Article |
Address correspondence to Michael G. Roth, The University of Texas Southwestern, 5323 Harry Hines Blvd., Dallas, TX 75930-9038. Tel.: (214) 648-3276. Fax: (214) 648-8856. email: michael.roth{at}utsouthwestern.edu
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Abstract |
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Key Words: phosphatidylinositol kinase; endocytosis; clathrin; transferrin receptor; phosphatidylinositol 4,5-bisphosphate
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Introduction |
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PIP2 might function for clathrin-mediated endocytosis in several ways. PIP2 might serve as a membrane identity marker, allowing proteins important for endocytosis to collect at the correct membrane surface. In this event, PIP2 might control where endocytosis occurs without influencing the rates of endocytosis, if the steady-state concentration of PIP2 at the plasma membrane supports maximum rates of endocytosis. PIP2, either directly or after phosphorylation to PIP3 (Gaidarov et al., 1996, 2001; Rapoport et al., 1997), might also act as an allosteric regulator of proteins important for endocytosis, and thus modulate the endocytic rate. Phosphatidylinositides including PIP2 increase the affinity of AP-2 proteins for the internalization signals in endocytic cargo (Rapoport et al., 1997), and the crystal structure of the AP-2 tetramer suggests that binding to phosphoinositides may be required to open the binding site for internalization signals (Collins et al., 2002).
In addition to proteins of the clathrin coat, many other proteins bind PIP2 at the plasma membrane, and there must exist some mechanism regulating competition between different processes. One way to accomplish this would be to increase the production of PIP2 locally by controlling the location and activity of a phosphatidylinositol phosphate kinase. Interactions between the kinase and components of a particular cellular function that is regulated by PIP2 would then serve to channel PIP2 into the pathway subserving that function.
Two classes of lipid kinases produce PIP2 (Fruman et al., 1998). Originally classified as PI4P5K type I and type II, type I enzymes were found to phosphorylate the 5 position of the abundant phosphatidylinositol 4-phosphate to produce PIP2, but type II enzymes are really PIP 4-kinases that produce PIP2 by phosphorylating the 4 position of phosphatidylinositol 5-phosphate, a lipid species about which little is currently known. There are three isoforms of type I enzymes; phosphatidylinositol phosphate 5-kinase (PIP5KI) , ß, and
. The mouse and human PIP5KI
and ß enzymes were named in a reciprocal manner (Ishihara et al., 1996; Loijens and Anderson, 1996), and in this report we will use the human nomenclature throughout. PIP5KI enzymes contain a highly conserved central catalytic domain of
400 residues and have nonconserved amino- and carboxyl-terminal sequences. The terminal domains contain information for dimerization of the enzyme (Galiano et al., 2002), and may serve other isoform-specific functions. PIP5KI enzymes and their yeast counterpart are negatively regulated by phosphorylation (Vancurova et al., 1999; Park et al., 2001). Two splice variants of PIP5KI
are produced, and the longer form, which predominates in brain, is found at adhesion plaques as well as at the plasma membrane (Di Paolo et al., 2002; Ling et al., 2002). PIP5KI
and
have been implicated in different specialized forms of endocytosis. PIP5KI
is the major producer of PIP2 at the synapse, a site of extremely active endocytosis (Wenk et al., 2001). A truncated form of PIP5KI
was identified through a genetic screen for cDNAs that could restore signaling through a mutant CSF-1 receptor (Davis et al., 1997). The truncated kinase apparently acted as a dominant-negative inhibitor of endocytosis, allowing the mutant receptor to persist at the plasma membrane. Overexpressed PIP5KI
increased endocytosis of activated EGF receptors and was coprecipitated with the receptors, whereas PIP5KIß was reported to have no effect on endocytosis of EGF (Barbieri et al., 2001). These observations are consistent with the ability of PIP5KI enzymes to regulate clathrin coat formation for at least certain endocytic events, and suggest that different isoforms may function in different cell types or for different endocytic activities.
Both endocytosis at the synapse and regulated endocytosis of growth factor receptors differ in a number of ways from the constitutive endocytosis of nutrient receptors, such as the transferrin receptor. Both use additional components compared with constitutive endocytosis. Endocytosis of synaptic vesicle components is an order of magnitude faster than clathrin-mediated endocytosis in other cell types, and the rate of endocytosis of growth factor and hormone receptors is acutely regulated. Thus, we were interested in determining if the rates of constitutive endocytosis might be regulated by PIP2 levels, and if so, by which enzyme. Using overexpression and RNAi to raise or lower the cellular concentration of each of the PIP5KI isoforms in HeLa or CV-1 cells, we found that increased PIP2 levels increased the rate of constitutive endocytosis. PIP5KIß was the major contributor to PIP2 levels and to clathrin-mediated endocytosis of the transferrin receptor. In contrast, increased expression of exogenous PIP5KI or increased transcription of endogenous PIP5KI
had no impact on endocytosis or cellular PIP2 levels.
