A Novel Interaction between the Juxtamembrane Region of the p55 Tumor Necrosis Factor Receptor and Phosphatidylinositol-4-phosphate 5-Kinase*

(Received for publication, September 9, 1996, and in revised form, November 25, 1996)

Alexander M. Castellino Dagger , Gregory J. Parker §, Igor V. Boronenkov §par , Richard A. Anderson §par ** and Moses V. Chao Dagger

From the § Department of Pharmacology, the  Program in Cellular and Molecular Biology, and the par  Department of Biomolecular Chemistry, University of Wisconsin Medical School, Madison, Wisconsin 53706 and the Dagger  Department of Cell Biology and Anatomy, Cornell University Medical College, New York, New York 10021

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

Tumor necrosis factor-alpha (TNF-alpha ) binding to its receptors leads to a diversity of biological responses. The actions of TNF are the result of the interaction of cytoplasmic proteins that bind directly to the intracellular domains of the two TNF receptors, p55 and p75. Here we report a novel interaction between the juxtamembrane region of the p55 TNF receptor and a newly discovered 47-kDa isoform of phosphatidylinositol-4-phosphate 5-kinase (PIP5K), a member of the enzyme family that generates the key signaling messenger, phosphatidylinositol 4,5-bisphosphate. The interaction was found to be specific for the p55 TNF receptor and was not observed with the p75 TNF receptor, the Fas antigen, or the p75 neurotrophin receptor, which are other members of the TNF receptor superfamily. In vitro experiments using recombinant fusion proteins verify the authenticity of the interaction between the p55 receptor and PIP5KIIbeta , a new isoform of PIP5K, but not the previously identified 53-kDa PIP5KIIalpha . Treatment of HeLa cells with TNF-alpha resulted in an increased PIP5K activity. These results indicate that phosphatidylinositol turnover may be linked to stimulation of the p55 TNF receptor and suggest that a subset of TNF responses may result from the direct association of PIP5KIIbeta with the p55 TNF receptor.


INTRODUCTION

Tumor necrosis factor-alpha (TNF-alpha )1 initiates its proliferative, differentiative, or cytotoxic actions on mammalian cells by binding to two transmembrane molecules, the p55 and p75 TNF receptors (1-3). The p55 receptor is responsible for many of the biological effects of TNF, including programmed cell death, cell differentiation, and cell proliferation (4-6). A major step in understanding the mechanism of the p55 receptor has been the identification of the interacting protein, TRADD, which accounts for signals leading to apoptosis and increased gene expression through NF-kappa B-mediated events (7). Likewise, the p75 receptor is capable of signal transduction through the association of ring finger proteins, such as TRAF1 and TRAF2, with the cytoplasmic domain of p75 (8, 9).

The interaction of TRADD with the p55 TNF receptor has revealed the importance of protein-protein interactions via a region of homology called the "death domain." This sequence has been found in a variety of transmembrane and cytosolic molecules and is usually localized at the C-terminal region of each protein. The functional significance of this domain has been demonstrated in studies with the p55 TNF receptor and the Fas antigen, which contain similar functional death domain sequences (10). The binding of p55 receptors to TRADD, a cytoplasmic protein containing a death domain, and the binding of the Fas antigen to FADD, an analogous protein, have been localized to an 80-amino acid region at the C terminus of both receptors. While overexpression of TRADD or FADD in heterologous cells leads to cell death (7, 11), deletions or mutations in the death domains abolish the ability of these molecules to participate in the initiation of apoptosis (6, 12).

A link to the interleukin converting enzyme/ced-3 protease family was made recently with the identification of a cysteine protease, FLICE/MACH, which interacts with the death effector domain of FADD (13, 14). The connection of the Fas antigen and the p55 TNF receptor with a member of the interleukin converting enzyme protease family provides a mechanism for how cell death signals are initiated by ligand-receptor interactions.

While cell death can be instigated by TNF-alpha and represents the major signaling function of the Fas antigen, TNF-alpha can participate in many other diverse activities, including the synthesis of proinflammatory mediators and cell proliferation and differentiation (15), neuroprotection (16), and synaptic transmission (17). The striking interactions of the C-terminal half of the intracellular domain of the p55 receptor raise the issue of the functional significance of the juxtamembrane region of the p55 receptor. This region is ~100 amino acids in length, is rich in proline residues, and is not required for TNF-mediated cellular cytotoxicity. Here we report the interaction of the juxtamembrane region of the p55 TNF receptor with phosphatidylinositol-4-phosphate 5-kinase (PIP5K), an enzyme that produces phosphatidylinositol 4,5-bisphosphate (PIP2). PIP5K is a significant enzymatic activity to be linked to TNF signaling since its product, PIP2, is a critical second messenger intermediate that also has many direct modulatory effects. Moreover, this study has identified a new member of the PIP5K family, PIP5KIIbeta . The association of the p55 receptor with PIP5K indicates how diverse functions are encoded in the TNF receptor structure to generate multiple signal transduction events after ligand binding.


EXPERIMENTAL PROCEDURES

Cell Culture

HeLa, U373, and MCF7 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. In experiments using TNF, the cells were transferred to serum-free medium when an 80% confluency was reached. The cells were serum-starved for 12-24 h before TNF was added at a final concentration of 100 ng/ml. Cells (10-cm dish) were harvested at appropriate time intervals, washed three times in cold phosphate-buffered saline, and lysed in 500 µl of cold lysis buffer (20 mM Tris, pH 7.5, 25 mM beta -glycerophosphate, 137 mM NaCl, 100 mM EDTA, 1% Triton X-100, 2 mM sodium pyrophosphate, 100 µM sodium vanadate, 0.5 mM LiCl, 1 µg/ml leupeptin, 2 µg/ml aprotinin, and 25 µg/ml phenylmethylsulfonyl fluoride). The cell debris was pelleted after a 20-min incubation on ice. The lysates were stored frozen at -70 °C.

Construction of Yeast Expression Vectors

The yeast expression vector pSD.04, containing the lexA DNA-binding domain (18), was used for these studies. All recombinant clones were generated by PCR using primers containing a 5'-NotI linker and a 3'-SpeI linker. The bait used in the primary screening protocol was Y1 (residues 204-280 of the human p55 receptor). Other regions of the human p55 receptor used were Y2 (residues 278-364), Y3 (residues 324-426), Y4 (residues 204-364), and Y5 (residues 204-426). Other constructs used the cytoplasmic domains of the Fas antigen, p75 TNF receptor, and p75 neurotrophin receptor. The primers for the baits were as follows: Y1, 5'-primer (5'-ATTTGCGGCCGCCATGTATCGCTACCAACGG-3') and 3'-primer (5'-CTAACTAGTTCAAGCCGCAAAGTTGGG-3'); Y2, 5'-primer (5'-ATTTGCGGCCGCTTTGCGGCTCCCCGCAGA-3') and 3'-primer (5'-CTAACTAGTTCACCCGTTCTGCAGCTC-3'); Y3, 5'-primer (5'-ATTTGCGGCCGCCACTGATGACCCCGCGACG-3') and 3'-primer (5'-CTAACTAGTTCATCTGAGAAGACTGGG-3'); Y4, 5'-primer (5'-ATTTGCGGCCGCCATGTATCGCTACCAACGG-3') and 3'-primer (5'-CTAACTAGTTCACCCGTTCTGCAGCTC-3'); Y5, 5'-primer (5'-ATTTGCGGCCGCCATGTATCGCTACCAACGG-3') and 3'-primer (5'-CTAACTAGTTCATCTGAGAAGACTGGG-3'); Fas antigen, 5'-primer (5'-ATTTGCGGCCGCCCGAAAGTACCGGAAAAGA-3') and 3'-primer (5'-CTAACTAGTTCACTCCAGACATTGTCC-3'); and p75 TNF receptor, 5'-primer (5'-AAATTTGCGGCCGCCATGACCCAGGTGAAAAAGAAG-3') and 3'-primer (5'-CTAACTAGTTTAACTGGGCTTCATCCCAGC-3').

