 antibody for 1.5 h at 4°C) using antiphosphotyrosine antibody (1:1,000; Transduction Laboratories). In both cases, cellular extracts were prepared in the presence of 0.5% NP-40. The blots were visualized using ECL system (Amersham Pharmacia Biotech).
The detection of phosphorylated EGFR and protein kinase B (PKB) proteins in A431 cells was performed before and after EGF stimulation of cells preincubated in the presence or absence of LY294002 as described above. The stimulation was terminated by washing with ice-cold PBS. Cell lysates were prepared using a lysis buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 0.2 mM PMSF, 0.5% NP-40, protease inhibitor cocktail, and phosphatase inhibitor cocktail) (Sigma-Aldrich) and the clarified lysates separated by SDS-PAGE and analyzed by Western blotting. The antiphosphotyrosine antibody (1:1,000; Transduction Laboratories) or antiphosphoPKB/Akt (Serine 473) antibody (1:1,000; New England Biolabs, Inc.) were used, followed by the incubation with an appropriate secondary antibody (antirabbit Ig HRP-linked whole antibody or antimouse Ig HRP-linked whole antibody, diluted 1:3,000) (Amersham Pharmacia Biotech).
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Results |
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Translocation of PLC to the Plasma Membrane: An Essential Role of the SH2 Domains
To analyze the cellular localization of PLC1 and PLC
2 in real time, molecules incorporating green (GFP) or red (RFP) fluorescence tags (placed either at the COOH or NH2 terminus of PLC) were constructed. The expression of all constructs in COS-7 or A431 cells resulted in the appearance of proteins of expected size (
170 kD). The constructs used in most of the experiments described here, which have an RFP tag at the COOH terminus of PLC
(PLC
1-RFP and PLC
2-RFP), are shown in Fig 1. Measurements of in vitro PLC activity in the extracts of transfected cells confirmed that the constructs encoded enzymatically active PLC
1 and PLC
2. The cell line that has been chosen to monitor cellular localization was A431-epidermoid carcinoma cell line, where the number of receptors responding to EGF is increased. Using biochemical methods, it has been shown previously that endogenous PLC
1 in this cell line redistributes from the cytosolic fraction to membrane fractions within minutes of EGF stimulation (
1 and PLC
2 were distributed throughout the cytoplasm and excluded from the nucleus and plasma membrane. After addition of EGF, the translocation to the plasma membrane was largely completed within 23 min for both PLC
1 and PLC
2 and was accompanied by a reduction of fluorescence in the cytoplasm (Fig 2). This similarity between PLC
1 and PLC
2 translocation is consistent with previous comparisons of the two isoforms in growth factor signaling (
1 in fibroblasts, was enhanced in cells overexpressing either PLC
1 or PLC
2; both enzymes could undergo phosphorylation by and interaction with the receptor. Further analysis of recordings shown in Fig 2 taken in 5-s intervals, revealed some differences between individual cells. However, in most cells the appearance of PLC
in some parts of the membrane could be detected after 1030 s. The area of the membrane where PLC
was present increased with time to cover most of the membrane surface. In the majority of cells, some further increases in the membrane recruitment could be seen between 2 and 3 min. This is illustrated in Fig 2 C and also by the kinetic analysis in Fig 2 D, using PLC
2 construct. The kinetic studies of translocation performed on fields of cells were consistent with the main conclusions described above, without significant differences observed between the two isoforms.
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Several domains, including the PH domain at the NH2 terminus (N-PH), two SH2 domains (N-SH2 and C-SH2), and the SH3 domain, have been implicated in subcellular targeting of PLC isozymes. The importance of the SH2 domains has been best documented and related to interactions with the receptor or adapter proteins (
family ("
-specific array," [
SA]) incorporates most of the domains that could play a role in translocation. The constructs of PLC
1 and PLC
2 containing only this region with the GFP tag placed at the NH2 terminus (GFP-
1SA and GFP-
2SA) have been made and found to express proteins of the predicted size (Fig 1). These constructs, in most cells, completed translocation to the plasma membrane within 23 min (Fig 3A and Fig B); these similar timecourses were further supported by kinetic studies, when giving an average translocation for a field of cells (Fig 3 D). However, as observed with the full-length constructs not all cells responded uniformly. In Fig 3 C, two cells from the same field were selected to illustrate these differences. In one cell, the interaction that spread all over the membrane surface could be seen after 3 min; whereas, in the other it was completed after
1 min, followed by extensive membrane ruffling (see 2 and 3 min timepoints). The extensive membrane ruffling could be seen in some but not all cells responding to EGF and was usually prominent at later timepoints.
