(Received for publication, September 16, 1996, and in revised form, October 24, 1996)
From the Genetic and biochemical studies have shown that
the phosphatidylinositol (PtdIns) 3-kinase encoded by the yeast
VPS34 gene is required for the efficient sorting and
delivery of proteins to the vacuole. A human homologue of the yeast
VPS34 gene product has recently been characterized as part
of a complex with a cellular protein of 150 kDa (Volinia, S., Dhand,
R., Vanhaesebroeck, B., MacDougall, L. K., Stein, R., Zvelebil, M. J.,
Domin, J., Panaretou, C., and Waterfield, M. D. (1995) EMBO J. 14, 3339-3348). Here, cDNA cloning is used to show that the
amino acid sequence of this protein, termed p150, is 29.6% identical
and 53% similar to the yeast Vps15p protein, an established regulator
of Vps34p. Northern blot analysis showed a ubiquitous tissue
distribution for p150 similar to that previously observed with PtdIns
3-kinase. Recombinant p150 associated with PtdIns 3-kinase in
vitro in a stable manner, resulting in a 2-fold increase in lipid
kinase activity. Addition of phosphatidylinositol transfer protein
(PI-TP) further stimulated the lipid kinase activity of the
p150·PtdIns 3-kinase complex 3-fold. A PtdIns 3-kinase activity could
also be co-immunoprecipitated from human cell lysates using anti-PI-TP
antisera. This observation demonstrates that an interaction between a
PtdIns 3-kinase and PI-TP occurs in vivo, which further
implicates lipid transfer proteins in the regulation of PtdIns 3-kinase
activity. These results suggest that the Vps15p·Vps34p complex has
been conserved from yeast to man and in both species is involved in
protein trafficking.
The functional identity of organelles is maintained largely by a
set of unique proteins that reside within them. As a result, the
accurate and efficient sorting of proteins is crucial for the proper
function of these organelles and ultimately of the cell itself. The
yeast vps mutants define a set of 50 genes whose functions
include those required for the sorting and delivery of soluble vacuolar
hydrolases from the late trans-Golgi network (TGN)1 to the vacuole, the yeast equivalent
of the mammalian lysosome (1). Other phenotypes shown by the
vps mutants involve a temperature-sensitive defect in growth
and defects in osmoregulation and in vacuole segregation at mitosis.
The VPS34 gene was originally identified as part of a
molecular complex responsible for the control of intracellular protein
trafficking in yeast (2, 3). The VPS34 gene product, Vps34p,
shows sequence similarity to phosphoinositide (PI) 3-kinases (2, 4).
Vps34p phosphorylates phosphatidylinositol (PtdIns) at the 3 The first mammalian PI 3-kinase identified, a heterodimer of a
regulatory p85 subunit and a catalytic p110 Other genetic studies in yeast have independently implicated
phosphoinositides in vesicular transport. Secretory protein transport from the Golgi complex to the plasma membrane in yeast is dependent upon the activity of a PtdIns/phosphatidylcholine phospholipid transfer
protein (PI-TP) encoded by the SEC14 gene (21, 22). A
mammalian PI-TP was identified as a cytosolic factor required for the
ATP-dependent priming of secretory granules in PC12 cells (23). PI-TP also participates in the synthesis of phosphoinositides by
presenting PtdIns to PtdIns 4-kinase (24, 25). Recent studies of the
biogenesis of immature secretory vesicles from the TGN in cell-free
systems have led to the isolation of mammalian PI-TP as a factor
required for vesicle budding (26). Such studies implicate mammalian
PI-TP in vesicular transport from the TGN. Furthermore, the requirement
of PI-TP in Golgi complex function may reflect a general role for this
protein in PtdIns substrate presentation (27) not only to PtdIns
4-kinase, but also to other PtdIns-specific lipid kinases such as the
human PtdIns 3-kinase.
In this study, we describe the cloning and expression of a human
150-kDa protein (p150), which is homologous to the yeast Vps15p protein
kinase. In addition, we report the association of p150 with the human
Vps34p homologue, PtdIns 3-kinase, and demonstrate the activation of
this p150·PtdIns 3-kinase complex when the substrate, PtdIns, is
presented in the presence of PI-TPs.
The probe used
for screening a U937 For Northern blotting, multiple human tissue
RNA blots (CLONTECH) were used. The 330-base pair PCR fragment used to
screen the cDNA library was labeled with
[ For the expression of p150 as a GST
fusion protein in insect Sf9 cells, the full-length open reading frame
(ORF) was inserted into pAcGEX-2T in three steps. First,
BamHI/NotI and EcoRI/KpnI sites were introduced by PCR at the 5 Jurkat 6 and U937 cells were
cultured in RPMI 1640 medium supplemented with 5% fetal calf serum.
Baculoviral plasmid DNA was cotransfected with Baculogold DNA
(Pharmingen) into Sf9 insect cells using Lipofectin (Life Technologies,
Inc.); recombinant virus was isolated and characterized as described
previously (28).
Sf9 cells were infected
at a density of 1 × 106/ml with recombinant
baculoviruses for 60 h. Cells were harvested and lysed in TBS-T
(10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1%
Triton X-100, 1 mM EDTA, phenylmethylsulfonyl fluoride, and
100 units/ml aprotinin), and the extract was incubated with
glutathione-Sepharose beads (Pharmacia Biotech Inc.) or antisera for
1 h at 4 °C. GST-tagged proteins bound to beads or
immunoprecipitates were washed four times with TBS-T, resolved by
SDS-PAGE, and visualized by Coomassie Blue or silver staining.
