From the Pacific Northwest Research Institute,
Seattle, Washington 98122 and Department of Pathobiology, University of
Washington, Seattle, Washington 98195, the § Fred Hutchinson
Cancer Research Center, Seattle, Washington 98109, and the
¶ Department of Pharmacology, Emory University School of Medicine,
Atlanta, Georgia 30322
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ABSTRACT |
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Protein kinases activated by
sphingosine or N,N'-dimethylsphingosine, but not by
other lipids, have been detected and are termed
sphingosine-dependent protein kinases (SDKs). These SDKs were previously shown to phosphorylate endogenous 14-3-3 proteins (Megidish, T., White, T., Takio, K., Titani, K., Igarashi, Y., and Hakomori, S. (1995) Biochem. Biophys. Res. Commun.
216, 739-747). We have now partially purified one SDK, termed SDK1,
from cytosol of mouse Balb/c 3T3(A31) fibroblasts. SDK1 is a serine
kinase with molecular mass 50-60 kDa that is strongly activated by
N,N'-dimethylsphingosine and sphingosine, but not by
ceramide, sphingosine 1-phosphate, or other sphingo-, phospho-, or
glycerolipids tested. Its activity is inhibited by the protein kinase C
activator phosphatidylserine. Activity of SDK1 is clearly distinct from
other types of serine kinases tested, including casein kinase II, the
and
isoforms of protein kinase C, extracellular
signal-regulated mitogene-activated protein kinase 1 (Erk-1), Erk-2,
and Raf-1. SDK1 specifically phosphorylates certain isoforms of 14-3-3 (
,
,
) but not others (
,
). The phosphorylation site was
identified as Ser* in the sequence Arg-Arg-Ser-Ser*-Trp-Arg in 14-3-3
. The
and
isoforms of 14-3-3 lack serine at this position,
potentially explaining their lack of phosphorylation by SDK1.
Interestingly, the phosphorylation site is located on the dimer
interface of 14-3-3. Phosphorylation of this site by SDK1 was studied
in 14-3-3 mutants. Mutation of a lysine residue, located 9 amino acids
N-terminal to the phosphorylation site, abolished 14-3-3 phosphorylation. Furthermore, co-immunoprecipitation experiments
demonstrate an association between an SDK and 14-3-3 in
situ. Exogenous N,N'-dimethylsphingosine stimulates
14-3-3 phosphorylation in Balb/c 3T3 fibroblasts, suggesting that SDK1 may phosphorylate 14-3-3 in situ. These data support
a biological role of SDK1 activation and consequent phosphorylation of
specific 14-3-3 isoforms that regulate signal transduction. In view of the three-dimensional structure of 14-3-3, it is likely that
phosphorylation by SDK1 would alter dimerization of 14-3-3, and/or
induce conformational changes that alter 14-3-3 association with other
kinases involved in signal transduction.
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INTRODUCTION |
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Glycosphingolipids, sphingolipids, and their catabolites have been implicated as modulators of signal transduction processes (for reviews, see Refs. 1-5). Sphingosine (Sph),1 the backbone component of all sphingolipids, was originally discovered as an inhibitor of protein kinase C (PKC), in contrast to the stimulatory effect of diacylglycerol (DAG) (6). The inhibitory effect of D-erythro-Sph and its N-methyl derivative (N,N'-dimethyl sphingosine; DMS) on PKC was stronger than other Sph stereoisomers and Sph derivatives (7). Subsequent studies have demonstrated effects of Sph and DMS on various protein kinases. Sph inhibits calmodulin-dependent kinase (8) and insulin receptor tyrosine kinase (9), but enhances casein kinase II (CKII) (10). DMS strongly enhances kinase activity of epidermal growth factor receptor (11) and Src kinase (12).
Sph has been suggested as a second messenger, since its low level is
increased upon stimulation of cells. Agonists such as platelet-derived
growth factor and insulin-like growth factor, cause activation of
ceramidase, an enzyme that converts ceramide to Sph and fatty
acids, resulting in an increase in Sph levels (2, 13). Phorbol ester
treatment of HL60 cells causes a 3-fold increase in Sph levels detected
by chemical method (14). DMS is generated from Sph by an enzyme present
in mouse brain (15), and mass spectrometric analysis indicated that DMS
is a minor component relative to Sph in HL60 cell extract; these
findings suggest a physiological significance for both Sph and DMS
(16). Exogenous Sph stimulates tyrosine phosphorylation of focal
adhesion kinase, paxillin, and Crk, and induces mitogenesis in Swiss
3T3 fibroblasts (2, 17-19). Sph also induces prominent phosphorylation of multiple endogenous proteins with molecular mass 18-165 kDa in
Jurkat cell extracts, suggesting the existence of multiple substrates
for Sph-activated kinases (20). Similar phosphorylation of multiple
proteins occurs in the presence of Sph or DMS in both cytosolic and
membrane extracts of mouse Balb/c 3T3(A31) fibroblasts (21).