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Results |
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Internalization assays were performed to determine whether PIP5KI expression levels would affect constitutive endocytosis. The expression of transferrin receptors in cells expressing PIP5KI proteins was examined by immunoblotting (unpublished data) and by fluorescence microscopy (Fig. 2 A), and was not changed compared with control cells, although the cells overexpressing PIP5KIß became more elongated than uninfected cells. Overexpression of either the 87-kD (unpublished data) or 90-kD isoforms had no effect on endocytosis of transferrin receptors, but overexpression of PIP5KI
and ß increased the rate of endocytosis of transferrin
30 and 100%, respectively (Fig. 2). To determine whether the observed differences between the
and ß isoforms were due to differences in protein expression levels, we immunoprecipitated the overexpressed proteins from lysates of cells 35S-labeled with amino acids. We found that both proteins were expressed at a very similar level (unpublished data).
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Discussion |
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We observed that overexpression of recombinant PIP5KI (both splice variants) did not change PIP2 levels or impact constitutive endocytosis, and the increase in endogenous PIP5KI
that occurred when expression of PIP5KIß was inhibited by siRNA did not compensate for the loss of PIP5KIß. Therefore, it is likely that PIP5KI
plays no role in constitutive endocytosis in either CV-1 cells or HeLa cells. Either this isoform is not active in these cell types, or it acts in a location different from the plasma membrane that does not provide a significant fraction of the total PIP2 production in these cells. We can conclude less about PIP5KI
. Overexpressing this isoform had less impact on PIP2 levels than did overexpressing PIP5KIß, although protein levels of PIP5KI
were slightly higher. A possibility consistent with our observations and those reported by Barbieri et al. (2001) is that a significant fraction of PIP5KI
may be sequestered and not available for constitutive endocytosis, perhaps by associating with growth factor receptors. Perhaps this fraction of PIP5KI
is not constitutively active, and only the remaining PIP5KI
is able to produce PIP2 and impact constitutive endocytosis. We cannot answer the question of whether PIP5KI
plays some role in constitutive endocytosis or can compensate for the loss of PIP5KIß, because siRNA oligonucleotides that inhibited PIP5KIß also lowered expression of PIP5KI
. Three separate oligonucleotide sequences of PIP5KIß that contained significant mismatches with PIP5KI
each had this effect, and we currently have no explanation for this.
Also, we observed that inhibiting the expression of one isoform of PIP5KI with siRNA resulted in increased expression of the other isoforms and, conversely, overexpression of one isoform can result in reduced expression of the other isoforms. Thus, transcription of PIP5KI genes is coordinately regulated in some fashion. In the case of siRNA ablation of PIP5KIß, which also slightly lowered PIP5KI, PIP5KI
mRNA increased threefold, but this did not translate into an active enzyme capable of rescuing total cellular PIP2 levels (Fig. 6). PIP2 levels did not decrease when PIP5KI
expression was lowered by siRNA, presumably because the increased expression of PIP5KIß compensated for the loss of
. Inhibition of expression of PIP5KI
also caused a twofold increase in the other two isoforms. Because we have no evidence that PIP5KI
is active in HeLa cells, either there is a mechanism that senses the presence of PIP5KI isoforms independently of their enzymatic activity, which suggests that they might share common interaction partners, or the sensing mechanism uses a PIP2 pool that is a small fraction of total cellular PIP2.
One difficulty for understanding the functions for PIP2 in endocytosis is that it interacts with a great variety of factors, including lipid-modifying enzymes, such as PLD1 (Liscovitch et al., 1994), and the actin cytoskeleton (Yin and Janmey, 2003), which plays an undefined role in constitutive receptor-mediated endocytosis (Fujimoto et al., 2000). Overexpression of any of the three isoforms of PIP5KI leads to reorganization of the actin cytoskeleton (Shibasaki et al., 1997; Ishihara et al., 1998; Rozelle et al., 2000; Yamamoto et al., 2001). In particular, overexpression of the PIP5KIß isoform in CV-1 cells promotes formation of stress fibers, and cells become elongated (Yamamoto et al., 2001). We have shown that actin-disrupting drugs did not prevent the PIP5KI-mediated increase in endocytosis of transferrin receptors; thus, the effects of PIP5KI for promoting stress fibers is separate from its effect on endocytosis. Our results suggest that one effect of PIP5KIß on constitutive endocytosis is to increase the fraction of endocytic adaptors bound to membranes that may in turn regulate the formation of clathrin-coated pits. We observed that the proportions of AP-2 proteins associated with membranes increased with increasing PIP2 levels. Cells overexpressing PIP5KIß also had more clathrin-coated pits at the plasma membrane. This observation suggests that the impact of increased PIP2 for increasing endocytic rates was preferentially on the processes for forming clathrin-coated pits, rather than those responsible for budding off clathrin-coated vesicles or that determine the occupancy of transferrin receptors in coated pits.