Recombinant PI-TP Antisera
against PtdIns 3-kinase were raised in rabbits and
affinity-purified as described previously (13). Antibody for detecting
PI-TP Jurkat cells were spun down and resuspended
at 2 × 107/ml in phosphate-free Dulbecco's modified
Eagle's medium supplemented with 1% dialyzed fetal calf serum. Cells
were then labeled with [32P]orthophosphate (Amersham
International) at 1 mCi/ml for 4 h at 37 °C. After this time,
radiolabeled cells were incubated in the presence and absence of
phorbol 12-myristate 13-acetate (10 ng/ml) for 20 min, washed once with
phosphate-buffered saline, and then solubilized with TBS-T. The
extracts were precleared with protein A-Sepharose followed by
immunoprecipitation with preimmune and PtdIns 3-kinase-specific
antisera. Precipitates were washed four times with TBS-T and resolved
by SDS-PAGE. Proteins were then transferred to Immobilon, which was
autoradiographed and then subjected to Western blotting as described
above. For analysis of p150 myristoylation, Sf9 cells (1 × 106) were infected with recombinant baculovirus and labeled
24 h postinfection for 16 h with
[9,10-3H]myristic acid (Amersham International) at 100 µCi/ml. Labeled cells were lysed with TBS-T, and extracts were
incubated with glutathione-Sepharose beads. Immobilized proteins were
washed four times with TBS-T and resolved by SDS-PAGE. The gel was
treated for fluorography using Amplify (Amersham International), dried, and autoradiographed.
The protein kinase activity of immobilized recombinant
GST-PtdIns 3-kinase, p150, or GST-p150·PtdIns 3-kinase complex was determined by incubating the immobilized protein with 30 µl of a
phosphorylation mixture containing 0.1 µCi of
[ PI
3-kinase assays were performed essentially as described (32), in a
volume of 50 µl containing 20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 3.5 mM MnCl2, 40 mM ATP (0.2 mCi of [ PI-TP (5 µg) was
preincubated with 1 mM PtdIns at 37 °C for 5 min.
Immunoprecipitated proteins or proteins immobilized on glutathione-Sepharose beads were then added in a volume of 300 µl of
TBS-T. Incubation was continued for 1 h at 4 °C. The resulting protein complexes were washed once with TBS-T and twice with
phosphate-buffered saline and resolved by SDS-PAGE. PI-TP was detected
by Western blotting using specific antibody.
Immunoprecipitations from human Jurkat 6 cells performed with an affinity-purified anti-PtdIns 3-kinase antibody
contain a 150-kDa protein in addition to PtdIns 3-kinase. The partial
amino acid sequence of the 150-kDa protein was determined, yielding five amino acid sequences: 1) kVF/TAIQDPXLP, 2) kLLALK 3)
kETFLSADEEk, 4) kVVTLLSDPENIVFK, and 5) kXLNMENRHLN (12). No
significant sequence similarities were identified when the p150 peptide
sequences were compared with the protein sequences of the Swissprot
data base. Using the program TFASTA (34), however, which compared the
p150 peptides with the six possible translations of the data base DNA
entries, the short anonymous expressed sequence tag (EST) HS8804 (human
clone HFBEP44) was shown to encode a sequence identical to peptide 1. The human HFBEP44 cDNA sequence was translated, and the resulting
amino acid sequence was used to rescreen the Swissprot data base using
the program FASTA (34). This new search identified the N terminus of
the yeast Vps15p protein kinase as having the greatest homology to the
predicted product of HFBEP44. Using specific primers and reverse
transcription-PCR, we amplified the HFBEP44 partial cDNA from U937
mRNA. This product was used as a probe to screen a
A comparison of the
sequences of p150 and yeast Vps15p using the program COMPARE (34)
defined three main areas of colinear homology between the two proteins
(Fig. 1, B and C): homology region i, amino acids
1-10; region ii, amino acids 11-262; and region iii, amino acids
400-700.
The N-terminal region i contains a consensus site for the attachment of
myristic acid. Myristic acid is cotranslationally added to an
N-terminal glycine residue of specific eukaryotic proteins via an amide
linkage following the removal of the initiating methionine to the
adjacent glycine residue (36). Analysis of the substrate specificity of
the yeast myristoyl-CoA:protein N-myristoyltransferase suggests that a consensus sequence exists for N-terminal myristoylation of
G1X2Z3Z4S5Z6
(where uncharged residues are indicated by X and neutral
residues by Z) (37). The Vps15p sequence of
(M)G1A2Q3L4S5L6
fits this consensus, suggesting that Vps15p is myristoylated in
vivo.
Homology region ii is conserved between p150 and Vps15p (47% identity
and 67% similarity) and shows similarity to the catalytic region of
Ser/Thr protein kinases. Both Vps15p and p150 contain the consensus
triplets APE and DFA, which are often found within the catalytic
domains of protein kinases (38, 39). The central core of homology
region ii (amino acids 100-200 of Vps15p and p150) contains a stretch
of amino acids that can be used as an indicator of substrate
specificity. The shared sequence DIKTEN within this stretch closely
resembles the consensus element DLKPEN, which is specific for Ser/Thr
protein kinases (40), suggesting that Vps15p and p150 are Ser/Thr
kinases. Indeed, Vps15p has been shown to be a functional Ser/Thr
kinase in that it is able to autophosphorylate on a serine residue
(5).