Phosphorylation is not stimulated by other lipids indicating existence
of specific protein kinases highly activated by Sph or DMS, but not by
ceramide, sphingosine 1-phosphate (Sph-1-P), or various other
sphingolipids, phospholipids, and glycerolipids. Protein kinases
activated by Sph and DMS are termed
"sphingosine-dependent protein kinases" (SDKs).
However, the number, identities, and substrates of these SDKs are
unclear. Using amino acid sequencing, we have identified several
cellular proteins that are phosphorylated in vitro by SDKs
including the 14-3-3 isoforms ,
, and
(21).
14-3-3 proteins comprise a family with a remarkable evolutionary
conservation extending to lower eukaryotes and plants. There are seven
distinct mammalian isoforms (,
,
,
,
,
, and
). 14-3-3 isoforms appear to modulate a large variety of functional proteins and enzymes, including lipid-activated kinases (22-24) such
as PKC, Raf-1, and phosphatidylinositol 3-kinase, and other kinases or
phosphatases involved in control of cell cycle, cell death, and
mitogenesis (25-27). 14-3-3 proteins are thought to function as
adaptor proteins that allow interaction between signaling proteins that
do not associate directly with each other (25). The association of
14-3-3 with different kinases in cytosol and membrane may contribute to
kinases activation during intracellular signaling (28, 29).
Platelet-derived growth factor and insulin-like growth factor induce
association of 14-3-3 with the death agonist BAD, preventing its
interaction with the membrane bound death agonist, BCL-XL,
and contributing to cell survival (30). These agonists also increase
Sph level, suggesting a link between 14-3-3 and Sph signaling. The
diverse effects of Sph and its derivatives on cell cycle progression,
cell death, and mitogenesis, and the similarity between Sph biological
effects and 14-3-3 protein functions, suggest that SDKs mediate the
biological role of Sph through 14-3-3 phosphorylation.
Here we report purification of one type of cytosolic SDK (SDK1) which
phosphorylates only certain isoforms (,
, and
) but not other
isoforms (
and
) of 14-3-3, or other proteins known to be
substrates of other SDKs. We also identified the site at which SDK1
phosphorylates 14-3-3. Although the physiological significance of
14-3-3 phosphorylation is unknown, there is an interesting possibility
that activation of SDK1 and consequent phosphorylation of 14-3-3, in
combination with an increase in the intracellular levels of Sph or DMS,
promotes or inhibits 14-3-3 dimerization, through which 14-3-3 activity
is regulated.
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EXPERIMENTAL PROCEDURES |
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Materials and Reagents
D-erythro-Sph (31), and
N,N'-dimethyl-D-erythro-Sph (7) were
synthesized chemically as described. Protein disulfide isomerase (PDI),
TPA, and dithiothreitol (DTT) were purchased from Calbiochem (San
Diego, CA). Columns with Q-Sepharose Fast Flow HR 10/10, Mono-S (HR
5/5), Mono-Q (HR 5/5), Sephacryl 200 (HR 10/30; for gel filtration),
and phenyl-Sepharose HR 5/5 were purchased from Pharmacia Biotech Inc.
(Pleasant Hill, CA). Molecular size standards for gel filtration were
from Sigma. Leupeptin, aprotinin, pepstatin, trypsin inhibitor,
4-(2-aminoethyl)-benzene-sulfonyl fluoride (AEBSF),
n-octyl--glucoside, Triton X-100, Tween 20, and
chemiluminescence kit were obtained from Boehringer Mannheim
(Indianapolis, IN). [
-32P]ATP (specific activity 3000 mCi/mmol) was from NEN Life Science Products Inc. (Boston, MA).
Recombinant bovine 14-3-3
isoform, and mutants
Arg60-Glu and Lys49-Glu, were prepared as
described (32). Expression and purification of recombinant 14-3-3 isoforms
,
,
, and
, are described
elsewhere.2 Recombinant PKM
of PKC
, purified from Sf9 cells infected with baculovirus
expressing recombinant human PKC
, was obtained from PanVera Corp.
(Madison, WI). PKC antibodies (Abs), myelin basic protein, and
prestained standards were purchased from Life Technologies, Inc. (Grand
Island, NY). CKII assay reagents and its peptide substrate RRRDDDSDDD,
and PKC assay reagents including myosin basic protein peptide
corresponding to residues 4-14, NKRPSNRSKYL, were from Amersham.
Reagents for SDS-PAGE, silver staining of SDS-PAGE, Bradford's for
protein determination, and nitrocellulose (NC) membrane were from
Bio-Rad. Dulbecco's modified Eagle's medium (DMEM) was from Irvine
Scientific (Santa Ana, CA). Fetal bovine serum was from HyClone
Laboratories (Logan, UT). T-Mat film (used for most experiments) and
Bio-Max film (used for identification of phosphorylation site) were
from Kodak (Tukwila, WA). Polyclonal Abs PAN specific to all 14-3-3 isoforms (catalog number sc-629), 14-3-3
(catalog number sc-1019),
14-3-3
(catalog number 732), extracellular signal-regulated
mitogene-activated protein kinase (Erk-1), and Erk-2 Abs were from
Santa Cruz Biotechnology (Santa Cruz, CA). Raf-1 Ab was from Upstate
Biotechnology Inc. (Lake Placid, NY). Ab to CKII was a generous gift
from Dr. E. Krebs (University of Washington). Calreticulin was a
generous gift from Dr. A. Helenius (Yale University).