For each PCR fragment, the reaction mixture (100 µl) contained the primers at 1 ng/µl; the DNA template at 0.1 ng/µl; 200 nM each dATP, dCTP, dGTP, and dTTP; 1 × Pfu buffer; and 1.25 units of cloned Pfu polymerase (Stratagene). The PCR conditions for Y1, Y2, and Y3 were as follows: 1 cycle at 85 °C for 8 min followed by 5 cycles at 94 °C for 2 min, annealing temperature of 30 °C for 1.5 min, and extension temperature of 72 °C for 1 min and 25 cycles at 94 °C for 2 min, 60 °C for 1.5 min, and 72 °C for 1 min. This was followed by a final cycle of 72 °C for 4 min. For the Fas antigen, the PCR conditions were similar, except the annealing temperature was 50 °C. For the p75 TNF receptor, the annealing temperature was 55 °C. The DNA templates were pCMVp55 and pCMVp75 (19) and pCMVmFas (20). These conditions yielded PCR fragments of the expected sizes. The PCR fragments were phenol/chloroform-extracted, ethanol-precipitated, and digested with NotI and SpeI before ligation in the lexA DNA-binding yeast expression vector. Transformants were screened by restriction digestion and verified by DNA sequencing.

Yeast Growth and Transformation

Saccharomyces cerevisiae strain S260, which contains a lexA operator-lacZ reporter gene, was maintained as described (18). The baits were transformed into S. cerevisiae (21), and the transformants were selected and maintained on Trp- plates and Trp- medium. Log phase cells (1010) were cotransformed with 150 µg of murine cerebellar library DNA constructed in the VP16 activation domain according to published procedures (21). The cotransformants were plated on Amersham Hybond-N filters laid on Ura-Trp- plates containing 2% glucose. After incubation at 30 °C for 48 h, the filters were transferred to similar plates containing 2% galactose and further incubated for 24 h to induce expression of the VP16-cDNA fusion proteins encoded by the library plasmids. Interacting proteins were detected by a qualitative colony beta -galactosidase activity assay as described (18). Colonies positive for beta -galactosidase were streaked on Ura-Trp- plates. The yeast plasmids were rescued by transforming into Escherichia coli and were subjected to DNA sequencing analysis (Sequenase).

Generation and Purification of GST Fusion Proteins

The cytoplasmic (residues 204-426), juxtamembrane (residues 204-337), and death domain (residues 340-426) regions of human p55 were amplified using PCR. The primers used were as follows: for the cytoplasmic domain, 5'-primer (5'-AAAGGATCCATGTATCGCTAC-3') and 3'-primer (5'-GAGTCGACCTCTGAGAAGACT-3'); for the juxtamembrane region, 5'-primer (5'-AAAGGATCCTGTATCGCTAC-3') and 3'-primer (5'-GAGTCGACCTCACACGTTCTC-3'); and for for the death domain, 5'-primer (5'-AAAGGATCCTTGCGCTGGAAG-3') and 3'-primer (5'-GAGTCGACCTCTGAGAAGACT-3'). A BamHI linker was incorporated into the 5'-primers, and a SalI linker was incorporated into the 3'-primers. PCRs were carried as described above using 30 cycles with a melting temperature of 94 °C for 2 min, an annealing temperature of 48 °C for 1 min, and an extension temperature of 72 °C for 2 min. The PCR fragments were digested with BamHI and SalI and ligated into pGSTag (22). Large-scale GST fusion proteins expressed from the various constructs were purified according to published procedures (23).

For making GST and MBP fusion proteins of clone 11 (c11), the yeast library plasmid corresponding to c11 was used as a template to amplify the library insert. In both cases, the 5'-primer (5'-AAAGGATCCGGTGGGGAATTCCCA-3') and the 3'-primer (5'-TATCTAGACTAGTCTAATCGATCTCGAGCCA-3') had a BamHI linker incorporated in the 5'-primer. The fragment was PCR-amplified as described above using the conditions described for the p75 TNF receptor. The fragment was digested with BamHI and XbaI and ligated separately into pGSTag and pMAL-c2 (New England Biolabs Inc.). The positive clones were tested for their ability to yield fusion proteins of the expected sizes after isopropyl-1-thio-beta -D-galactopyranoside induction (0.5 mM). The GST fusion protein was purified as described (23), and the MBP fusion protein was purified using the protocol provided by New England Biolabs Inc.

Generation of Anti-GST-c11 and Anti-MBP-c11 Antibodies

The GST-c11 and MBP-c11 fusion proteins were denatured in SDS and used as immunogens to generate polyclonal antibodies (Pocono Farms).

Isolation of PIP5KIIbeta cDNA

In a data base search, the PIP5KIIalpha sequence (GenBankTM/EMBL accession number U14957[GenBank]) matched the HFBEP52 (GenBankTM/EMBL accession number T07883[GenBank]) expressed sequence tag from the human infant brain cDNA library with 70.5% identity. This 2.7-kb clone was obtained and sequenced (Sequenase Version 2.0, U. S. Biochemical Corp.). A 2.0-kb EcoRI fragment corresponding largely to the 3'-untranslated region of this clone was used to screen 8 × 105 plaques from a human fetal brain lambda DR2 cDNA library (CLONTECH). The plaques were transferred to nitrocellulose filters and hybridized with the probe at 42 °C in 5 × SSPE, 5 × Denhardt's solution, 50% formamide, 0.1% SDS, and 100 µg/ml salmon sperm DNA. These filters were washed at high stringency (65 °C, 0.01 × SSC and 0.01% SDS) for 20 min. Eleven positive clones were obtained, plaque-purified, and converted to their respective phagemids. Upon sequencing, two clones were found to extend the sequence of HFBEP52 in both the 5'- and 3'-directions. Additional sequencing of the 5'-region was done on an Applied Biosystems Model 373 sequencer at the University of Wisconsin Biotechnology Center. This revealed a putative start site within the context of a Kozak consensus sequence (24) that was preceded by upstream stop codons in all three reading frames. This evidence demonstrated that a full-length open reading frame for PIP5KIIbeta had been obtained.