The central region of both p150 and Vps15p (homology region iii) shows
similarity to a repeating unit found in a number of proteins including
the 65-kDa regulatory subunit (PR65) of protein phosphatase 2A. The
PR65 subunit consists of 15 leucine-rich imperfect repeating units of
38-40 amino acids (41) that produce a rod-like structure important for
intersubunit interactions in protein phosphatase 2A (42). Thus, the two
PR65-related leucine-rich repeats found in Vps15p and p150 might also
function as protein-protein interaction modules.
Region iv at the C terminus of p150 (amino acids 1000-1300) contains
WD repeat units, which were first found in the Northern blot analysis using poly(A)+ RNA
isolated from human tissues revealed that the cDNA encoding p150
hybridized to a 5.5-kilobase species, which is similar in size to the
isolated cDNA (Fig. 2). p150 is detectable in the
majority of the human tissues analyzed, with the highest levels present
in skeletal muscle, liver, ovary, testis, and thymus and the lowest
levels in kidney and lung.
To initiate the biochemical characterization of p150, Sf9 cells were
infected with a baculovirus encoding a GST-p150 fusion protein.
Affinity purification on glutathione-Sepharose beads revealed a 180-kDa
protein representing p150 tagged with the 27-kDa GST moiety.
Co-infection with GST-p150 and a PtdIns 3-kinase baculovirus demonstrated that PtdIns 3-kinase associates with GST-p150 (Fig. 3A) and that the resulting complex possesses
PI 3-kinase activity (Fig. 3B). Therefore, the GST tag at
the N terminus of p150 did not significantly alter its ability to bind
PtdIns 3-kinase or affect lipid kinase activity. Similarly, when Sf9
cells were co-infected with viruses expressing GST-PtdIns 3-kinase and
p150 (Fig. 3C) or PtdIns 3-kinase and p150 (Fig.
3D), a p150·PtdIns 3-kinase complex could be detected. In
contrast, p150 was not observed when GST-p110
Consistent with the presence of an N-terminal myristoylation
site, yeast Vps15p is myristoylated in vivo (45, 46). To analyze whether p150 was also myristoylated in vivo, Sf9
cells were infected with baculovirus to express either GST-PtdIns
3-kinase or the GST-p150·PtdIns 3-kinase complex and radiolabeled
with tritiated myristic acid. Subsequent autoradiography of
affinity-purified GST-p150·PtdIns 3-kinase showed that p150 was
specifically labeled, suggesting that it is modified by the addition of
myristic acid in vivo (Fig.
4A).
Since both Vps15p and Vps34p exist as phosphoproteins in
vivo (5), the phosphorylation state of the p150·PtdIns 3-kinase complex in [32P]orthophosphate-radiolabeled U937 and
Jurkat cells was investigated. The cells were incubated in the presence
or absence of phorbol 12-myristate 13-acetate, and then lysates were
prepared and immunoprecipitated with antisera specific to PtdIns
3-kinase (Fig. 4B). In U937 cells, both PtdIns 3-kinase and
p150 appear to be phosphorylated. Stimulation with phorbol 12-myristate
13-acetate did not result in a significant increase in the
phosphorylation of PtdIns 3-kinase or p150. In Jurkat cells, PtdIns
3-kinase and p150 were also found to be phosphorylated. Furthermore, a
120-kDa phosphoprotein was also co-immunoprecipitated from these cell
lysates. We were, however, unable to observe the in vitro
autophosphorylation of PtdIns 3-kinase (13) or p150 (data not shown),
which has previously been reported for Vps34p and Vps15p (5). As a
complementary approach, the protein kinase activities of the
recombinant GST-p150·PtdIns 3-kinase complex toward peptide and
protein substrates were investigated (Table I). These
results demonstrate that the GST-p150·PtdIns 3-kinase complex is able
to phosphorylate protein kinase C substrate and myelin basic protein.
Interestingly, this protein kinase activity displays a cation
preference for Mn2+, which is also observed for the Vps34p
and Vps15p protein kinase activities (5).
Phosphorylation of different substrates by GST-p150·PtdIns 3-kinase
in the presence of Mg2+ or Mn2+
Ludwig Institute of Cancer Research,
Department of Biochemistry
and Molecular Biology,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
-position
of the inositol ring, but not PtdIns(4)P or PtdIns(4,5)P2
(5); hence, Vps34p is a PtdIns-specific 3-kinase. This lipid kinase
activity is of physiological relevance since point mutations in the
Vps34p kinase domain result in defective vesicle-mediated transport,
strongly suggesting a role for PtdIns(3)P as a signal transducer in
protein trafficking (6). The severe defect in sorting displayed by
another vps mutant, termed vps15, suggested that
the VPS15 and VPS34 gene products act at the same step of the vacuolar protein-sorting pathway. Subsequent biochemical studies revealed that Vps34p and Vps15p, a 160-kDa Ser/Thr protein kinase, exist as a complex in vivo (7). Vps15p recruits
Vps34p to the membrane of the Golgi complex and enhances Vps34p PtdIns 3-kinase activity. Mutational inactivation of Vps15p protein kinase activity stops its association with Vps34p, blocks activation of Vps34p
lipid kinase activity, and prevents recruitment of Vps34p to the
appropriate membrane site (5, 7).