Cell Culture and Stimulation with TPA
Confluent fibroblasts treated with TPA under serum-free
conditions were used for purification of SDK1 from cytosolic extracts, since intracellular Sph level was increased while PKC activity was
reduced under these growth conditions (33, 34). Mouse Balb/c 3T3(A31)
cells (American Type Culture Collection, Rockville, MD) were cultured
in DMEM supplemented with 10% fetal bovine serum, in a humidified
atmosphere containing 10% CO2 and 90% air, at 37 °C.
Number of passages was limited to five. Cells (5 × 105) were plated on 150-mm dishes, cultured for 6-8 days
until confluence, washed 3 × with serum-free DMEM, incubated in
the same medium for 3 h and then in DMEM containing 200 nM TPA in 0.01% dimethyl sulfoxide for 3-4 h. Next, cells
were washed with ice-cold phosphate-buffered saline without
Ca2+ and Mg2+, harvested, and kept frozen at
80 °C until utilized for enzyme purification.
Purification of SDK1
All purification procedures were carried out at 0-4 °C, and
buffers used were filtered through membrane and degassed under vacuum
immediately prior to column chromatography. The following buffer
systems were used. Buffer A consisted of 20 mM Tris buffer (pH 8.5), 1 mM EDTA. Buffer B was Buffer A plus 10 mM DTT, 10 mM sodium fluoride, and 0.1 mg/ml
AEBSF. Buffer C was Buffer A plus 10 mM DTT, 10 mM sodium fluoride, and 1 M KCl. Buffer D
consisted of Buffer A with pH adjusted to 5.8-6.0, plus 10 mM DTT, 10 mM sodium fluoride, and 0.2%
n-octyl--glucoside. Buffer E was Buffer D with pH
adjusted to 7.5 and Buffer F consisted of Buffer E plus 100 mM NaCl.
Step 1: Preparation of Cell Extract--
Aliquots of packed
frozen cells (2.5 ml, 100 mg) were thawed on ice and suspended in 45 ml
of homogenizing buffer consisting of 20 mM Tris (pH 7.5),
0.5 mM EDTA, 0.5 mM EGTA, 12 mM
sodium fluoride, 10 mM -mercaptoethanol, 10 µg/ml
leupeptin, 10 µg/ml aprotinin, 10 µg/ml trypsin inhibitor, 10 µg/ml pepstatin, 0.5 mg/ml AEBSF, and 5% glycerol. Homogenization
was performed by 40 strokes in an ice-cooled, tight-fitting Dounce
homogenizer. The homogenate was centrifuged at 500 × g
for 10 min, and the supernatant was centrifuged at 100,000 × g for 60 min. The supernatant (cytosol) was subjected to
further purification of SDK1, and activity was tested as described
under "Standard SDK1 Activity Assay."
Step 2: Q-Sepharose Column Chromatography--
45 ml of
cytosolic fraction was mixed with 2.5 ml of 10% Triton X-100, agitated
at 4 °C for 1 h, and immediately loaded on a Q-Sepharose column
(1 × 10.5 cm) pre-equilibrated with Buffer B and washed with 4 volumes of Buffer B. In order to separate SDK1 activity from other
kinases, proteins were then eluted by a combination of stepwise changes
and linear gradient changes of NaCl concentration (0 to 1.0 M), as indicated by a dotted line in Fig.
1A. The linear gradients were from 0 to 0.2 M
NaCl for 15 min, 0.2-0.35 M for 85 min, constant at 0.35 M for 33 min, 0.35-1 M for 24 min, and finally
constant at 1 M for 18 min. Elution was performed at a flow
rate of 0.5 ml/min, and 1.5-ml fractions were collected and subjected
to standard SDK1 assay. Fractions containing SDK1 activity were pooled
and dialyzed 4-6 h against 4 liters of Buffer A containing 10 mM -mercaptoethanol.
Step 3: Phenyl-Sepharose Column Chromatography--
A
phenyl-Sepharose column was pre-equilibrated with Buffer C. KCl (final
concentration 1 M) was added to the pooled fractions showing SDK1 activity at step 2 (after Q-Sepharose column
chromatography). The pooled fractions were loaded on the
phenyl-Sepharose column in Buffer C and washed with 4 column volumes of
Buffer C. Proteins were eluted at a flow rate of 0.5 ml/min by
decreasing KCl. The concentration was decreased from 1 to 0.5 M for 20 min, maintained at 0.5 M for 14 min,
decreased from 0.5 to 0 M for 56 min (Fig. 1C),
and 1-ml fractions were collected. Flow-through (FT) (unbound) fractions were pooled and dialyzed against 4 liters of Buffer A
containing 10 mM -mercaptoethanol for at least 6 h.