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Materials and methods |
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Transfection, adenovirus, and SV40 infections
Recombinant adenovirus expressing an HA-tagged version of mPIP5KI was obtained from Y. Shibasaki (University of Tokyo, Tokyo, Japan). Adenoviruses expressing the myc-tagged type PIP5KI
, ß-galactosidase, or GFP were constructed using the AdEasyTM Adenoviral Vector System (Stratagene). A plasmid encoding the 90-kD splice variant of PIP5KI
was obtained from the Kazusa DNA Research institute (KIAA0589), myc-tagged and subcloned into pCMV5. A cDNA encoding an HA-tagged 87-kD splice variant of mPIP5KI
(Ishihara et al., 1998) was obtained from Dr. P. De Camilli with permission of Dr. Y. Oka (Tohoku University, Sendai, Japan), and was expressed using the plasmid vector pcDNA3.1. For all experiments using adenovirus vectors, cells were infected at a multiplicity of infection of 10. After 2 h, cells were washed and then cultured in fresh DME containing 10% FCS for 36 h. For experiments expressing the HA Y543 mutant, cells were infected with recombinant SV40 virus in suspension for 30 min on ice followed by culture in complete growth medium for 26 h. For experiments using transient transfection, cells were transfected with pCMV5-PIP5KI
and pEGFP-C3 (CLONTECH Laboratories, Inc.) at a ratio of 3 to 1, using LipofectAMINETM according to the manufacturer's instructions (Invitrogen).
RNA interference
siRNA oligonucleotides were designed according to the protocol provided by Dharmacon Research, Inc. In brief, sequences of the type AA(N19) (N = any nucleotide) from the ORF of the targeted mRNA were selected and subjected to a BLAST® search (National Center for Biotechnology Information database) against the human genome sequence to ensure the specificity of targeting. RNA oligonucleotides encoding both the sense and antisense of the target were synthesized by the Center for Biomedical Inventions (University of Texas Southwestern Medical Center at Dallas, Dallas, TX), and annealed after a protocol from Dharmacon Research, Inc. The siRNA sequence targeting PIP5KI (GenBank/EMBL/DDBJ accession no. U78575) was from position 19231943. The siRNA targeting PIP5KIß (GenBank/EMBL/DDBJ accession no. NM_003558) encoded bases 11141135. The oligonucleotide targeting PIP5KI
(GenBank/EMBL/DDBJ accession no. XM_047620) encoded bases 619639. An oligonucleotide corresponding to nucleotides 695715 of the firefly luciferase (GenBank EMBL/ DDBJ accession no. U31240) was used as a negative control. On d 1, HeLa cells were plated in 6-well plates at 3040% confluency in antibiotic-free DME supplemented with 10% (vol/vol) FCS, 10 mM Hepes, and 1 mM sodium pyruvate. On d 2, siRNA was introduced into cells using OligofectAMINETM reagent according to the manufacturer's instructions (Life Technologies), with 10 µl of 20 µM siRNA and 4 µl transfection reagent/well. On d 4, cells were lysed and analyzed by Western blotting.
Quantitative real-time PCR
Total RNA was extracted from HeLa cells transfected with siRNAs using the total RNA/mRNA isolation reagent RNA STAT-60TM (TEL-TEST, Inc.). RNA samples were treated with DNase I (RNase-free; Roche), and were reverse-transcribed with random hexamers using SuperScriptTM II RNase H-reverse transcriptase to generate cDNA. Primer Express software (PerkinElmer) was used to design primers for cyclophilin (used as internal control; GenBank/EMBL/DDBJ accession no. XM_057194), forward TGCCATCGCCAAGGAGTAG, reverse TGC ACA GAC GGT CAC TCA AA; PIP5KI, forward TCAAAGGCTCAACCTACAAACG, reverse TTTAAATGTGGGAAGAGGCTTCT; PIP5KIß, forward GAAACGGTGCAATTCAATCG, reverse TCCTGGCTAATTGAGGACACA; and PIP5KI
, forward TGTCGCCTTCCGCTACTTC, reverse GGCTCATTGCACAGGGAGTAC. Primers were validated through analysis of template titration and dissociation curves to establish both linearity of the reaction and production of a single product. PCR assays were conducted on a sequence detection system (Prism® 7000; Applied Biosystems). The 20-µl final reaction volume contained 50 ng reverse-transcribed RNA, 150 nM of each primer, and 10 µl SYBR® Green PCR Master Mix (Applied Biosystems). PCR reactions were performed in triplicate, and relative RNA levels were determined by the comparative Ct method (User Bulletin No. 2; PerkinElmer).