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To further analyze which domains within the SA region could be involved in translocation, point mutations were introduced into the N- and C-SH2 domains. Replacements of Arg586 (N-SH2) and Arg694 (C-SH2) in PLC
1 and Arg564 (N-SH2) and Arg672 (C-SH2) in PLC
2 have been shown previously to interfere with the function of these enzymes (
2SA or PLC
2-RFP (GFP-
2SA R564A, GFP-
2SA R672A, and PLC
2 R564A-RFP), did not reduce protein expression (Fig 1). As shown in Fig 4, these mutations completely prevented translocation to the membrane after the addition of EGF.
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Analysis of Requirements for the NH2-terminal PH Domain
To assess the role of the PH domain in the translocation of PLC, this domain from both PLC
1 and PLC
2 was expressed in isolation as a GFP fusion protein (GFP-
1N-PH and GFP-
2N-PH) (Fig 1). In addition, the deletion and point mutations within the NH2-terminal PH domain have been introduced in the context of the full-length PLC
2 protein (Fig 1). When analysis of the isolated PLC
PH domains was performed, the PH domain from PLC
1 was used for comparison. As shown previously in several cell types (
1 PH domain associated with the membrane of unstimulated cells (Fig 5 A). In contrast, GFP-
1N-PH and GFP-
2N-PH were not present at the membrane of unstimulated cells and the staining observed in the cytoplasm and nucleus; the later was more prominent for the GFP-
2N-PH. After EGF stimulation, the PH domain constructs could be seen associated with the membrane (Fig 5 A) as observed previously for the PLC
1 PH domain in PDGF-treated fibroblasts (
SA constructs.
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For further analysis, we attempted to introduce deletion and point mutations of the PH domain in the context of the full-length protein. Attempts to delete the PH domain of PLC2 proved incompatible with the expression of the soluble functional protein (it was exclusively in the pellet and inactive), suggesting its essential structural role. Instead, mutations were introduced in the PH domain based on a previous study, restricted to the isolated PH domain of PLC
1 (
1 and PLC
2 have similar sequences and identical positions of positively charged residues in this region, the same replacement has been introduced (57TADK60 replaced by 57DDDE60) into the full-length PLC
2; in addition, the alanine substitutions have also been made (mutation of 56KTADK60 to 56AAAAA60). Unlike the deletion of the PH domain, the expression of these mutants (PHacPLC
2-RFP and PHalaPLC
2-RFP) was the same as for the wild-type PLC
2 (Fig 1). The translocation of PLC
2 incorporating the substitutions in the N-PH was analyzed after stimulation of A431 cells with EGF. The membrane translocation of the wild type and the proteins incorporating the mutations could be clearly seen 3 min after stimulation. This is illustrated by the acidic substitution in Fig 5 B. Further kinetic analysis did not show any substantial changes (not shown). Tyrosine phosphorylation of these mutants was also comparable with the wild-type construct (Fig 6 B), and based on the analysis of PLC
1 constructs where sequence specific antiphosphotyrosine antibody is available (Fig 6 A), the phosphorylation is likely to occur at physiologically relevant sites.
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Finding that the N-PH domain may not play a role in the initial translocation to the membrane suggested that it could be important for some other steps involved in the stimulation of PLC. To test this, we used a PLC
-deficient B cell line (PLC
2-/DT40). After stimulation of B cell antigen receptor, an increase in intracellular free Ca2+ concentrations has been attributed to highly expressed PLC
2 in normal B lymphocytes (
1 or PLC
2 in the PLC
2-/DT40 cells (
in these cells (
2 fully restored the calcium responses in PLC
2-/DT40 cells stably transfected with this construct (Fig 5 C, right). Analysis of the stable cell lines obtained with the full-length constructs incorporating mutations within the PH domain (PHacPLC
2 and PHalaPLC
2), demonstrated the same calcium responses as seen with the wild-type PLC
2 (Fig 5 C, right). The same experimental system, stimulation of DT40 cells with the M4 antibody, was used to test a possible dominant-negative function of the isolated PH domain, which has been suggested in previous studies using fibroblasts stimulated with PDGF (
2N-PH (lane 2), the calcium responses were comparable with the responses in the wild-type DT40 cells (lane 1).
Colocalization of PLC with the EGFR Visualized Using Red and Green Fluorescent Tags
The GFP-tagged EGFR construct (EGFR-GFP) used in this study, has been analyzed previously and shown to encode a stable protein that undergoes phosphorylation and internalization after stimulation of transfected COS cells with EGF (-specific domain array of PLC
2, using a combination of red and green fluorescent tags expressed in the same cell.