Step 4: Mono-S Column Chromatography-- Dialyzed FT and bound phenyl-Sepharose fractions (diluted 4 times in buffer D) were passed separately through Mono-S column. SDK1 activity from both pools was eluted in FT fraction, and minimal SDK1 activity was bound to Mono-S.
Step 5: Mono-Q Column Chromatography-- Mono-S FT fractions of both pools were subjected to Mono-Q ion-exchange chromatography. A Mono-Q (HR 5/5) column was equilibrated with buffer E, and proteins were loaded at a flow rate of 1 ml/min. The column was washed with 6 ml of buffer E. In order to separate SDK1 activity from major PKC activity, elution was performed by applying a combined stepwise and linear gradient of NaCl (0 to 1.0 M) at a flow rate of 0.25 ml/min as indicated by the dotted line in Fig. 1D. The linear gradients were from 0 to 0.2 M NaCl for 10 min, constant at 0.2 M for 10 min, from 0.2 to 0.35 M for 30 min, constant at 0.35 M for 20 min, from 0.35 to 1 M for 10 min, and finally constant at 1 M for 10 min. Fractions (0.65 ml) were collected, and active fractions were pooled. Data obtained from the phenyl-Sepharose-bound pool is shown in Fig. 1D. A similar pattern was obtained from the phenyl-Sepharose-unbound pool (data not shown).
Step 6: Size Exclusion Chromatography--
In order to estimate
the Mr of SDK1, a sample (0.5 ml) obtained from
a Mono-Q column of phenyl-Sepharose-unbound pool was analyzed by gel
filtration. The Sephacryl S-200 column (1 × 30 cm; total column
volume 22.5 ml) was equilibrated with Buffer F. The sample was loaded
at a flow rate of 0.5 ml/min for 3 min and eluted at a flow rate of 0.1 ml/min, and 0.5-ml fractions were collected. Standard SDK1 assay was
performed, except that reactions were for 30 min in the presence of 1 µM ATP (1.4 × 105 cpm/pmol). Gel
filtration protein standards -amylase (200 kDa), bovine serum
albumin (66 kDa), carbonic anhydrase (29 kDa), and cytochrome
c (12.4 kDa) were fractionated immediately after SDK1 fractionation, and the Mr of SDK1 was estimated
(Fig. 1E).
Standard SDK1 Activity Assay
Sph and DMS both activate SDK1; however, DMS was used for
detection of SDK1 activity during purification since it cannot be metabolized to either ceramide or Sph-1-P. SDK1 activity during purification was estimated by standard assay utilizing 0.1 µg of
14-3-3 and 100 µM DMS in 0.5% octyl-
-glucoside.
SDK1 activity required Mg2+ with optimal concentration of
15 mM (Mn was ineffective), and decreased as ionic strength
increased. An IC50 of 150 mM NaCl was defined
at purification steps 2 and 3. The total reaction volume of the
standard assay system was 30 µl, i.e. 20 µl of
substrate/kinase/sphingolipid mixture and 10 µl of ATP solution. The
mixture contained 5- or 10-µl kinase fractions eluted from column
chromatography (containing 5.0-0.02 µg of protein), 3 µl of 14-3-3 (1 µg of 14-3-3 in 50 mM Tris), 3 µl of 30 mM DTT, 6 or 1 µl of 50 mM Tris buffer (pH 7.5), and 3 µl of DMS (1 mM DMS in 5%
octyl-
-glucoside) or vehicle (5% octyl-
-glucoside) alone. The
reaction was initiated by addition of 10 µl of ATP solution
consisting of 75 µM ATP, 45 mM magnesium acetate, and 2.5 µCi of [
-32P]ATP in 50 mM Tris-HCl (pH 7.5). The reaction mixtures were incubated at 30 °C for 15 min. The reactions were stopped by addition of 10 µl of 4 × concentrated Laemmli's SDS-PAGE sample buffer and heating at 100 °C for 10 min. Phosphorylation reactions were
resolved on 12% SDS-PAGE, and gels were stained with Coomassie
Brilliant Blue R-250 (0.1%) in 50% methanol and 10% acetic acid,
destained, dried, and subjected to autoradiography for 1-24 h. Control
assays were made using reaction without DMS, 14-3-3 substrate, or
kinase. The 28-kDa band was excised and the amount of 32P
incorporated into the 14-3-3 (28 kDa) substrate was determined by
scintillation counting. SDK1 activity was calculated from the amount of
32P incorporated into the substrate, taking into account
the specific activity of the radioisotope and reaction time. Values
obtained from gel without phosphorylated protein (blank) were
subtracted from each determination.
The substrate specificity of SDK1 toward 14-3-3 isoforms, 14-3-3 mutants, and other SDK substrates (PDI, calreticulin) were tested in the presence of 120 nM protein. Other lipids and vehicles were used as indicated in the figure legends.