Northern Blotting

A human multiple tissue Northern blot (CLONTECH) was probed with a 1.7-kb KpnI fragment of HFBEP52, which lies entirely within the 3'-untranslated region of PIP5KIIbeta . The DNA fragment was gel-purified following restriction enzyme digestion and labeled by random priming with [alpha -32P]ATP and Klenow DNA polymerase. Hybridization and washing were performed as outlined by the manufacturer, and the membrane was exposed to film overnight.

Expression of PIP5KIIbeta in E. coli

The 1.2-kb open reading frame of PIP5KIIbeta was amplified by PCR (30 cycles at 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 3 min followed by an extension temperature of 72 °C for 10 min) using native Pfu polymerase (Stratagene) and the following primers: 5'-GCCGCCGC<UNL>GGATCC</UNL>CATGTCGTCC-3' (forward) and 5'-GCTGAAGGTA<UNL>CTCGAG</UNL>AACTACG-3' (reverse). The forward primer was designed to anneal over the start codon (in boldface) and to introduce a BamHI site (underlined) just upstream of the start site, while the reverse primer was designed to anneal over the stop codon (in boldface) and to introduce a XhoI site (underlined) just downstream of the termination codon. These PCR products were sucloned into the BamHI and XhoI sites of pBluescript SK(-) (Stratagene). The entire insert was sequenced to confirm that no errors had been introduced by PCR. The BamHI/XhoI fragment was then subcloned into pET28b (Novagen) for the purpose of expressing a hexahistidine fusion version of PIP5KIIbeta (His-PIP5KIIbeta ).

E. coli strain BL21(lambda DE3) was transformed with either pET28b containing the PIP5KIIbeta open reading frame or just pET28b. For the initial expression, these strains were grown at 37 °C to A600 = 0.6, induced with 1 mM isopropyl-1-thio-beta -D-galactopyranoside, and incubated at 37 °C for an additional 3 h. The cells were pelleted and resuspended in 50 mM Tris-HCl, pH 8.0, and 2 mM EDTA. Next, 4000 units of Ready-lyse solution (Epicentre Technologies Corp.) was added to the resuspended cells, and Triton X-100 was added to a final concentration of 0.1%. After incubating the cells at 30 °C for 15 min, they were sonicated at 70 watts twice for 10 s. These lysates were centrifuged at 12,000 × g for 15 min at 4 °C, and the supernatant was decanted. The pellet was dissolved in 1 × SDS-PAGE sample buffer, while an aliquot of the supernatant was mixed with an equal volume of 2 × sample buffer. All samples were boiled for 10 min and analyzed by SDS-PAGE in the presence of 250 mM beta -mercaptoethanol.

His-PIP5KIIbeta was purified by Ni2+ chelate chromatography. The strain containing the His-PIP5KIIbeta open reading frame was grown at 30 °C to A600 = 0.6, induced with 1 mM isopropyl-1-thio-beta -D-galactopyranoside, and incubated at 30 °C for an additional 3 h. The cells were centrifuged and resuspended in 8 ml of 1 × binding buffer (20 mM Tris-HCl, pH 7.9, 500 mM NaCl, and 5 mM imidazole). After sonication with three 20-s pulses at 70 watts, the extract was centrifuged at ~39,000 × g for 20 min at 4 °C and passed through a 0.45-µm syringe filter. An aliquot of this extract was set aside, and the rest was purified on a 2.5-ml gravity-packed column of His-Bind resin (Novagen). Bound proteins were eluted in three 5-ml fractions. Each of these fractions was dialyzed overnight at 4 °C against 2.0 liters of phosphate-buffered saline.

Kinase Activity Assays

The supernatants of the crude fraction of His-PIP5KIIbeta and of partially purified His-PIP5KIIbeta were assayed for phosphatidylinositol-4-phosphate 5-kinase activity as described previously (25) with some modifications. Kinase activity was assayed in 50-µl reactions done for 20 min at room temperature in a final concentration of 50 mM Tris-HCl, pH 7.6, 0.5 mM EGTA, 10 mM MgCl2, 160 µM phosphatidylinositol 4-phosphate (Sigma), 50 µM ATP, and 10 µCi of [gamma -32P]ATP. The lipids were extracted, and the labeled products were separated by thin layer chromatography and detected by autoradiography.

Western Blotting---Proteins were transferred to nitrocellulose (Micron Separations, Inc.) following SDS-PAGE. Antibodies were incubated with the membrane for 1 h at room temperature following blocking with 5% powdered milk in phosphate-buffered saline containing 0.2% Tween 20. The primary antibody was raised against the MBP-c11 fusion protein and was detected by chemiluminescence using a horseradish peroxidase-conjugated antibody (Santa Cruz Biotechnology, Inc.) and LumiGLO substrates (Kierkegaard and Perry Laboratories, Inc.).

In Vitro Binding

GST fusion proteins containing the cytoplasmic, the juxtamembrane, or the death domain of the p55 fusion protein were purified on glutathione-Sepharose beads, and 100 µl of the fusion protein on the glutathione beads was incubated with 3 µg of purified MBP-c11. The reaction volume was brought up to 500 µl with 50 mM Tris, pH 7.5, 200 mM NaCl, 20 mM EDTA, and 0.1% Nonidet P-40. After incubation at 4 °C for 1 h, the slurry was washed three times with the same buffer. The slurry was then boiled in SDS-PAGE buffer, and the samples were loaded on a 10% SDS-polyacrylamide gel and electrophoresed at 100 V until the dye front reached the bottom of the gel. The gel was then transferred to an Immobilon-P transfer membrane (Millipore Corp.), and immunoblotting was carried out using chemiluminescence. The primary antibody was the rabbit anti-MBP antibody (New England Biolabs Inc.) used at a 1:1000 dilution. The secondary antibody was a peroxidase-conjugated goat anti-rabbit IgG (Sigma) used at a 1:15,000 dilution.

Immunoprecipitation

Cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Near confluency, the cells were washed three times with phosphate-buffered saline, scraped, and centrifuged. The cells were lysed in 500 µl of radioimmune precipitation assay buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton, and 0.4% SDS) supplemented with protease inhibitors. Lysates were incubated on ice for 30 min; the cell debris was removed by centrifugation; and an aliquot of the lysate was used to determine protein concentration using the Bio-Rad reagent. Lysates (4 mg) were subjected to immunoprecipitation in a final volume of 500 µl of radioimmune precipitation assay buffer. Antibodies against PIP5K (protein A-purified) that were coupled to Affi-Gel-10 beads (Bio-Rad) were added to the lysate, and the mixture was incubated at 4 °C for 1 h. In separate reactions, lysates were incubated either with 100 µl of protein A-purified anti-p55 antibodies (Genzyme Corp.) coupled to Affi-Gel-10 beads or with nonspecific goat serum. The immunoprecipitate was washed three times in radioimmune precipitation assay buffer and boiled for 30 s in SDS-PAGE buffer. The sample was subjected to electrophoresis on a 10% SDS-polyacrylamide gel. Chemiluminescence was carried out using the ECL procedure (Amersham Corp.).