Cells coexpressing the EGFR-RFP and the array of domains from PLC2 as a GFP fusion protein,
2SA-GFP, were analyzed first (Fig 7). Before stimulation, EGFR-RFP was present at the plasma membrane (Fig 7 A, right, top and bottom). Although high levels of expression of the GFP-
2SA construct could be seen throughout the cytoplasm of COS-7 cells, the plasma membrane association could not be detected before EGF stimulation (Fig 7 A, left, top and bottom). The merge of the enlarged areas was red due to presence of only the EGFR fluorescent tag. After stimulation, the GFP-
2SA construct first appeared in some parts of the membrane (similar to distribution in A431 cells in Fig 2) and spread across the membrane surface within 1 min (Fig 7B, left, top and bottom). As seen in the merge, the membrane color has become yellow, due to colocalization with the green fluorescence from
2SA-GFP after EGF stimulation. Similar translocation and membrane colocalization after the EGF treatment have been observed in cells expressing red-tagged receptor and green full-length PLC
2 or the EGFR-GFP and PLC
2-RFP constructs. In the later case, the color of the merge was changed from green to yellow (data not shown).
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In some cells and in particular at later timepoints after stimulation, extensive ruffling could be observed. In cells where ruffling was prominent, the EGFR distribution (followed by the green fluorescent tag) appeared to be uneven, with the majority of the receptor present in areas of membrane ruffles (Fig 8 A). Distribution of the full-length PLC2 (with the red fluorescence tag) in the same cell demonstrated that PLC
2 was localized in areas where EGFR was present. However, PLC
2-RFP did not cover all areas of the membrane where EGFR-GFP could be detected, and as seen in Fig 8 A some areas contained only EGFR. However, it is difficult to distinguish between different possibilities that could explain this distribution. The possibilities include complex formation in "preferred" areas of the membrane or a quick dissociation and separate internalization of PLC
2.
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The internalization of the EGFR and PLC2, coexpressed in a single cell, could also be studied after EGF stimulation. The appearance of small intracellular vesicles could be seen within minutes after stimulation (Fig 8 A), which subsequently increased in size (Fig 8 B); this was visualized for both EGFR-GFP (right, top and bottom) and PLC
2-RFP (left, top and bottom). As shown in Fig 8 B,
15 min after EGF stimulation, most of the EGFR was associated with the migrating intracellular vesicles shown previously to represent endosomes undergoing fusion and forming vesicular swellings (
2 was entirely associated with the intracellular vesicles (Fig 8 B, left, top and bottom). Further analysis of the areas containing the intracellular vesicles revealed colocalization of PLC
2-RFP and EGFR-GFP constructs in many of these structures. However, some vesicles seem to contain only EGFR or only PLC
2. These data suggest that the internalization paths of EGFR and PLC
2 could be, at least in part, separate.
Localization of PI 4,5-P2
In addition to biochemical studies, the metabolism and localization of PI 4,5-P2 have been monitored using reagents that specifically bind to this inositol lipid. In particular, the PH domain of PLC1 has been shown to bind PI 4,5-P2 present in the plasma membrane (
1 PH domain (or the catalytically inactive full-length PLC
1), the membrane localization could be seen before and after stimulation with EGF (Fig 5 A). Antibody specific to PI 4,5-P2 was also used, and in this case the staining was more clearly seen in the membranes of stimulated cells. In addition, staining of perinuclear area and Golgi could be seen in some cells using the antibody and was not strictly dependent upon cell stimulation (Fig 9a and Fig b). As reported previously (
1 PH domain, but its localization was less prominent in these structures than in the plasma membrane. This difference could be due to the different affinities or accessibility of the two probes. Nevertheless, in cells with membrane ruffles where the distribution of GFP-PLC
1 was uneven it could be clearly seen that staining with the antibody to PI 4,5-P2 completely colocalized with the green fluorescence in these structures. As shown in Fig 9 C, at the timepoints when some of the PLC
remained on the membrane although the larger portion has been internalized into intracellular vesicles, PI 4,5-P2 and PLC
colocalized only at the membrane. This can be concluded from direct comparisons of PLC
distribution and staining with antiPI 4,5-P2 antibody in the same cell (Fig 9 C) and from observations that PLC
1 (the full-length or the PH domain) or the antibody to PI 4,5-P2 did not show internalization into intracellular vesicles at times when PLC
was present in this compartment. This suggests that after internalization, PI 4,5-P2 was not available to PLC
, even if the enzyme could still be phosphorylated and/or present in a complex with the receptor.