subunit, was purified and cloned from bovine brain (4, 8, 9). Subsequently, the use of PCR
and degenerate oligonucleotides based on the kinase domain sequences of
p110
and Vps34p made it possible to identify several forms of PI
3-kinase from mammals and Drosophila (10-12). The
identification of human (13),
Drosophila,2 soybean (15),
Arabidopsis (16), and Dictyostelium (17) Vps34p
homologues indicates that the amino acid sequence and enzymatic activity of this lipid kinase have been conserved through evolution and
can be assigned to a distinct PI 3-kinase class (18). The human PtdIns
3-kinase has amino acid sequence homology (37% identity and 53%
similarity) and similar biochemical characteristics to the yeast enzyme
Vps34p, suggesting that it is a Vps34p homologue (13). In mammalian
cells, the use of the potent PI 3-kinase inhibitors wortmannin and
LY294002, which also inhibit PtdIns 3-kinase (13), causes mammalian
lysosomal marker enzymes analogous to those studied in yeast to be
mistargeted (19, 20). These findings implicate a PI 3-kinase activity
in vesicular transport from the Golgi complex to lysosomes. The
identification of a human Vps34p homologue and the effect of PI
3-kinase inhibitors on protein sorting strongly suggest that a PI
3-kinase, which may be the PtdIns 3-kinase, is involved in protein
trafficking.
Cloning and Sequencing of the p150 cDNA
ZAP cDNA library was amplified by PCR. The
forward and reverse primers 5
-CCAGATCCTTTCTGTAG and
5
-GAATGGACGGGTACTGATGC, respectively, were designed based on the human
clone HFBEP44. To facilitate subcloning of the amplified fragment, an
EcoRI restriction site was incorporated into the 5
-end of
each primer. PCR was carried out using a Perkin-Elmer DNA thermal
cycler for 30 cycles of 94 °C for 1 min, 55 °C for 15 s, and
72 °C for 30 s. The 50-µl reaction volume contained the U937
cDNA library, 10 mM Tris-HCl, pH 8.0, 50 mM
KCl, 1.5 mM MgCl2, 200 µM dNTP,
0.1 mM forward and reverse primers, and 2 units of
Taq DNA polymerase (Life Technologies, Inc.). The 330-base pair PCR product was cloned into the EcoRI site of
pBluescript SK. After sequencing, the PCR fragment was labeled with
[
-32P]dCTP to a specific activity of 109
cpm/µg of DNA using random primers (Amersham International) and was
used to screen a U937
ZAP cDNA library (Stratagene).
Approximately 3 × 106 plaques of the library were
plated and transferred to nylon filters (Hybond-N, Amersham
International). They were hybridized with the
[
-32P]dCTP-labeled probe for 16 h at 65 °C in
0.5 M sodium phosphate, pH 7.2, 7% SDS, and 1 mM EDTA, pH 8.0. After washing with 0.5 × SSC (1 × SSC = 0.15 M NaCl and 0.015 M sodium
citrate) and 0.1% SDS twice for 20 min at 60 °C, the filters were
exposed to x-ray film for 48 h at
70 °C. Following two rounds
of screening, one positive clone was plaque-purified, and the insert
(pSKp150) was rescued using the helper phage ExAssist (Stratagene). The
nucleotide sequence was determined from the double-stranded pSKp150
plasmid. The nucleotide sequence of one strand was determined by Lark
Sequencing Technology, Inc. using the dideoxy chain termination method
(53), a modified T7 DNA polymerase, and
[
-35S]thio-dATP. The complementary strand was
sequenced on an ABI 370A sequencer using the Taq DyeDeoxy
Terminator Cycle Sequencing system (ABI Advanced Biotechnologies,
Inc.).
-32P]dCTP by random priming as described above.
Hybridization was carried out using ExpressHyb (CLONTECH);
hybridization and washing were carried out according to the
manufacturer's instructions.
- and 3
-ends, respectively, of
nucleotides 1-350 of the ORF. The forward and reverse primers 5
-C
A
ATGGGAAATCAGCTTGCTGGC and
5
-CG
GC
GAATGGACGGGTACTGAT (restriction sites are underlined), respectively, were used in a
30-cycle PCR of 94 °C for 30 s, 48 °C for 15 s, and
72 °C for 30 s. The final volume of 100 µl contained 40 ng of
pSKp150, 20 mM Tris-HCl, pH 8.8, 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, 200 mM dNTP, each primer at 0.1 mM, and 1 unit of Vent DNA polymerase (New England Biolabs Inc.). This was subcloned into
pAcGEX-2T cut with BamHI and EcoRI to create
pAcGEX-2T/p150-(1-350). Second, nucleotides 141-3487 were introduced
by digesting pSKp150 with StuI and KpnI and
inserting this fragment into pAcGEX-2T/p150-(1-350) digested with the
same enzymes, to create pAcGEX-2T/p150-(1-3487). Finally, this
construct was digested with KpnI to allow insertion of
nucleotides 3488-4077, the final PCR-generated
KpnI-digested 590-base pair fragment. This PCR product was
created using the forward primer 5
-C
ATGGCTTGTTGGGA
(KpnI site is underlined) and the reverse primer
3
-C
A
TTATTTCCACACCTT
(KpnI/NotI sites are underlined) using the PCR
conditions described above. The introduction of NotI sites
at the 5
- and 3
-ends allowed the full-length 4077-base pair ORF from
this construct to be subcloned into the NotI-digested
baculovirus vector pVL1393 (Invitrogen), thereby enabling expression of
p150 without the GST tag.