RESULTS

The p55 TNF receptor is a transmembrane protein with four extracellular cysteine-rich repeats and an intracellular domain of 222 amino acids. While the sequences responsible for mediating cytotoxicity (6) and aggregation of the p55 receptor (26) have been localized to the death domain in the C-terminal half of the cytoplasmic tail, the functional significance of the juxtamembrane region has not been fully defined.

To identify cellular proteins that bound specifically to the juxtamembrane domain of the p55 TNF receptor, a recombinant LexA fusion protein was generated that contained amino acids 204-280 (Y1), beginning at the end of the transmembrane domain and extending to the middle of the cytoplasmic domain. Y1 did not contain sequences representing the death domain of p55. The Y1 domain was then used as the bait in the yeast two-hybrid screen.

A mouse cerebellar cDNA library was used to screen for proteins that interacted specifically with the Y1 region. The library was engineered in a yeast expression vector containing VP16 as the activation domain (18). Colonies were obtained following cotransformation of the S260 yeast strain with the Y1-LexA construct, and the cDNA library colonies were analyzed by beta -galactosidase activity measurements. Of 1.5 × 107 cotransformants, 20 cDNAs were found to be positive using beta -galactosidase activity as a measure of the activation of the Y1-LexA construct. After tests for specificity and DNA sequence analysis were conducted, one positive clone of 0.7 kb in size (c11) was pursued for further analysis.

The yeast two-hybrid assay was further used to test the specificity of the interaction. For this purpose, four other LexA fusion proteins containing different regions of p55 were generated. These proteins are schematically represented in Fig. 1. Each construct (called Y2, Y3, Y4, and Y5) encoded different segments of the intracellular domain of p55, including the juxtamembrane region, the C-terminal death domain (Y3), or both regions (Y4, Y5). The interaction of c11 with the p55 receptor was found to be confined to sequences representing the juxtamembrane region. Cotransformation of yeast strain S260 with each of these constructs and the plasmid harboring c11 rescued from the initial screening indicates that p55 constructs containing the death domain (Y3) or LexA alone did not yield any beta -galactosidase activity, whereas other constructs containing p55 juxtamembrane sequences (Y1, Y4, and Y5) gave positive beta -galactosidase activity (Fig. 1).


Fig. 1. Specificity of the TNF receptor interaction. Proteins representing the cytoplasmic domain of p55 were tested with truncated PIP5K (clone 11) in the yeast two-hybrid system. Y1, Y2, Y3, Y4, and Y5 represent different cytoplasmic regions of the p55 TNF receptor (p75-TNFR). The colony assay for beta -galactosidase activity of the cotransformants indicates the interaction between the bait, cloned in the lexA DNA-binding domain, and c11, one of the library clones constructed in the VP16 activation domain. The entire cytoplasmic domains of other receptors in the same family were also used in the specificity tests. WT, wild-type; TM, transmembrane domain; p75-NGFR, p75 nerve growth factor receptor.
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This analysis was also extended to the cytoplasmic regions of the p75 TNF receptor, the Fas antigen, and the p75 neurotrophin receptor. No interactions were detected between c11 and LexA constructs containing these receptor sequences (Fig. 1). In support of this observation is the fact that there is little similarity in sequence between these family members in the juxtamembrane region. Therefore, the yeast two-hybrid results indicate that the protein expressed from the c11 cDNA specifically associates with the juxtamembrane domain of the p55 TNF receptor.

The cDNA Encodes Phosphatidylinositol-4-phosphate 5-Kinase

Sequence analysis revealed that the mouse c11 cDNA clone encoded a protein that was highly homologous to human PIP5KII cloned previously (25). The c11 cDNA sequence showed 77% (protein level: 83%) identity to the published sequence of PIP5K cloned from a human placental cDNA library (Fig. 2B). The PIP5K enzymes have been defined as types I and II on the basis of their elution from a phosphocellulose ion exchange column. These kinases differ in size, kinetic properties, and differential sensitivity to heparin, spermine, and phosphatidic acid (27, 28). The placental PIP5K sequence, encoded by a 4.1-kb mRNA, may now be identified as the type IIalpha isoform (25), which was previously isolated as a 53-kDa enzyme (28, 29).



Fig. 2. Nucleotide and predicted amino acid sequences of PIP5KIIbeta and comparison of the amino acid sequences of PIP5KIIbeta , PIP5KIIalpha , and c11. A, the first ATG (boxed) of the open reading frame is within a Kozak consensus sequence (24) and is preceded by an in-frame stop codon. The open reading frame is 1248 base pairs, encoding a protein of 416 residues. The in-frame stop codons are in boldface. The underlined sequence was used for Northern blotting, while the sequence in italics was used to screen the lambda DR2 cDNA library. The shaded sequence corresponds to c11, detected via the yeast two-hybrid screen. B, the translated amino acid sequences of PIP5KIIbeta and PIP5KIIalpha are compared with the 202-amino acid protein generated from the c11 clone. The alignment was generated using the PileUp program (Genetics Computer Group). Residues conserved in at least two sequences are in boxed in black.
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A search of the sequence data bases with the DNA sequence of PIP5KIIalpha revealed a putatively transcribed sequence that was highly identical to the query sequence. This clone, HFBEP52, was obtained, sequenced, and used to screen a human fetal brain lambda DR2 cDNA library. Several different cDNAs were obtained, and the composite sequences from these partial cDNAs predicted an open reading frame of 1248 base pairs, encoding a 416-amino acid protein with a calculated molecular mass of 47,378 Da (Fig. 2A). Based on this composite sequence, the full-length open reading frame was amplified by PCR from the human fetal brain lambda DR2 cDNA library. The open reading frame was found to be 77.8% identical to PIP5KIIalpha at both the nucleotide and protein levels.

Based on this similarity, this new clone has been designated as PIP5KIIbeta . The murine clone c11 isolated in the yeast two-hybrid screen is 90.3% identical to PIP5KIIbeta at the nucleotide level and 99.5% identical at the amino acid level. Because of the level of identity, the c11 cDNA corresponds to a newly identified isoform of PIP5K, referred to as PIP5KIIbeta . A comparison of the amino acid sequences of PIP5KIIalpha , PIP5KIIbeta , and c11 appears in Fig. 2B.

Using a probe representing the 3'-untranslated region of HFBEP52, a discrete 6.3-kb mRNA, distinct from the alpha -isoform 4.1-kb mRNA (25), was detected (Fig. 3). Identical results were obtained by hybridization with the c11 cDNA (data not shown). Hence, PIP5KIIbeta is encoded by a 6.3-kb mRNA, while PIP5KIIalpha is represented by a 4.1-kb mRNA. Northern blot analysis also revealed that a probe derived from the 5'-coding region of PIP5KIIalpha also detected the 6.3-kb transcript at lower stringency (data not shown). This observation was expected, considering the degree of identity between the two isoforms. Abundant levels of messages were found in heart, placenta, kidney, and pancreas, whereas lung and liver displayed lower levels. Although both the 4.1- and 6.3-kb transcripts are highly expressed in brain, a major difference is that skeletal muscle is more enriched for beta -isoform mRNAs than the alpha -isoform.