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In agreement with previous observations (1 PH domain did not reveal detachment of this domain from the membrane at times (12 min) of I 1,4,5-P3 formation and calcium release triggered by growth factors (data not shown). This could be due to a smaller portion of PI 4,5-P2 hydrolyzed when compared with the system where the detachment could be seen (
Effects of the PI 3-Kinase Inhibitor LY294002
The effects of PI 3-kinase inhibitors, wortmannin, and a more specific LY294002 compound on I 1,4,5-P3 formation and calcium mobilization have been observed after PDGF stimulation, resulting in the reduction of these responses (1 activity and bind to the N-PH domain and/or C-SH2 domain (
1 phosphorylation and the receptor binding (
translocation, we first analyzed inhibition of phosphorylation of PKB within the range commonly tested in cells (050 µM) and demonstrated that even higher concentrations (50 µM) did not inhibit EGFR phosphorylation nonspecifically, which would otherwise interfere with the translocation (Fig 10 A). Even with higher concentrations of LY294002, translocation of PLC
2-RFP to the membrane could be seen (Fig 10 B, left) and was comparable to the timecourse of translocation in untreated cells. Similarly, no effect of LY294002 has been seen on the translocation of GFP-
1 and -
2SA or
1- and
2N-PH constructs (data not shown). Furthermore, the appearance of PI 4,5-P2 in membrane ruffles was also unaffected by LY294002 treatment (Fig 10 B, right).
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The calcium responses to the stimulation by a growth factor (for example, PDGF) could usually be detected after 2040 s with a duration of 23 min (20 to 50%) (Fig 10 C). Since inhibition of PI 3-kinase activity (shown by PKB phosphorylation, Fig 10 A) seems to be complete at 10 µM LY294002, it is possible that the binding to other targets could be responsible for the effects on calcium responses using 50 µM LY294002.
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Discussion |
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To follow the cellular localization of PLC in real time, cells expressing PLC
containing a fluorescent tag were stimulated by EGF known to activate this enzyme after binding to EGFR (
, which is in unstimulated cell present in the cytoplasm, undergoes several stages of subcellular redistribution, including fast but progressive attachment to the plasma membrane, followed by concentration in areas of extensive membrane ruffling and incorporation into intracellular vesicular structures.
As shown in Results, the initial translocation first occurred in some parts of the plasma membrane and then spread to all areas where the receptor was present. This gradual appearance at different parts of the membrane is unlikely to be due to differences in the amounts of the receptor, since it could be observed in cells where the receptor molecules containing the fluorescent tag were evenly distributed around the membrane. Use of the deletion and point mutations demonstrated that all determinants for PLC translocation to the plasma membrane are within the region unique for the PLC
family, which is well conserved between the two isoforms and is likely to involve the function of both SH2 domains (Fig 3 and Fig 4). A requirement for both SH2 domains of PLC
is consistent with a previous in vitro binding study of isolated SH2 domains to phosphorylated peptides from EGFR, suggesting simultaneous binding to two different parts of the receptor (binding of N-SH2 to pY1173 in the EGFR and binding of C-SH2 to pY992 in the EGFR) (
phosphorylation in cells stimulated with EGF (
in binding to the receptor or adapter proteins has been shown for signaling via PDGF receptor (
activity in the presence of the PDGF receptor peptide supported the possibility that binding to the receptor could cause allosteric changes and stimulate PLC activity (
binding to the receptor via its SH2 domains may not be restricted to translocation to the membrane and, together with PLC
phosphorylation, could contribute to stimulation of PLC
activity. The colocalization of PLC
and the receptor observed at early timepoints at the plasma membrane, where the presence of the PI 4,5-P2 substrate can also be visualized, seem to coincide with the peak of calcium responses, thus suggesting that the enzyme is fully active (Fig 5, Fig 7, and Fig 10).
The experiments using the isolated SA demonstrated that it contains all of the determinants required for translocation. Together with the mutations in the N-PH domain, these data do not support the possibility that the PH domainmediated translocation precedes the interaction with the receptor or contributes to the stability of the complex, as suggested in earlier studies (
phosphorylation and the enzymereceptor complex formation detected by coimmunoprecipitation (
may be important for activation. However, understanding of molecular basis and specificity of this potential link would require further studies. Regarding the role of the N-PH domain, its contribution to this process could differ, depending on the specific signaling pathway or PLC
isoform. In contrast to the suggested involvement of this domain in PLC
1 signaling in response to PDGF (
2 PH domain (shown to block PI 3,4,5-P3 binding to the PH domain of PLC
1) did not interfere with the function of PLC
2 (Fig 5 C).