Kinetic and Stoichiometric Analysis of SDK1 Activity
Kinetics of SDK1 activity were determined as explained in the
Fig. 5, A and B. The stoichiometry of
phosphorylation was estimated in order to determine how many moles of
phosphate are incorporated in 1 mol of 14-3-3. The phosphorylation
reactions were scaled up from 30 to 150 µl and assayed as above,
except that 50 µl of purified SDK1, 2 µg of 14-3-3 , and 500 µM ATP with specific activity of 7000 cpm/pmol were
incubated for 4 h. To prevent depletion of the reaction
components, 40 µl of mixture containing the same proportion of SDK1,
DMS, DTT, and ATP as above was added after the first 2 h.
Determination of PKC and CKII Activity
The Ca2+- and phospholipid-dependent PKC
activities present in cytosolic extract (5 µg) or fractions yielded
from column chromatography (5 µl) were assayed by a modification of
the method described previously (35), using a PKC assay kit (Amersham),
that utilizes PKC-specific substrate (synthetic peptide MBP4-14) which
is phosphorylated with the labeled phosphate group from
[-32P]ATP. Reactions were performed in the presence of
kinase fraction and substrate, and in the presence or absence of kinase
cofactors Ca2+ and PS/TPA. Samples (5 µl) were incubated
in a buffer containing 50 mM Tris-HCl (pH 7.5), 90 µM peptide, 3 mM DTT, 1 mM
CaCl2, L-
-phosphatidyl-L-serine
(PS) dispersed in 0.3% Triton X-100 (8 mol % of Triton X-100), 2 µg/ml TPA, 15 mM magnesium acetate, and 100 µM ATP (containing 0.2 µCi of
[
-32P]ATP) at 30 °C. After a 10-min reaction at
30 °C, radiolabeled peptide was separated from unincorporated
32P by binding to affinity phosphocellulose paper. Degree
of phosphorylation was determined by liquid scintillation counting.
Enzyme activity was calculated from the amount of 32P
incorporated into the peptide, taking into account radioisotope specific activity and reaction time.
CKII activity present in cytosolic extract (5 µl) or fractions
yielded from column chromatography was assayed by a modification of the
method described previously (36) using a CKII assay kit (Amersham) with
CKII specific substrate, a synthetic peptide. Samples (5 µl) were
incubated in the presence of 250 µM peptide and 150 mM NaCl in 16 mM MOPS (pH 7.2), and reactions
were initiated by addition of a mixture of 200 µM ATP, 10 mM MgCl2, and 0.2 µCi of
[-32P]ATP in 10 mM HEPES pH 7.5. After a
30-min incubation at 30 °C, radiolabeled peptide was separated from
unincorporated 32P by binding to affinity phosphocellulose
paper.
Chemical Synthesis of Hexadecapeptide Containing Phosphorylation Site
The peptide KNVVGARRSSWRVITT was synthesized by the Fmoc/PyBop
method in 15 µmol scale utilizing a Shimadzu PSSM-8 synthesizer (Shimadzu Corp., Kyoto, Japan). Generated peptides were purified by
preparative reverse phase high performance liquid chromatography on a C18 column (20 × 250 mm, Nakarai Tesque, Japan) at a flow rate of 10 ml/min, with a linear gradient of acetonitrile (0 to 60%
for 45 min) in 0.1% trifluoroacetic acid, utilizing a Gilson auto-LC
system (France). Purity and authenticity of the peptide was checked by
reverse phase high performance liquid chromatography and electrospray
ionization (ESI) mass spectrometry (ESI voltage 4.3 kV, scan range
m/z 500-3000/3 s) utilizing a triple stage quadrupole mass
spectrometer combined with ESI model bTSQ-700 (Thermo Quest Finnigan,
San Jose, CA). Molecular ion m/z 1830 was detected together
with additional sodium ion. This work was performed by Kimie Murayama
and Tsutomu Fujimura, Central Laboratory of Medical Sciences, Juntendo
University School of Medicine, Japan.
Phosphoamino Acid Analysis and Two-dimensional Phosphopeptide
Mapping of 14-3-3
One µg of 14-3-3 was phosphorylated by SDK1 as described
under "Standard SDK1 Activity Assay," except that the specific activity of [
-32P]ATP was 6.6 × 105
cpm/pmol. Reactions were continued for 90 min, and the products were
separated on SDS-PAGE and transferred to NC membranes. Proteins were
visualized by staining with Amido Black (0.2% (w/v) in methanol/acetic acid, 45:10) and subjected to autoradiography. The phosphorylated 14-3-3 band was excised, treated with 1% polyvinylpyrolidone-40 in 100 mM acetic acid at 37 °C for 1 h, washed 5 times
with water, and digested with 10 µg of trypsin, endoarginylpeptidase
(ArgC), or endolysylpeptidase (LysC) in 50 mM ammonium
bicarbonate at 37 °C for 16-24 h. After the first 2 h, an
additional 10 µg of protease was added to ensure complete cleavage.