, PI-TP
, and Sec14p were expressed and purified
as described (29, 30).
and PI-TP
on immunoblots was raised in rabbits as
described (31). For immunoprecipitating PI-TP, a mouse monoclonal antibody was used. For immunoprecipitations, cell lysates were clarified by centrifugation at 14,000 × g for 10 min
and incubated with antibody for 2 h at 4 °C. Immunoprecipitates
were collected on protein A-Sepharose or protein G-Sepharose beads
(Pharmacia Biotech Inc.) and washed four times with TBS-T. Proteins
were extracted in 2 × Laemmli sample buffer, separated by
SDS-PAGE, and transferred to polyvinylidene difluoride membrane
(Millipore Corp.). Blots were incubated with antibody in
phosphate-buffered saline containing 0.05% (w/v) Tween 20 and 5%
(w/v) dried milk. Immunoreactive bands were visualized using
horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibody
and an enhanced chemiluminescence kit (Amersham
International).
-32P]ATP, 30 mM Tris-HCl, pH 8.0, 20 mM MgCl2 or MnCl2, and 3.0 mg/ml protein or peptide substrate. The substrates used were as follows: protein kinase C substrate (PLSRTLSVATAKK), myelin basic protein, Kemptide (LRRASLG), tyrosine protein kinase substrate (RRLIGDAGYAARG), p34cdc2 protein kinase substrate (ADAQHATPPKKKRKEDPKDF),
histone, and syntide 2 (PKTPKKAKKL). After 30 min of incubation at
30 °C, the kinase reaction was stopped by spotting 25 µl of the
supernatant on phosphocellulose P-81 paper. [
-32P]ATP
was separated from the labeled substrate by washing the P-81 filter
papers five times for 5 min with 75 mM
H3PO4. The papers were dried, and the
radioactivity incorporated into protein and peptide substrates was
determined by Cerenkov counting.
-32P]ATP), and 1 mM phosphoinositide (PtdIns, PtdIns(4)P, or
PtdIns(4,5)P2; Sigma). After preincubating
the enzyme and lipid for 20 min at 4 °C, each reaction was initiated
by the addition of ATP and allowed to proceed for 15 min at room
temperature. To examine the effect of PI-TP on lipid kinase activity,
PI-TP or Sec14p (0-2.6 µM final concentration) was
preincubated with 1 mM PtdIns at 37 °C for 5 min and
then added to recombinant PtdIns 3-kinase for 20 min on ice before
addition of ATP. Assays were terminated with acidified chloroform/methanol, and the lipids were extracted and resolved by TLC.
For standard assays using PtdIns as substrate, the solvent used was
chloroform, methanol, and 4 M ammonium hydroxide
(45:35:10). For separation of PtdIns(3)P, PtdIns(3,4)P2,
and PtdIns(3,4,5)P3, propan-1-ol and 2 M acetic
acid (65:35) were used as the solvent system. For separation of
PtdIns(3)P from PtdIns(4)P, a borate buffer system was used (33).
PtdIns 4-kinase assays were performed on TBS-T A431 cell lysates in a
volume of 50 µl containing 10 mM MgCl2, 25 mM Tris-HCl, pH 7.5, 70 µM ATP (0.2 µCi of
[
-32P]ATP), and 1 mM PtdIns. Reactions
were carried out as described above for PI 3-kinase assays.
Radioactivity was analyzed by autoradiography or quantified using a
PhosphorImager (Molecular Dynamics, Inc.).
Cloning of p150
ZAP cDNA
library from the human cell line U937. One 5-kilobase clone was
isolated and found to contain sequence identical to the HFBEP44 PCR
product. This 5-kilobase cDNA contained a 4-kilobase ORF with a
putative start codon preceded by a 5
-Kozak consensus site (35) and an
in-frame stop codon. The ORF encodes a protein of 1358 amino acids
(Fig. 1A), including all five of the
sequenced peptides with a calculated molecular mass of 150,000 Da. The
predicted amino acid sequence of this protein shows homology to yeast
Vps15p over its entire length (29.6% identity and 53% similarity)
(Fig. 1A) and is hereafter referred to as human p150.
Fig. 1.
Comparison of human p150 and yeast Vps15p
predicted protein sequences. A, the optimal alignment of
p150 (upper sequence) and Vps15p (lower sequence)
was obtained using the GAP program (University of Wisconsin Genetics
Computer Group package) (34). Identical residues are indicated by
vertical lines, and conserved residues by colons.
Peptides isolated by microsequencing analysis are
underlined. B, shown is a dot plot comparison of
Vps15p (1455 amino acids; horizontal axis) and p150 (1358 amino acids; vertical axis) using the COMPARE program
(University of Wisconsin Genetics Computer Group package) (34).
C, shown is a schematic representation of conserved regions
of amino acid sequence present in both Vps15p and p150. (i)
represents an N-terminal myristoylation consensus site; (ii)
represents a Ser/Thr protein kinase domain; (iii) represents a region with homology to the 65-kDa regulatory subunit of protein phosphatase 2A. (iv) of p150 represents a region containing
WD repeat motifs. This consensus is less defined in the Vps15p
sequence.
[View Larger Versions of these Images (71 + 12K GIF file)]
-subunit of
heterotrimeric G-proteins (43). These repeats have a region of variable
length (6-94 amino acids) followed by a core of more constant length
(23-41 amino acids), which is bracketed by two characteristic pattern
elements, a conserved His and WD:
X6-94[HX23-41WD]. p150 contains four such repeats: 1)
H1102-(WD)1124-1125, 2)
H1137-(WD)1168-1169, 3)
H1193-(WD)1213-1214, and 4)
H1243-(WD)1268-1269. Analysis of Vps15p (44)
indicates that repeating elements similar to but not matching the
consensus pattern for WD repeats are also present at the C terminus of
this protein. WD motifs are thought to mediate protein-protein
interactions and have been implicated in the regulation of various
cellular functions including cell division, cell fate determination,
gene transcription, and vesicle fusion (44).