Fig. 3. Human multiple tissue Northern blot. A human multiple tissue Northern blot with 2 µg of poly(A)+ RNA/lane was hybridized with a 32P-labeled 1.7-kb KpnI fragment derived from the 3'-untranslated region of HFBEP52 (PIP5KIIbeta ) according to the manufacturer's instructions (CLONTECH).
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Enzymatic Activity

To verify the enzymatic activity of PIP5KIIbeta , a cDNA containing the coding region of PIP5KIIbeta was expressed as a hexahistidine fusion protein (Fig. 4A). The recombinant His-PIP5KIIbeta protein was partially purified by Ni2+ affinity chromatography and found to be ~51 kDa, as measured by SDS-PAGE (Fig. 4B). This molecular mass is ~3 kDa larger than the native enzyme due to the hexahistidine tag and linker sequence, which would make the size of the wild-type protein 47 kDa. This is consistent with the calculated molecular mass of 47.4 kDa. The purified His-PIP5KIIbeta protein exhibited phosphatidylinositol-4-phosphate 5-kinase activity (Fig. 4C). As a control, lysates from cells containing the empty vector lacked kinase activity (Fig. 4C), whereas lysates expressing PIP5KIIbeta displayed enzymatic activity (data not shown).


Fig. 4. Expression, purification, detection, and kinase activity of recombinant PIP5KIIbeta . A, PIP5KIIbeta expressed in E. coli as a hexahistidine fusion protein and purified from cell lysates by Ni2+-charged chromatography. The samples were separated by SDS-PAGE and stained with Coomassie Blue. Lane 1, E. coli lysate from an induced strain containing the empty vector; lane 2, E. coli lysate from an induced strain containing His-PIP5KIIbeta cDNA; lane 3, affinity-purified recombinant hexahistidine fusion of PIP5KIIbeta (5 µg). B, Western blot with antibodies raised against the MBP-c11 fusion protein. The lanes are the same described for A, except that lane 3 (His-PIP5KIIbeta ) contained 4 ng of protein. C, phosphatidylinositol-4-phosphate 5-kinase activity of recombinant His-PIP5KIIbeta . Lane 1, affinity-purified His-PIP5KIIbeta ; lane 2, E. coli lysate from an induced strain harboring the empty vector; lane 3, recombinant His-PIP5KIIalpha .
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In Vitro Interaction

To verify the interaction of PIP5KIIbeta with the p55 receptor in vitro, two separate approaches were taken. In the first case, the truncated PIP5KIIbeta cDNA (c11) isolated from the yeast two-hybrid screen was fused in frame with the coding sequences for MBP. The resulting fusion protein was expressed and purified from E. coli. To test for binding in vitro, the purified MBP-PIP5KIIbeta fusion protein (Fig. 5A, right panel, lane 4) was incubated with glutathione-Sepharose beads and GST fusion proteins representing different regions of the p55 TNF receptor. After incubation, the reaction was extensively washed, separated by SDS-PAGE, and analyzed by Western blotting using antibodies against MBP. These antibodies do not cross-react with the GST protein alone, PIP5K, or p55 receptor proteins (data not shown). The PIP5KIIbeta fusion protein interacted with GST fusion proteins containing the juxtamembrane region (residues 204-337) and the entire cytoplasmic region (residues 204-426) of the p55 receptor (Fig. 5A). The full-length p55 cytoplasmic fusion protein was somewhat less reactive. However, the fusion protein containing the p55 death domain did not exhibit any binding to MBP-PIP5KIIbeta . This provides independent evidence that the interaction between PIP5KIIbeta and p55 TNF receptor is restricted to the juxtamembrane region.


Fig. 5. Binding of p55 TNF receptor cytoplasmic domains to PIP5K. A, the flow chart indicates the experimental scheme (left panel). GST fusion proteins containing the cytoplasmic region (residues 204-426) (right panel, lane 1), the juxtamembrane region (residues 204-337) (lane 2), or the death domain region (residues 340-426) (lane 3) of the p55 TNF receptor were purified on glutathione-Sepharose beads and incubated with the maltose-binding fusion protein of clone c11 (PIP5KIIbeta ). Lane 4, input PIP5KIIbeta protein. The beads were washed, and the products were run on an SDS-polyacrylamide gel followed by an immunoblot with anti-MBP-c11 antibodies. B, hexahistidine-tagged PIP5KIIalpha or PIP5KIIbeta expressed and purified from E. coli was incubated with the GST fusion proteins containing either the death domain or the cytoplasmic domain of the p55 TNF receptor on glutathione beads. The reaction was washed, and the products were separated by SDS-PAGE and immunoblotted with anti-MBP-c11 antibodies. The amounts of PIP5KIIbeta (lane 5) and PIP5KIIalpha (lane 6) used are indicated.
[View Larger Version of this Image (25K GIF file)]


In a separate approach, an analogous experiment was undertaken with recombinant His-PIP5KIIbeta or His-PIP5KIIalpha proteins (Fig. 5B, lanes 5 and 6). The immunoblot indicates that PIP5KIIbeta binds to the cytoplasmic region, and not to the death domain. No interaction was detected between p55 cytoplasmic sequences and PIP5KIIalpha . Taken together, these results suggest that the juxtamembrane region of the p55 TNF receptor interacts specifically with PIP5KIIbeta .

Co-immunoprecipitation

To determine if the p55 TNF receptor interacts with PIP5KIIbeta in vivo, extracts of two different cell lines, MCF7 and HeLa cells, were prepared and immunoprecipitated either with antibodies against the p55 receptor or with antibodies directed against MBP-c11 coupled to Affi-Gel-10 beads. Following immunoprecipitation with anti-p55 antibodies, Western blot analysis using anti-PIP5KIIbeta (c11) antibodies was carried out. A 47-kDa protein, representing PIP5KIIbeta , was detected after immunoprecipitation with anti-p55 antibodies, indicating that the p55 TNF receptor directly associated with PIP5KIIbeta in HeLa and MCF7 cells (Fig. 6A). Further immunoblotting verified that this 47-kDa isoform was expressed in the cell lines (Fig. 6B). Mock immunoprecipitation reactions with nonspecific goat serum (Fig. 6C) indicated the co-immunoprecipitation of PIP5K and p55 was specific. These results indicate that PIP5KIIbeta is associated with the p55 TNF receptor in two TNF-responsive cell lines.