Peptides were recovered from NC membrane by washing in 20%
acetonitrile, dried, washed with water, and dried 4 times to remove
ammonium bicarbonate. 32P recovered in the supernatants
from these digestions was 80, 70, or 50% for the three enzymes,
respectively. For phosphoamino acid analysis, tryptic peptides were
hydrolyzed in 6 N HCl at 110 °C for 2 h, and analyzed (37).
The digested peptides were subjected to two-dimensional phosphopeptide mapping (37). Briefly, the peptides were separated according to charge by electrophoresis at pH 1.9 at 1.2 kV for 25 min in the first dimension, followed by separation according to hydrophobicity by chromatography in 65% isobutyric buffer (10 h) in the second dimension. After visualization by autoradiography (Bio-Max film), 32P-labeled phosphopeptides were extracted from the cellulose plates in 200 µl (pH 1.9) of buffer by vortex, followed by centrifugation. Phosphopeptides in the supernatant were dried, washed 3 times with water, and subjected to either a second analytical digestion or manual sequencing by Edman degradation (37). The degradation products were resolved at pH 3.5 at 1.3 kV for 16 min, and visualized by autoradiography.
For performic acid oxidation, performic acid was prepared by mixing 10 µl of H2O2 (30%) in 90 µl of HCOOH (98%). The solution was incubated on ice for 1 h before use. Digested phosphopeptides were recovered from NC membrane, dried once, incubated in H2O2/HCOOH on ice for 2 h, washed, and analyzed as described above for unoxidized peptides.
In vitro Phosphorylation of Synthetic Peptide by PKM of
PKC
1 µl of solution containing 5.4 ng of PKM (specific activity,
800 fmol/min/ng) was incubated with 1 mM synthetic peptide
in 14 µl of kinase buffer containing 50 mM Tris-HCl (pH
7.5), 1 mM peptide, 3 mM DTT, 1 mM
CaCl2, PS dispersed in 0.3% Triton X-100 (8 mol/% of
Triton X-100), 2 µg/ml TPA, 15 mM magnesium acetate, and
1 µM ATP (containing 2 µCi of
[-32P]ATP) for 1 h at 30 °C. The synthetic
peptide was omitted in control reaction. The phosphorylated peptide was
separated from unreacted ATP by thin layer electrophoresis at pH 3.5, 1.6 kV, for 16 min. After visualization by autoradiography at room
temperature for 10 min, phosphopeptides were extracted from the plate
in pH 1.9 buffer and dried in a Speed Vac. The purified phophopeptide was digested with trypsin as described for phosphorylated 14-3-3 proteins.
Western Blotting with Isoform-specific Anti-14-3-3 Abs
Cell homogenates or samples obtained from column fractions were
resolved on SDS-PAGE and transferred to NC membranes. The membranes
were incubated at room temperature in 20 mM Tris-HCl (pH
7.4), 200 mM NaCl, 3% bovine serum albumin, and 10% horse serum for 30 min, incubated with specific antiserum for 1-2 h, and
washed. The blot was incubated with secondary Ab for 45 min, and
developed with enhanced chemiluminescence. The PAN Ab was raised to a
peptide found in all 14-3-3 isoforms, KSELVQKAKLAEQAERYDD, corresponding to amino acids 3-21 of human 14-3-3 . The
Ab was
raised to peptide CAGDDKKGIVDQSQQAY, corresponding to amino acids
134-149 of human 14-3-3
. The
Ab was raised to peptide TSDSAGEECDAAEGAEN, corresponding to amino acids 229-245 of rat 14-3-3
. Mouse 14-3-3
is identical to human 14-3-3
, and therefore these terms are used interchangeably in the literature (21, 38, 39); we
will refer to them as
. For Western blot analysis, 14-3-3 Abs and
CKII Abs were used at 1:2000 dilution, kinase suppressor of
ras (KSR) and PKC Abs at 1:1000 dilution, and Raf, Erk1, and Erk2 Abs at 1:500 dilution.
Immunoprecipitation and in Vitro Kinase Assay
Cells were homogenized with 40 strokes in Dounce homogenizer in
lysis buffer containing TEN buffer (50 mM Tris, pH 7.5, 5 mM EDTA, 100 mM NaCl, and 50 mM
sodium fluroide) plus 30 µg/ml leupeptin, 30 µg/ml aprotinin, 30 µg/ml trypsin inhibitor, 30 µg/ml pepstatin, 50 mM
AEBSF, 0.5% Triton X-100. Proteins were solubilized by incubation at
4 °C for 30 min. Lysates were centrifuged in Eppendorf centrifuge at
4 °C for 15 min. The supernatant (6 mg) was diluted 1:2 in lysis
buffer without Triton X-100 and 1 µg of anti-14-3-3 Ab was added at
4 °C. After 1 h, Protein A-agarose was added for 10-14 h.