Fig. 2.
Northern blot analysis of p150. A
Northern blot (CLONTECH) of human poly(A)+ RNA was
hybridized with full-length p150 cDNA radiolabeled with [-32P]dCTP. Lanes 1-16 contain 2 µg of
RNA from pancreas, kidney, skeletal muscle, liver, lung, placenta,
brain, heart, peripheral blood leukocytes, colon, small intestine,
ovary, testis, prostate, thymus, and spleen, respectively.
Hybridization was performed at 65 °C using the ExpressHyb protocol
(CLONTECH). The positions of RNA molecular size markers (in kilobase
pairs (kb)) are indicated.
[View Larger Version of this Image (75K GIF file)]
was affinity-purified
from Sf9 cells coexpressing GST-p110
and p150 (data not shown),
indicating that p150 associates specifically with the PtdIns-specific
PtdIns 3-kinase and does not associate with p110
.
Fig. 3.
Expression and association of p150 with
PtdIns 3-kinase. Sf9 cells were infected with recombinant
baculovirus, and after 60 h, GST fusion proteins were
affinity-purified from cell lysates using glutathione-Sepharose beads
or immunoprecipitated with antibody and collected on protein
A-Sepharose beads. Immobilized proteins were washed and analyzed by
SDS-PAGE. A, Sf9 cells were infected with either GST-p150
alone (lane 1) or GST-p150 and PtdIns 3-kinase
(PtdIns 3-K) (lane 2); the expressed proteins
were affinity-purified and analyzed by SDS-PAGE and Coomassie Blue
staining. B, PI 3-kinase assays were performed in the
presence of Mn2+ using PtdIns as substrate on GST-PtdIns
3-kinase (lane 1), GST-p150 (lane 2), and
GST-p150·PtdIns 3-kinase (lane 3). PI(3)P,
(PtdIns(3)P). C, Sf9 cells were infected with either
GST-PtdIns 3-kinase (lane 1) or GST-PtdIns 3-kinase and p150
(lane 2). The recombinant proteins were affinity-purified
and analyzed by SDS-PAGE and Coomassie Blue staining. D,
using specific antisera, PtdIns 3-kinase was immunoprecipitated from
Sf9 cells infected with PtdIns 3-kinase alone (lane 1) or
PtdIns 3-kinase and p150 (lane 2). The expressed proteins
were analyzed by SDS-PAGE and silver staining.
[View Larger Version of this Image (47K GIF file)]
Fig. 4.
Post-translational modification of PtdIns
3-kinase and p150. A, in vivo myristoylation of
p150. Sf9 cells were infected with recombinant baculovirus and either
GST-PtdIns 3-kinase alone (lane 1) or GST-PtdIns 3-kinase
and p150 (lane 2) for 24 h and then labeled with
[3H]myristic acid for 16 h at 27 °C. GST fusion
proteins were affinity-purified from labeled lysates using
glutathione-Sepharose beads. Immobilized proteins were washed and
resolved by SDS-PAGE. Following fluorographic processing, radioactive
bands were visualized by autoradiography. B, in
vivo phosphorylation of PtdIns 3-kinase (PtdIns 3-K)
and p150. U937 and Jurkat cells (2 × 107
cells/sample) were radiolabeled with [32P]orthophosphate
for 4 h at 37 °C and then treated in the presence and absence
of phorbol 12-myristate 13-acetate (PMA; 10 ng/ml) as
described under "Experimental Procedures." Using antisera specific to PtdIns 3-kinase, protein complexes were immunoprecipitated from U937
cells (lanes 2 and 3) and Jurkat cells
(lane 5). Controls were performed using preimmune serum on
radiolabeled U937 (lane 1) and Jurkat (lane 4)
cells. Immunoprecipitates were washed extensively, resolved by
SDS-PAGE, and transferred to Immobilon. Radioactive proteins were
visualized by autoradiography.
[View Larger Version of this Image (53K GIF file)]
-32P]ATP as described under "Experimental
Procedures." All substrates used were at a final concentration of 3.0 mg/ml. Background phosphorylation has been subtracted to show the
phosphorylation attributable to the GST-p150·PtdIns 3-kinase complex.
Substrate
GST-p150·PtdIns 3-kinase activity
Mg2+
Mn2+
cpm/assay
Protein kinase C substrate
58
10,363
Myelin basic protein
3328
17,631
Kemptide
44
2458
Tyrosine protein kinase substrate
170
2183
p34cdc2 protein kinase substrate
93
1325
Histone
2127
3305
Syntide 2
7480
6032
To investigate whether p150 could regulate the enzymatic
activity of PtdIns 3-kinase, insect cells were infected with
baculoviruses to express PtdIns 3-kinase or both p150 and PtdIns
3-kinase. Proteins were immunoprecipitated with anti-PtdIns 3-kinase
antibody, and the resulting immune complexes were assayed for lipid
kinase activity. Each lipid kinase assay was performed in the presence
of Mn2+ using PtdIns as substrate (as described under
"Experimental Procedures"). Quantitation of the phosphorylated
lipid products using a PhosphorImager (Fig.
5A) demonstrated that the lipid kinase
activity of PtdIns 3-kinase increases 2-fold when associated with p150.
Western blotting using anti-PtdIns 3-kinase antibodies on
immunoprecipitates produced in parallel to those used above (Fig.
5B) showed that equal amounts of the enzyme were used.