Membrane fractionation
Cells were homogenized in 0.2 M sucrose buffered with 20 mM Hepes (pH 7.3) by 25 strokes in a pre-chilled steel homogenizer, and were centrifuged at 960 g for 15 min at 4°C. The supernatant was then centrifuged at 128,000 g for 60 min at 4°C. Supernatants and pellets were separated and stored at -80°C.
Endocytosis assays
Cells infected with adenoviruses or transfected with RNAi oligos were rinsed and incubated in serum-free medium for 30 min to remove any residual transferrin, and then were exposed to 50 µg/ml transferrin conjugated with Alexa® Fluor 488 or 633 at 37°C for the times indicated. Internalization was stopped by chilling the cells on ice. External transferrin was removed by washing with ice-cold serum-free DME and PBS, whereas bound transferrin was removed by an acid wash in PBS at pH 5.0 followed by a wash with PBS at pH 7.0. The fluorescence intensity of internalized transferrin was measured by flow-cytometry using FACScanTM or FACSCaliburTM (Becton Dickinson) instruments, and the average intensity of 10,000 cells was recorded for each time point. Data are normalized to the increase in mean fluorescence of cells infected with adenovirus expressing ß-galactosidase. We confirmed that the uptake of fluorescent transferrin was receptor mediated by measuring the uptake of fluorescent transferrin in the presence of a 100-fold excess of nonfluorescent holotransferrin under the same experimental conditions. No increase in cell-associated fluorescence was obtained in the presence of excess competing nonfluorescent transferrin. Cell viability was measured by staining with propidium iodide.
Internalization of influenza mutant HA Y543
Endocytosis of an internalization-competent HA mutant was measured as described previously (Lazarovits and Roth, 1988). In brief, CV1 cells were infected with recombinant SV40 virus encoding the HA Y543 mutant. 26 h after SV40 infection, cells were washed and 35S-labeled with amino acids for 30 min at 37°C. The labeling medium was then replaced with DME, and after an additional incubation of 2 h at 37°C, the cells were exposed to polyclonal anti-HA antibody on ice for 45 min. Antibody was removed, cells were washed, and were then incubated at 37°C for 0, 2, 4, and 8 min. Cells were then returned to ice, treated with 100 µg/ml trypsin for 45 min, and lysed in the presence of 200 µg/ml soybean trypsin inhibitor and a cocktail of protease inhibitors. The HA protein bound to antibody was immunoprecipitated with protein ASepharose and was resolved by SDS-PAGE. The internalized HA was calculated as the fraction that became resistant to trypsin cleavage and was expressed as percentage of total HA.
PIP2 analysis
Cells were labeled with 40 µCi/ml [32P]orthophosphoric acid for 4 h in phosphate-free DME plus 0.5% FBS. Lipids were extracted from the cells with a 4:10:5 mixture of CHCl3:CH3OH:1N HCl and were resolved by TLC, visualized by autoradiography, and quantified by densitometry. Lipid standards (Avanti Polar Lipids, Inc.) were detected by iodine vapors.
Fluorescence microscopy
Cells on coverslips were washed with PBS and fixed in 3.7% PFA for 15 min. The fixative was removed and cells were washed with DME and permeabilized with 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.25% gelatin, 5 mM EDTA, 0.02% NaN3, 0.05% NP-40, and 0.05% Triton X-100. Nonspecific binding sites were blocked with PBS containing 1% BSA before incubating samples with primary and secondary antibodies for 1 h at RT. Coverslips were mounted on glass slides with Aqua-Polymount (Polysciences) and visualized with a microscope (Axioplan; Carl Zeiss MicroImaging, Inc.) fitted with a confocal scanning head (MRC600; Bio-Rad Laboratories) and a Kr/Ar laser (Bio-Rad Laboratories) or with a confocal microscope (model 510; Carl Zeiss MicroImaging, Inc.).
EM
Control and infected cells were processed for EM following standard procedures using osmium tetroxide and lead citrate for staining. Clathrin-coated pits were visualized and counted at a magnification of 70,000 in an electron microscope (model 1200EX; JEOL USA, Inc.). Digital images of each cell were stored for analysis, and the length of plasma membrane on which coated pits were counted was quantified using Image J 1.27z software (National Institutes of Health, Bethesda, MD).
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Acknowledgments |
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This work was supported by a grant from the Pew Foundation Latin American Fellows Program (to D. Padrón), grant GM37547 from the National Institutes of Health and the Diane and Hal Brierley Chair in Biomedical Research (to M.G. Roth), and grant GM51112 from the National Institutes of Health (to H. Yin).
Submitted: 7 February 2003
Accepted: 2 July 2003
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