Immunoprecipitates were washed 2 times with TEN buffer containing 0.1%
Triton X-100 at room temperature, 1 time with TEN buffer, and then
incubated for 5 min at room temperature in TEN buffer containing 0.5%
n-octyl--glucoside and washed (this last step was
repeated). The final wash was with 50 mM Tris and 50 mM NaCl. Pellets were resuspended in 150 µl of 50 mM Tris (pH 7.5) and 6 mM DTT. The suspension
(15 µl) was subjected to standard SDK1 assay, except that DTT was not
added, and reactions were performed in the presence of 1 µM ATP (1.4 × 105 cpm/pmol) for 30 min.
Metabolic 32P Labeling and Immunoprecipitation
3T3(A31) fibroblasts were incubated in phosphate-free DMEM
containing dialyzed 10% fetal bovine serum for 3 h. The cells
were labeled in serum-free, phosphate-free DMEM containing
[32P]orthophosphate (0.045 mCi/ml) for 9 h. During
the last 20 min cells were challenged with 2 or 10 µM DMS
in 0.1% ethanol (stock 1 mM DMS was dissolved in 10%
ethanol), or with 200 nM TPA. Cells were washed with
phosphate-buffered saline without Ca2+ and Mg2+
and harvested with 0.5 mM EDTA at 4 °C. Cell viability
was not affected under these conditions. Cells were sonicated in lysis buffer (10 mM Tris, pH 7.5, 5 mM EDTA, 50 mM sodium fluoride, 50 µg/ml leupeptin, 50 µg/ml
aprotinin, 50 µg/ml trypsin inhibitor, 0.1 mg/ml AEBSF, 3 mM -mercaptoethanol, 1% Triton X-100), and incubated at
4 °C for 1 h for protein solubilization. Lysates were pelleted
in an Eppendorf centrifuge at 4 °C for 15 min. Samples were
denaturated at 70 °C for 30 min, centrifuged, and supernatants were
diluted 1:4 in the above buffer containing 0.2 M NaCl,
without Triton X-100. PAN anti-14-3-3 Ab complexed with immobilized
Protein A/G (Santa Cruz) was added, and 14-3-3 was immunoprecipitated at 4 °C for 3 h. The immunoprecipitates were washed 3 times
with TEN buffer containing 0.05% Tween 20 and once with buffer
containing 50 mM Tris (pH 7.5) and 100 mM NaCl.
Samples were resuspended in 2 times Laemmli sample buffer, separated by
SDS-PAGE, and transferred to a NC membrane. Equal recovery of 14-3-3 proteins was confirmed by probing the filter with 14-3-3 Ab and
enhanced chemiluminescence. Chemiluminescent substrate was removed by
washing in 50 mM Tris (pH 7.5) and 100 mM NaCl,
and by exposure to light for 1 h. The NC membrane was subjected to
autoradiography to detect radioactive 14-3-3.
![]() |
RESULTS |
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Purification and Separation of SDK1-containing Fraction--
SDK1
was purified by sequential column chromatography, and detected using
100 µM DMS as activator and 14-3-3 as substrate (Fig.
1). SDK1 activity was eluted at 260-350
mM NaCl from a Q-Sepharose column at purification step 2 (Fig. 1A). SDK1 phosphorylated 14-3-3
,
,
, and an
endogenous 28-kDa protein, which was eluted between 300 and 350 mM NaCl (Fig. 1B). The 28-kDa protein most
likely consists of 14-3-3 isoforms, since it was detected with PAN
14-3-3 Ab by Western blotting (data not shown). In some cases a minor SDK1 activity peak was eluted from the column at 0.1 M
NaCl, without change in the elution pattern of the major SDK1 peak,
which was purified further.
|
Sph and DMS Are the Only Sphingolipids That Activate SDK1--
The
effects of various concentrations of Sph and DMS delivered in ethanol
or n-octyl--glucoside, and their ability to activate purified SDK1 and consequently phosphorylate 14-3-3
, were examined. Purified SDK1 was capable of phosphorylating 14-3-3 in the presence of
Sph or DMS, but not in the presence of ethanol or
n-octyl-
-glucoside alone. Other controls lacking kinase
or 14-3-3 failed to activate phosphorylation of 14-3-3 (data not
shown). Very low concentrations (5 µM Sph or 25 µM DMS) were required for maximum SDK1 activation when
ethanol was used as vehicle (Fig.
2A). A sigmoidal kinetic curve
was observed when Sph or DMS were solubilized in ethanol for both
14-3-3
and
assay (data not shown). However, this phenomenon
disappeared when the vehicle was changed from ethanol to
n-octyl-
-glucoside, and higher concentrations of Sph or
DMS (100 µM) were necessary for SDK1 activation (Fig.