Fig. 6. Coimmunoprecipitation of PIP5KIIbeta with the p55 TNF receceptor. Lysates from HeLa and MCF7 cells were prepared, and 2 mg of lysate was immunoprecipitated with anti-p55 TNF receptor antibodies (Genzyme Corp.) (A), with anti-PIP5KIIbeta antibodies generated from the MBP-c11 fusion protein coupled to Affi-Gel-10 beads (B), or with nonspecific serum (C). The immunoprecipitates (IP) were run on an SDS-polyacrylamide gel, transferred to polyvinylidene difluoride membrane, and immunoblotted with anti-PIP5KIIbeta antibodies.
[View Larger Version of this Image (14K GIF file)]


Activation of PIP5K

PIP5K is a pivotal enzyme in phosphoinositide metabolism since it gives rise to PIP2, the parent molecule for the production of 1,2-diacylglycerol, inositol 1,4,5-trisphosphate, and phosphatidylinositol 3,4,5-trisphosphate. These lipid second messengers are involved in mitogenic responses to polypeptide growth factors and G proteins through phospholipase Cbeta (30, 31). To investigate whether the enzymatic activity of PIP5K is relevant to TNF-mediated signaling, the activity of PIP5K was determined in cell lysates of TNF responsive cells, HeLa, MCF7, and U373.

Cells were treated with 100 ng/ml TNF-alpha , and lysates were prepared as described under "Experimental Procedures." To determine the activity of the beta -isoform, cell lysates were first immunodepleted of the PIP5KIIalpha isoform using antibody sc-1330 (Santa Cruz Biotechnology, Inc.) raised against the N-terminal peptide of PIP5KIIalpha . Western blot analysis indicated that the immunodepleted lysates contained the beta -isoform (data not shown). Using phosphatidylinositol 4-phosphate as a substrate in the presence of [gamma -32P]ATP, the activity of PIP5K was measured (Fig. 7). After 30 min of TNF treatment, a significant increase in the level of PIP2 was detected. The ligand-dependent activation of PIP5KIIbeta in TNF-responsive cells demonstrates that one potential signaling mechanism for TNF-alpha may be in the induction of the phosphatidylinositol pathway.


Fig. 7. PIP5K activity of HeLa, MCF7, and U373 cells treated with TNF-alpha . Cells were treated with 100 ng/ml TNF-alpha for 0, 5, 10, 15, 30, and 60 min, and lysates were prepared (total). The lysates were immunodepleted of PIP5KIIalpha and subjected to PIP5K measurements using phosphatidylinositol 4-phosphate as a substrate. PIP2 generated from the reaction (see "Experimental Procedures") was visualized by autoradiography. The values for the total lysates versus the depleted lysates were normalized relative to their respective zero time points. Similar results were obtained in duplicate assays.
[View Larger Version of this Image (19K GIF file)]



DISCUSSION

Multiple signaling pathways have been characterized for TNF-alpha , a prominent cytokine produced by macrophages. Since TNF mediates divergent responses ranging from inflammatory, cytotoxic, and metabolic functions, it is likely that many second messengers are responsible for TNF-dependent signaling. These may include increased tyrosine phosphorylation, production of ceramide from the hydrolysis of sphingomyelin, activation of the mitogen-activated kinase cascade and p38 stress-activated kinase, production of arachidonic acid, and activation of protein kinase C and phosphatidylcholine-specific phospholipase C (3, 15). The mechanism of TNF in initiating cell death has been clarified by the identification of receptor adaptor proteins, such as TRADD, which are required for apoptotic signal transduction. The binding of the p55 receptor to TRADD occurs via domains located at the C terminus of both proteins, which can lead to the recruitment of FLICE/MACH proteases (13, 14).

Here we have identified a distinctive protein interaction between the juxtamembrane region of the p55 TNF receptor and a phosphatidylinositol lipid kinase, PIP5KIIbeta . This is the first identified enzymatic activity directly associated with the TNF receptor. The PIP5K enzyme is responsible for the phosphorylation of phosphatidylinositol 4-phosphate, giving rise to PIP2. PIP2 can be further hydrolyzed to inositol 1,4,5-trisphosphate and 1,2-diacylglycerol by phospholipase C, or it can be phosphorylated by phosphatidylinositol 3-kinases to generate another lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate. The interaction of the TNF receptor with PIP5K therefore reveals a potential link to several potent second messenger systems and cellular activities, including vesicular transport, endocytosis, and lysosomal function (32).

In addition to the activation of protein kinase C by diacylglycerol and the mobilization of intracellular calcium by inositol 1,4,5-trisphosphate, phosphoinositide metabolites contribute to other cellular activities that may be relevant to TNF signaling. The binding of PIP2 to pleckstrin homology domains may influence localization of pleckstrin homology domain-containing proteins to the membrane. PIP2 binds to cytoskeletal proteins such as gelsolin and profilin that can influence cytoskeletal remodeling (33). Also, clathrin coat-associated proteins, such as AP-2, bind avidly to inositol polyphosphates (34). In the case of AP-2, the binding of inositol phosphate blocks its clathrin coat assembly properties.

Other growth factor receptor systems have been linked to PIP5K activities. EGF stimulation of A431 cells results in an increase in the activity of lipid kinases associated with the cytoskeleton (35). Indeed, co-immunoprecipitation experiments using antibodies against the EGF receptor indicate that phosphatidylinositol 4-kinase and PIP5K activities are directly associated with the EGF receptor. Interestingly, these lipid kinases also interact in the juxtamembrane region of the EGF receptor near the ATP-binding site of the catalytic tyrosine kinase domain (36). Treatment with EGF increases the activities of these phosphoinositide kinases, phosphatidylinositol 4-kinase and PIP5K. One potential function of this association may be to aid the mitogenic responses to EGF stimulation (37).

A mitogenic role for PIP5K is also supported by evidence showing that monoclonal antibodies against PIP2 block cell proliferation in response to platelet-derived growth factor and bombesin (38) and are growth inhibitory for S. cerevisiae (39). Moreover, PIP5K activity has been directly linked to proliferation and malignancy (40). The increase in PIP5K activity observed following TNF treatment suggests that the known mitogenic effects of TNF upon cells (41) may be facilitated by increased PIP2 production through the association of PIP5K with the p55 receptor.

How can the same receptor system give rise to such divergent biological responses such as cell death and cell proliferation? Through its interaction with the p55 and p75 receptors, TNF can potentially generate second messengers, such as ceramide (42) and proinflammatory metabolites (15). The activity of PIP5K versus other signaling activities may determine whether cells respond to TNF by cell proliferation or apoptosis. Discrete actions by TNF may be dictated by the presence or absence of cellular proteins, such as TRADD or PIP5K. Other proteins, such as FADD, RIP, and TRAF2, can be recruited as a result of these initial interactions with TRADD (43, 44). When multiple proteins are present, the affinity of binding with the receptor will undoubtedly dictate the choice of the signaling pathway. It is important to note that the binding of PIP5KIIbeta to the p55 receptor was not observed with other family members, such as Fas and the p75 neurotrophin receptor, and that PIP5KIIbeta interacted preferentially over PIP5KIIalpha (Fig. 5C). These interactions would be expected to take place only in specific cell types, thereby providing the diversity in signaling potential by this cytokine family.

