(Received for publication, September 9, 1996, and in revised form, November 25, 1996)
From the § Department of Pharmacology, the
¶ Program in Cellular and Molecular Biology, and the
Department of Biomolecular Chemistry, University of Wisconsin
Medical School, Madison, Wisconsin 53706 and the
Department of Cell Biology and Anatomy, Cornell
University Medical College, New York, New York 10021
Tumor necrosis factor- (TNF-
) 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 PIP5KII
, a new
isoform of PIP5K, but not the previously identified 53-kDa PIP5KII
.
Treatment of HeLa cells with TNF-
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
PIP5KII
with the p55 TNF receptor.
Tumor necrosis factor- (TNF-
)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-
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- and represents the major
signaling function of the Fas antigen, TNF-
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, PIP5KII
.
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.
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
-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.
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 TransformationSaccharomyces
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
-galactosidase activity assay
as described (18). Colonies positive for
-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).
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-
-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.
The GST-c11 and MBP-c11 fusion proteins were denatured in SDS and used as immunogens to generate polyclonal antibodies (Pocono Farms).
Isolation of PIP5KIIIn a data base search, the
PIP5KII 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
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 PIP5KII
had been obtained.
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
PIP5KII
. The DNA fragment was gel-purified following restriction
enzyme digestion and labeled by random priming with
[
-32P]ATP and Klenow DNA polymerase. Hybridization and
washing were performed as outlined by the manufacturer, and the
membrane was exposed to film overnight.
The 1.2-kb open reading
frame of PIP5KII 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
CATGTCGTCC-3
(forward) and
5
-GCTGAAGGTA
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 PIP5KII
(His-PIP5KII
).
E. coli strain BL21(DE3) was transformed with either
pET28b containing the PIP5KII
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-
-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
-mercaptoethanol.
His-PIP5KII was purified by Ni2+ chelate chromatography.
The strain containing the His-PIP5KII
open reading frame was grown at 30 °C to A600 = 0.6, induced with 1 mM isopropyl-1-thio-
-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.
The supernatants of the crude
fraction of His-PIP5KII and of partially purified His-PIP5KII
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 [
-32P]ATP. The
lipids were extracted, and the labeled products were separated by thin
layer chromatography and detected by autoradiography.
Western BlottingProteins 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.).
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.
ImmunoprecipitationCells 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.).
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 -galactosidase activity measurements. Of 1.5 × 107 cotransformants,
20 cDNAs were found to be positive using
-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
-galactosidase activity, whereas other constructs containing p55
juxtamembrane sequences (Y1, Y4, and Y5) gave positive
-galactosidase activity (Fig. 1).
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-KinaseSequence 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 II isoform (25), which was previously
isolated as a 53-kDa enzyme (28, 29).
A search of the sequence data bases with the DNA sequence of PIP5KII
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
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
DR2 cDNA library. The open reading frame
was found to be 77.8% identical to PIP5KII
at both the nucleotide
and protein levels.
Based on this similarity, this new clone has been designated as
PIP5KII. The murine clone c11 isolated in the yeast two-hybrid screen is 90.3% identical to PIP5KII
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 PIP5KII
. A comparison of the amino acid sequences of PIP5KII
, PIP5KII
, 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
-isoform 4.1-kb mRNA
(25), was detected (Fig. 3). Identical results were
obtained by hybridization with the c11 cDNA (data not shown).
Hence, PIP5KII
is encoded by a 6.3-kb mRNA, while PIP5KII
is
represented by a 4.1-kb mRNA. Northern blot analysis also revealed
that a probe derived from the 5
-coding region of PIP5KII
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
-isoform mRNAs than the
-isoform.
Enzymatic Activity
To verify the enzymatic activity of
PIP5KII, a cDNA containing the coding region of PIP5KII
was
expressed as a hexahistidine fusion protein (Fig.
4A). The recombinant His-PIP5KII
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-PIP5KII
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 PIP5KII
displayed enzymatic activity (data not
shown).
In Vitro Interaction
To verify the interaction of PIP5KII
with the p55 receptor in vitro, two separate approaches were
taken. In the first case, the truncated PIP5KII
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-PIP5KII
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 PIP5KII
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-PIP5KII
. This provides independent evidence that the
interaction between PIP5KII
and p55 TNF receptor is restricted to
the juxtamembrane region.
In a separate approach, an analogous experiment was undertaken with
recombinant His-PIP5KII or His-PIP5KII
proteins (Fig. 5B, lanes 5 and 6). The immunoblot
indicates that PIP5KII
binds to the cytoplasmic region, and not to
the death domain. No interaction was detected between p55 cytoplasmic
sequences and PIP5KII
. Taken together, these results suggest that
the juxtamembrane region of the p55 TNF receptor interacts specifically
with PIP5KII
.
To determine if the p55 TNF receptor
interacts with PIP5KII 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-PIP5KII
(c11) antibodies was carried out. A 47-kDa
protein, representing PIP5KII
, was detected after
immunoprecipitation with anti-p55 antibodies, indicating that the p55
TNF receptor directly associated with PIP5KII
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 PIP5KII
is associated with the p55 TNF receptor in two TNF-responsive cell lines.
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 C
(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-, and lysates were prepared as
described under "Experimental Procedures." To determine the
activity of the
-isoform, cell lysates were first immunodepleted of
the PIP5KII
isoform using antibody sc-1330 (Santa Cruz
Biotechnology, Inc.) raised against the N-terminal peptide of
PIP5KII
. Western blot analysis indicated that the immunodepleted
lysates contained the
-isoform (data not shown). Using
phosphatidylinositol 4-phosphate as a substrate in the presence of
[
-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 PIP5KII
in TNF-responsive cells demonstrates that one potential signaling mechanism for TNF-
may be in the induction of the phosphatidylinositol pathway.
Multiple signaling pathways have been characterized for TNF-, 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, PIP5KII. 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 PIP5KII to the p55 receptor
was not observed with other family members, such as Fas and the p75
neurotrophin receptor, and that PIP5KII
interacted preferentially
over PIP5KII
(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 C (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 PIP5KII
(amino acids 307-329) and PIP5KII
(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 C
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 PIP5KII 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 C
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-
. 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U85245[GenBank].
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.