PI-TP Increases the PI 3-Kinase Activity of the GST-p150·PtdIns 3-Kinase Complex
Mammalian PI-TP has been identified as a factor
required for the formation of secretory vesicles from the TGN and has
been shown to play a role in lipid substrate presentation to PtdIns 4-kinase (24, 26). To investigate the possible effect of PI-TP on
PtdIns 3-kinase activity, the GST-p150·PtdIns 3-kinase complex expressed in Sf9 cells was used in lipid kinase assays in the absence
or presence of various PI-TPs. Before starting the reaction, PI-TP,
PI-TP
, or Sec14p protein (0-2.6 µM) that had been
preincubated with 1 mM PtdIns was added to the complex in a
dose-dependent manner. The radiolabeled PtdIns(3)P produced
was extracted and quantitated using PhosphorImager analysis. Enzymatic
activity was expressed as a percentage change over that detected in
reactions performed in the absence of PI-TP (Fig. 6).
The results showed that a dose-dependent increase in lipid
kinase activity took place upon the addition of PI-TP. At
concentrations over 1 µM, both PI-TP
and PI-TP
exhibited slight inhibitory effects. Sec14p gave the greatest increase
in PtdIns 3-kinase activity (375%); PI-TP
gave a 315% increase,
and PI-TP
increased activity by 275%. To investigate if PI-TP
physically associated with the GST-p150·PtdIns 3-kinase complex,
recombinant PI-TP
was incubated with either GST protein or
GST-p150·PtdIns 3-kinase, and the associated proteins were collected
on glutathione-Sepharose beads. Western blot analysis indicated that
PI-TP
associated with the GST-p150·PtdIns 3-kinase complex (Fig.
6D, lane 3), but not with GST alone (lane
2).
A PtdIns-specific PI 3-Kinase Associates with PI-TP in Vivo
To investigate whether PI-TP associates with a PtdIns
3-kinase in vivo, PI-TP was immunoprecipitated from Jurkat
cells and assayed for kinase activity toward PtdIns in the presence of
Mn2+. Extracted lipids were separated using a borate
solvent system (33), which separates PtdIns(4)P from PtdIns(3)P (Fig.
7A). A lipid kinase activity
co-immunoprecipitating with PI-TP was detected and found to be a PI
3-kinase and not a PI 4-kinase (Fig. 7A). We were unable to
detect PtdIns 3-kinase by Western blot analysis of the PI-TP
immunoprecipitates; this could be due to a lack of sensitivity, which
may fail to show the presence of low levels of PtdIns 3-kinase in these
immunoprecipitates. To investigate the substrate specificity of this
PI-TP-associated PI 3-kinase further, PI-TP was immunoprecipitated from
Jurkat cells, and the immune complex was subjected to PI 3-kinase
assays in the presence of Mg2+ or Mn2+ using
PtdIns, PtdIns(4)P, or PtdIns(4,5)P2 as substrate (Fig. 7B). The PI 3-kinase activity associated with PI-TP
primarily used PtdIns as substrate and exhibited a cation preference
for Mn2+. This biochemical profile parallels that of the
human Vps34p homologue, PtdIns 3-kinase, which uses PtdIns as its
preferred substrate and has a cation preference for Mn2+.
These results demonstrate biochemically that PI-TP associates with a
PtdIns-specific 3-kinase in vivo.
The Ser/Thr protein kinase encoded by the VPS15 gene and the PtdIns 3-kinase encoded by the VPS34 gene have previously been shown to be required for the efficient sorting and delivery of proteins to the yeast vacuole (2, 45, 47). The yeast Vps34p lipid kinase, unlike other PI 3-kinases, can only use PtdIns as substrate. A recently characterized human PtdIns 3-kinase has been shown to have extensive sequence homology to Vps34p and to display a substrate specificity for PtdIns, suggesting that the major components of the yeast VPS intracellular trafficking complex are conserved between the species (13).
In this study, we describe the cloning and molecular characterization of human p150. This protein was initially observed in complex with human PtdIns 3-kinase when immunoprecipitated from Jurkat cells using affinity-purified anti-PtdIns 3-kinase antisera (13). Human p150 and yeast Vps15p share sequence homology (29.6% identity and 43% similarity) at the amino acid level (Fig. 1A), which can be divided into four main areas of interest (Fig. 1, B and C). The N-terminal region represents a site for the attachment of myristic acid. This modification has been demonstrated biochemically for Vps15p (46), and in the present study, we confirm this for p150 (Fig. 4A). Myristoylation of p150 might allow its targeting to the cytoplasmic face of the Golgi membrane in mammalian cells, thereby allowing PtdIns 3-kinase to gain access to PtdIns in the membrane. The second area of homology is also located at the N terminus and has sequence similarity to the catalytic region of Ser/Thr protein kinases. Both Vps15p and Vps34p have been shown to be phosphorylated in vivo and can autophosphorylate in vitro (5). Autophosphorylation activity could not be demonstrated for either the human PtdIns 3-kinase (13) or p150. PtdIns 3-kinase and p150 were shown to be phosphorylated in vivo using both U937 and Jurkat cells. In addition, a 120-kDa phosphorylated protein was also co-immunoprecipitated from Jurkat cells; this is similar in size to a protein previously observed to be associated in PtdIns 3-kinase immunoprecipitates from Jurkat cell lysates (13). Despite the inability of p150 and PtdIns 3-kinase to mediate autophosphorylation in vitro, a complex of both enzymes was able to phosphorylate exogenous peptide and protein substrates in a Mn2+-dependent manner. The preference for Mn2+ resembles the Mn2+ dependence of Vps34p and Vps15p for autocatalytic activity. These findings support the hypothesis that similar regulatory mechanisms exist for the protein kinase activity of the yeast and human proteins. Homology region iii of both p150 and Vps15p is a domain that could be involved in protein-protein interactions, possibly mediating binding to PtdIns 3-kinase (Fig. 1C). Region iv of p150 contains WD motifs; these repeat elements are also thought to mediate protein-protein interactions. The absence of similar consensus WD motifs in Vps15p may impart functional or regulatory differences between the two proteins.