Additionally, phospholipase Cgamma (45, 46) and phosphatidylinositol 3-kinase (47) contain intrinsic SH3 domains or are associated with other SH3 domain-containing proteins. These SH3 domains interact with proline-rich regions and are thought to form specific protein-protein interactions. Both PIP5KIIalpha (amino acids 307-329) and PIP5KIIbeta (amino acids 323-344) contain proline-rich clusters that are highly homologous to the consensus sequence of SH3 domain targets (48, 49). Such an interaction between phospholipase Cgamma or phosphatidylinositol 3-kinase and PIP5Ks could account for the observed coupling of PIP2 production to its utilization by these enzymes. Similar interactions involving signal-generating enzymes have been observed for many mitogenic receptors (50, 51).

The PIP5K enzymes exist in multiple isoforms, which are found in brain, placenta, and erythrocytes. These isoforms have various immunoreactivities, kinetic properties, and molecular masses (28, 52, 53). Moreover, PIP5Ks are unique in that they possess almost no homology to kinase motifs present in other phosphatidylinositol, protein, and lipid kinases (25). Two yeast genes, MSS4 and FAB1, display a similar catalytic domain as PIP5KII. Interestingly, these genes are involved in cell cycle progression and chromosome segregation in S. cerevisiae. PIP5K is likely to play a central role in phosphoinositide signal transduction for a variety of growth conditions.

Recently, it has been shown that another phosphatidylinositol kinase is indirectly involved in TNF-promoted signaling (54). Stimulation of 3T3-L1 adipocytes with TNF promoted the phosphorylation of the IRS-1 insulin receptor substrate, which can bind to phosphatidylinositol 3-kinase. The results reported here demonstrate a direct interaction of the p55 receptor with a specific member of the PIP5K family, which is involved in many signaling pathways (55).

The finding of a specific association between PIP5KIIbeta and the p55 TNF receptor and the stimulation of PIP2 production following TNF production suggest that this receptor system may be closely linked to the control of protein kinase C and calcium-dependent enzymatic activities. Moreover, the involvement of phospholipase Cgamma and phosphatidylinositol 3-kinase in the breakdown and modification of PIP2 suggests that receptor tyrosine kinase-linked pathways may be biochemically and physically linked to the action of cytokine receptors, such as those for TNF-alpha . The regulated synthesis and breakdown of PIP2 may serve as an important crossroad to merge several seemingly diverse signal transduction pathways. Further investigation into the cellular functions regulated by activation of phosphatidylinositol kinases will likely provide insights into the mechanisms of how receptor-generated signals are transduced and integrated.


FOOTNOTES

*   This work was supported by a grant from the Aaron Diamond Foundation (to A. M. C.), by National Institutes of Health Grant CA56490 and by a grant from the Dorothy Rodbell Cohen Foundation (to M. V. C.), and by National Institutes of Health Grants GM38906 and GM51968 (to R. A. A.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U85245[GenBank].


**   To whom correspondence should be addressed. Tel.: 608-262-3753; Fax: 608-262-1257; E-mail: raanders{at}facstaff.wisc.edu.
1    The abbreviations used are: TNF-alpha , tumor necrosis factor-alpha ; PIP5K, phosphatidylinositol-4-phosphate 5-kinase; PIP2, phosphatidylinositol 4,5-bisphosphate; PCR, polymerase chain reaction; GST, glutathione S-transferase; MBP, maltose-binding protein; kb, kilobase pair(s); PAGE, polyacrylamide gel electrophoresis; EGF, epidermal growth factor; c11, clone 11.

Acknowledgments

We thank Brian Hilbush, Cornelia Kurshner, James Morgan, and Alexandra Chittka for assistance with the yeast interaction assay; Patrizia Casaccia-Bonnefil and Jeanne Oh for technical assistance; and Joost Loijens for assistance with the kinase assays.