In cells characterized by extensive ruffling prominent after several minutes of stimulation, PLC, EGFR, and PI 4,5-P2 were all localized in the most dynamic areas at the cell periphery (Fig 8 and Fig 9). Previous studies have suggested that cytoskeletal proteins are included in these structures. For example, the PI 4,5-P2bound PLC
1 PH domain and F actin colocalize in membrane ruffles (
could be bound to EGFR in these structures since PLC
has not been detected in any parts of the membrane where the receptor was not present (Fig 8). The presence of the substrate in the same structures (Fig 9) suggests the possibility that it could be hydrolyzed by the enzyme. Furthermore, earlier studies have demonstrated that only phosphorylated PLC
could hydrolyze PI 4,5-P2 bound to profilin (
Previous experiments using EGFR-GFP have enabled studies of EGFR endocytosis and visualized fragile endosomal structures ( in the same cell-expressing EGFR, demonstrated a similar pattern of endosomal movements where swelling and fusion were taking place followed by the formation of larger perinuclear structures (Fig 8). However, colocalization of PLC
and the receptor may be only partial (Fig 8), and PLC
appeared to be completely separated from its substrate (Fig 9). Therefore, in the case of PLC
the incorporation into intracellular vesicles is likely to lead to downregulation of the enzyme activity. This is further supported by findings that growth factor stimulation results in an early and transient peak of calcium release (Fig 10;
was hyperphosphorylated, suggesting that the main site of interaction and phosphorylation with EGFR is in the plasma membrane (
signaling.
In this study, the two PLC isoforms were compared (Fig 1 Fig 2 Fig 3, Fig 5, and Fig 6) and found to be remarkably similar in all aspects of EGFR-triggered translocation. Although PLC
1 has been more extensively studied in this context, it has been demonstrated that at least in transfected cells both PLC
1 and PLC
2 can bind to and be activated by growth factor receptors (
2 have shown that in addition to high levels of expression in some hematopoietic cells, this isoform is present in many different cell types and tissues (
1 and PLC
2 could signal in pathways via growth factor receptor and nonreceptor tyrosine kinases. When isolated domains from PLC
1 and PLC
2 were compared, again the
SA regions and the PH domains demonstrated similarity between the isoforms (Fig 3 and Fig 5). In the case of the PH domains, as shown earlier for the PLC
1 PH domain (
SA region from both isoforms followed kinetics of the full-length proteins and further mutagenesis (not performed in the study by
isoforms.
In conclusion, the data presented here obtained from the real time fluorescence imaging have provided new insights into a sequence of changes in subcellular localization and function of PLC after stimulation with EGF. Together with experimental evidence obtained previously using different experimental approaches, the following model could be suggested. First, PLC
undergoes rapid translocation to the plasma membrane. This step requires direct interaction with the activated receptor via the SH2 domains and leads to subsequent stimulation of PLC activity, resulting in an increase in the rate of PI 4,5-P2 hydrolysis present in the membrane. PLC
, that still could be in a complex with the receptor and active, is in some cells localized in membrane ruffles that are prominent at later times after stimulation. The internalization of PLC
into endosome-like structures is likely to result in the downregulation of PLC activity, which may no longer have access to its substrate.
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Footnotes |
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1 Abbreviations used in this paper: EGFR, EGF receptor; SA, PLC
-SA; GFP, green fluorescent protein; I, inositol; I 1,4,5-P3, I 1,4,5-trisphosphate; NGF, nerve growth factor; PDGF, platelet-derived growth factor; PH, pleckstrin homology; PI, phosphatidylinositol; PI 4,5-P2, PI 4,5-bisphosphate; PI 3,4,5-P3, PI 3,4,5-trisphosphate; PI-PLC, phosphoinositide-specific PLC; RFP, red fluorescent protein; SA, specific domain array; SH, Src homology.
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Acknowledgements |
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We are grateful to Dr. Kyoko Fukami for the antiPI 4,5-P2 antibody, Dr. Fred Wouters for the EGFR-GFP construct, Dr. Ron Kriz for the cDNAs of PLC1 and PLC
2, Dr. Tomohiro Kurosaki for the expression vector pApuro, and Dr. Tim Howkin for help with stable DT40 cells.
This work was supported by grants from The Cancer Research Campaign.
Submitted: 28 November 2000
Revised: 20 March 2001
Accepted: 20 March 2001
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References |
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