Co-infection experiments in Sf9 cells demonstrated that recombinant
p150 protein is able to associate with PtdIns 3-kinase; the inability
of p150 to interact with mammalian p110 shows that this binding is
specific to PtdIns 3-kinase. The p150-PtdIns 3-kinase interaction
results in a 2-fold stimulation of lipid kinase-specific activity.
These findings are consistent with those reported from yeast studies
where strains deleted for VPS15 are extremely defective in
Vps34p lipid kinase activity (7). Formation of the p85/p110 PI 3-kinase
heterodimer does not lead to lipid kinase activation. In contrast, when
p85
associates with p110
in vitro, a decreased lipid
kinase activity is observed, which correlates with serine phosphorylation of the p85
adaptor subunit (48).
The amino acid homology of PtdIns 3-kinase and p150 to yeast Vps34p and Vps15p, respectively, suggests that function could also be conserved through evolution. This implicates the p150·PtdIns 3-kinase complex in the regulation of mammalian vesicle formation and protein traffic from the TGN to organelles such as the lysosome that are functionally analogous to the yeast vacuole. The observation that wortmannin causes mistargeting of procathepsin D from the Golgi complex to the plasma membrane in mammalian cells implicates a PI 3-kinase in intracellular protein sorting (19, 20). Additionally, wortmannin has been observed to affect the localization of lysosomal type I integral membrane glycoproteins in cells, suggesting a role for PI 3-kinase activity in regulating membrane traffic late in the endocytic pathway (49).
In mammalian cells, the 3-phosphorylated products of the p85/p110 PI
3-kinase, such as PtdIns(3,4)P2 and
PtdIns(3,4,5)P3, have been postulated to serve as
second messenger molecules in regulating cell growth and proliferation
(50). Recent studies suggested that PtdIns(3)P is likely to have an
independent physiological role since levels of
PtdIns(3,4,5)P3 and PtdIns(3,4)P2, but not PtdIns(3)P, increase rapidly following agonist stimulation. As the
production of PtdIns(3)P remains constant, this phospholipid may play a
constitutive role in mammalian cells, facilitating the formation of
vesicles (27). This would suggest that a PtdIns-specific 3-kinase is
required in vesicle and protein traffic. Phospholipid metabolism has
already been demonstrated to regulate protein traffic through the
secretory pathway. Sec14p, which acts as a PI-TP, is required for Golgi
function in yeast (21). Mammalian PI-TP isoforms have also been
identified as factors required for the formation of secretory vesicles
from the TGN (26). Although yeast Sec14p has little sequence homology
to mammalian PI-TP at the amino acid level, it is able to substitute
for mammalian PI-TP in secretory vesicle formation. This suggests that
there is conservation of binding and exchange functions and perhaps
three-dimensional structure (26). Furthermore, PI-TP has been shown to
have a role in substrate presentation to PtdIns 4-kinase. This lipid kinase is thought to be involved in the regulation of intracellular protein traffic because it colocalizes with secretory vesicles and
insulin-responsive membrane vesicles in rat adipocytes (51). Additionally, PtdIns 4-kinase activity is required for the stimulated secretion of granule-associated catecholamine from chromaffin cells
(52).
Our findings show that PI-TP may also present PtdIns to other lipid
kinases. Addition of PI-TP stimulated the lipid kinase activity of the
p150·PtdIns 3-kinase complex in a dose-dependent manner.
Of the two mammalian PI-TPs, PI-TP provided the greater stimulation
of PtdIns 3-kinase activity. Stimulation of this lipid kinase may occur
as a result of a stable interaction since we show that a PI 3-kinase
activity is present in PI-TP immunoprecipitates from Jurkat cells.
Further characterization indicates that this PI-TP-associated PI
3-kinase could be PtdIns 3-kinase since it has a substrate preference
for PtdIns and a cation preference for Mn2+, which matches
the biochemical profile of the human Vps34p homologue. This interaction
is consistent with immunofluorescence studies showing the PI-TP
isoform to be preferentially associated with the Golgi system (14).
In this study, we have described the identification of p150, the human homologue of yeast Vps15p. Its ability to act as an adaptor protein for PtdIns 3-kinase demonstrates that the p150·PtdIns 3-kinase complex is likely to be functionally equivalent to the yeast Vps15p/Vps34p protein-sorting system. The lipid transfer protein PI-TP associates with the p150·PtdIns 3-kinase complex both in vitro and possibly in vivo, resulting in the activation of lipid kinase activity. We propose that the formation of this heterotrimeric complex plays a role in protein trafficking in mammalian cells through the generation of PtdIns(3)P.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) Y08991[GenBank].
We thank the following people for materials
provided during the course of this work: Vytas A. Bankaitis for the
Sec14p expression construct, Philip Swigart for recombinant
PI-TP and PI-TP
, and Andrew Ball for PI-TP-specific antisera. We
also thank Khatereh Ahmadi, Lindsay MacDougall, Sally Leevers, and Rob
Stein for constructive criticism of the manuscript.