REFERENCES

  1. Bazzoni, F., and Beutler, B. (1996) N. Engl. J. Med. 334, 1717-1725 [Free Full Text]
  2. Smith, C. A., Farrah, T., and Goodwin, R. G. (1994) Cell 76, 959-962 [Medline] [Order article via Infotrieve]
  3. Tartaglia, L. A., and Goeddel, D. V. (1992) Immunol. Today 13, 151-153 [CrossRef][Medline] [Order article via Infotrieve]
  4. Rothe, J., Lesslauer, W., Loetscher, H., Lang, Y., Koebel, P., Kontgen, F., Althage, A., Zinkernagel, R., Steinmetz, M., and Bluethmann, H. (1993) Nature 364, 798-802 [CrossRef][Medline] [Order article via Infotrieve]
  5. Pfeffer, K., Matsuyama, T., Kundig, T. M., Wakeham, A., Kishihara, K., Shahinian, A., Wiegmann, K., Ohashi, P. S., Kronke, M., and Mak, T. W. (1993) Cell 73, 457-467 [Medline] [Order article via Infotrieve]
  6. Tartaglia, L. A., Ayres, T. M., Wong, G. H. W., and Goeddel, D. V. (1993) Cell 74, 845-853 [Medline] [Order article via Infotrieve]
  7. Hsu, H., Xiong, J., and Goeddel, D. V. (1995) Cell 81, 495-504 [Medline] [Order article via Infotrieve]
  8. Rothe, M., Sarma, V., Dixit, V. M., and Goeddel, D. V. (1995) Science 269, 1424-1427 [Medline] [Order article via Infotrieve]
  9. Rothe, M., Wong, S. C., Henzel, W. J., and Goeddel, D. V. (1994) Cell 78, 681-692 [Medline] [Order article via Infotrieve]
  10. Chinnaiyan, A. M., Tepper, C. G., Seldin, M. F., O'Rourke, K., Kischkel, F. C., Hellbardt, S., Krammer, P. H., Peter, M. E., and Dixit, V. E. (1996) J. Biol. Chem. 271, 4961-4965 [Abstract/Free Full Text]
  11. Chinnaiyan, A. M., O'Rourke, K., Tewari, M., and Dixit, V. M. (1995) Cell 81, 505-512 [Medline] [Order article via Infotrieve]
  12. Itoh, N., and Nagata, S. (1993) J. Biol. Chem. 268, 10932-10937 [Abstract/Free Full Text]
  13. Boldin, M. P., Goncharov, T. M., Goltsev, Y. V., and Wallach, D. (1996) Cell 85, 803-815 [Medline] [Order article via Infotrieve]
  14. Muzio, M., Chinnaiyan, A. M., Fischkel, F. C., O'Rourke, K., Shevchenko, A., Ni, J., Scaffidi, C., Bretz, J. D., Zhang, M., Gentz, R., Mann, M., Krammer, P. H., Peter, M. E., and Dixit, V. M. (1996) Cell 85, 817-827 [Medline] [Order article via Infotrieve]
  15. Heller, R. A., and Kronke, M. (1994) J. Cell Biol. 126, 5-9 [Medline] [Order article via Infotrieve]
  16. Cheng, B., Christakos, S., and Mattson, M. P. (1994) Neuron 12, 139-153 [Medline] [Order article via Infotrieve]
  17. Tancredi, V., D'Arcangelo, G., Grassi, F., Tarroni, P., Palmieri, Santoni, A., and Eusebi, F. (1992) Neurosci. Lett. 146, 176-178 [CrossRef][Medline] [Order article via Infotrieve]
  18. Dalton, S., and Treisman, R. (1992) Cell 68, 597-612 [Medline] [Order article via Infotrieve]
  19. Hsu, K., and Chao, M. V. (1993) J. Biol. Chem. 268, 16430-16436 [Abstract/Free Full Text]
  20. Orlinick, J. R., and Chao, M. V. (1996) J. Biol. Chem. 271, 8627-8632 [Abstract/Free Full Text]
  21. Kurschner, C., and Morgan, J. I. (1995) Mol. Cell. Biol. 15, 246-254 [Abstract]
  22. Ron, D., and Dressler, H. (1992) BioTechniques 13, 866-869 [Medline] [Order article via Infotrieve]
  23. Darnay, B. G., Reddy, S. A. G., and Aggarwal, B. B. (1994) J. Biol. Chem. 269, 20299-20304 [Abstract/Free Full Text]
  24. Kozak, M. (1991) J. Cell Biol. 115, 887-903 [Abstract]
  25. Boronenkov, I. V., and Anderson, R. A. (1995) J. Biol. Chem. 270, 2881-2884 [Abstract/Free Full Text]
  26. Song, H. Y., Dunbar, J. D., and Donner, D. B. (1994) J. Biol. Chem. 269, 22492-22495 [Abstract/Free Full Text]
  27. Jenkins, G. H., Fisette, P. L., and Anderson, R. A. (1994) J. Biol. Chem. 269, 11547-11554 [Abstract/Free Full Text]
  28. Bazenet, C. E., Ruano, A. R., Brockman, J. L., and Anderson, R. A. (1990) J. Biol. Chem. 265, 18012-18022 [Abstract/Free Full Text]
  29. Ling, L. E., Schulz, J. T., and Cantley, L. C. (1989) J. Biol. Chem. 264, 5080-5088 [Abstract/Free Full Text]
  30. Exton, J. H. (1994) Annu. Rev. Physiol. 56, 349-369 [CrossRef][Medline] [Order article via Infotrieve]
  31. Rhee, S. G., and Choi, K. D. (1992) J. Biol. Chem. 267, 12393-12396 [Free Full Text]
  32. DeCamilli, P., Emr, S. D., McPherson, P. S., and Novick, P. (1996) Science 271, 1533-1539 [Abstract]
  33. Lassing, I., and Lindberg, U. (1988) Exp. Cell Res. 174, 1-15 [Medline] [Order article via Infotrieve]
  34. Ye, W., Ali, N., Bernbenek, M. E., Shears, S. B., and Lafer, E. M. (1995) J. Biol. Chem. 270, 1564-1568 [Abstract/Free Full Text]
  35. Payrastre, B., Bergen en Henegouwen, P. M. P., Breton, M., den Hartigh, J. C., Plantavid, M., Verkleij, A. J., and Boonstra, J. (1991) J. Cell Biol. 115, 121-128 [Abstract]
  36. Cochet, C., Filhol, O., Payrastre, B., Hunter, T., and Gill, G. N. (1991) J. Biol. Chem. 266, 637-644 [Abstract/Free Full Text]
  37. Kauffmann-Zeh, A., Klinger, R., Endemann, G., Waterfield, M. D., Wetzker, R., and Hsuan, J. J. (1994) J. Biol. Chem. 269, 31243-31251 [Abstract/Free Full Text]
  38. Matuoka, K., Fukami, K., Nakanishi, O., Kawai, S., and Takenawa, T. (1988) Science 239, 640-643 [Medline] [Order article via Infotrieve]
  39. Uno, I., Fukami, K., Kato, H., Takenawa, T., and Ishikawa, T. (1988) Nature 333, 188-190 [CrossRef][Medline] [Order article via Infotrieve]
  40. Singhal, R. L., Prajda, N., Yeh, Y. A., and Weber, G. (1994) Cancer Res. 54, 5574-5578 [Abstract]
  41. Lachman, L. B., Brown, D. C., and Dinarello, C. A. (1987) J. Immunol. 138, 2913-2916 [Abstract/Free Full Text]
  42. Adam, D., Wiegmann, K., Adam-Klages, S., Ruff, A., and Kronke, M. (1996) J. Biol. Chem. 271, 14617-14622 [Abstract/Free Full Text]
  43. Hsu, H., Shu, H.-B., Pan, M.-P., and Goeddel, D. V. (1996) Cell 84, 299-308 [Medline] [Order article via Infotrieve]
  44. Hsu, H., Huang, J., Shu, H.-B., Baichwal, V., and Goeddel, D. V. (1996) Immunity 4, 387-396 [Medline] [Order article via Infotrieve]
  45. Sieh, M., Batzer, A., Schlessinger, J., and Weiss, A. (1994) Mol. Cell. Biol. 14, 4435-4442 [Abstract]
  46. Kohda, D., Hatanaka, H., Odaka, M., Mandiyan, V., Ullrich, A., Schlessinger, J., and Inagaki, F. (1993) Cell 72, 953-960 [Medline] [Order article via Infotrieve]
  47. Wang, J., Auger, K. R., Jarvis, L., Shi, Y., and Roberts, T. M. (1995) J. Biol. Chem. 270, 12774-12780 [Abstract/Free Full Text]
  48. Gout, I., Dhand, R., Hiles, I. D., Fry, M. J., Panayotou, G., Das, P., Truong, O., Totty, N. F., Hsuan, J., Booker, G. W., Campbell, I. D., and Waterfield, M. D. (1993) Cell 75, 25-36 [Medline] [Order article via Infotrieve]
  49. Cheadle, C., Ivashchenko, Y., South, V., Searfoss, G. H., French, S., Howk, R., Ricca, G. A., and Jaye, M. (1994) J. Biol. Chem. 269, 24034-24039 [Abstract/Free Full Text]
  50. Seger, R., and Krebs, E. G. (1995) FASEB J. 9, 726-735 [Abstract/Free Full Text]
  51. Noh, D. Y., Shin, S. H., and Rhee, S. G. (1995) Biochim. Biophys. Acta 1242, 99-113 [CrossRef][Medline] [Order article via Infotrieve]
  52. Divecha, N., Brooksbank, C. E. L., and Irvine, R. F. (1992) Biochem. J. 288, 637-642 [Medline] [Order article via Infotrieve]
  53. Brooksbank, C. E. L., Hutchinigs, A., Butcher, G. W., Irvine, R. F., and Divecha, N. (1993) Biochem. J. 291, 77-82 [Medline] [Order article via Infotrieve]
  54. Guo, D., and Donner, D. B. (1996) J. Biol. Chem. 271, 615-618 [Abstract/Free Full Text]
  55. Loijens, J. C., Boronenkov, I. V., Parker, G. J., and Anderson, R. A. (1996) Adv. Enzyme Regul. 36, 115-140 [CrossRef][Medline] [Order article via Infotrieve]

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