2B). The fact that 14-3-3 phosphorylation reached a plateau
in the presence of n-octyl-
-glucoside implies that this
detergent prevents secondary effects, such as 14-3-3 dimerization or
14-3-3 oligomerization during SDK1 reaction.
n-Octyl-
-glucoside also increased SDK1 activity 5-fold
relative to ethanol. These results suggest that Sph is the primary
activator of SDK1 and that this activation is not mediated by a
detergent effect. In contrast, SDK1 was not activated by other closely
related sphingolipids such as Sph-1-P, C18-ceramide, PS,
lyso-GM3, and oleoyl-lysophosphatidic acid (Fig. 2C).
|
Substrate Specificity of SDK1--
SDK1 was activated by DMS,
resulting in phosphorylation of 14-3-3 ,
, or
, but not of
or
(Fig. 3A). This trend
was already evident in crude cytosolic fraction, and was unchanged after sequential chromatography including S200 column (purification step 6) (Fig. 3B). For the
isoform, the DMS requirement
was less stringent than for
or
, as seen in various steps of
purification (Fig. 3B). These results suggest that 14-3-3
is phosphorylated by a kinase less sensitive to DMS, and that this
kinase was removed by gel filtration on S200 column (step 6).
|
SDK1 Is a Kinase Distinct from CKII, PKCs, Erks, Raf, and
Ca2+-dependent Kinases--
To determine
whether SDK1 corresponds to known protein kinases, the elution profile
of known kinases determined by either immunoblotting or specific kinase
assay was compared with the elution profile of SDK1 activity.
Ca2+- and phospholipid-dependent PKC activity
(mostly PKC) and CKII activity were measured in fractions obtained
by Q-Sepharose ion-exchange chromatography. CKII activity was
completely separated from SDK1 activity by fractionation on a
Q-Sepharose column (Fig. 1A). PKC activity partially
overlapped SDK1 activity, but was completely removed after Mono-Q
chromatography (Fig. 1ID). Abs to PKC
, Raf, and KSR did not
recognize any proteins in SDK1 peak (data not shown). We further
confirmed these results for SDK1 purified from cells before treatment
with TPA. The elution pattern of SDK1 activity from the Q-Sepharose
column was compared with elution of PKC
, Erk1, Erk2, CKII, and Raf,
as determined by immunoblotting. PKC
, Erk1, Erk2, and CKII proteins
were detected in some fractions; however, their antibodies lacked
immunoreactivity with the SDK1 peak (Fig.
4A).
|
Kinetics of SDK1 Activity--
Reactions with various
concentrations of 14-3-3 and SDK1 revealed a linear relationship
between velocity of phosphorylation and quantity of both 14-3-3 and
SDK1. The velocity of phosphorylation was positively correlated with
substrate concentration (60-480 nM), without a plateau.
However, when substrate concentration was 60 nM, the
reaction was linear for higher concentrations of kinase (0-1000 ng)
(Fig. 5A). Phosphorylation of
14-3-3
was very low at 44 °C and optimal at 30 °C (Fig.
5B). The velocity of phosphorylation was linear and
positively correlated with reaction time (Fig. 5B).
Stoichiometric analysis of 14-3-3
phosphorylation activated by SDK1
was performed using a large quantity of substrate, higher concentration
of ATP, and prolonged incubation period (2-4 h). Approximately 55 and
60 pmol of 32P was incorporated into 60 pmol of 14-3-3
during 2 and 4 h incubation, respectively, and phosphorylation was
completed after 4 h (Fig. 5C). Similar results were
obtained in four separate experiments. These findings indicate that
SDK1 phosphorylates only a single Ser residue in 1 mol of 14-3-3 monomer.
|
Identification of Phosphorylation Site--
In order to understand
the molecular basis of SDK1 specificity toward particular 14-3-3 isoforms, we identified the phosphorylation site on 14-3-3 . 14-3-3
was phosphorylated by SDK1 in the presence of DMS, and its
phosphoamino acid content was determined. Phosphorylation occurred only
at serine (Fig. 6A). A single
phosphorylated peptide was released by trypsin digestion of
phosphorylated 14-3-3
(Fig. 6B). These results are
consistent with the stoichiometric analysis indicating a maximum of 1 mol of phosphate/mol of monomer of 14-3-3, which suggestes a single
phosphorylation site. The likely identity of this site was deduced from
the following properties. First, performic acid oxidation of the
tryptic digest gave four labeled phosphopeptides, indicating the
presence of performic acid-susceptible amino acids (i.e.
Cys, Trp, or Met) in the phosphopeptide (Fig. 6D). Second,
digestion of phosphorylated 14-3-3
with endoarginylpeptidase C
(ArgC), which cleaves C-terminal to Arg, released the same
phosphopeptide as trypsin, which cleaves C-terminal to Arg or Lys (Fig.
6C). In contrast, digestion of phosphorylated 14-3-3
with endolysylpeptidase (LysC), which cleaves C-terminal to Lys,
released different phosphopeptides with slower migration (data not
shown). This suggests that Arg residues are closer than Lys residues on
both sides of the phosphorylated Ser. Third, the tryptic phosphopeptide
had a high positive charge-to-mass ratio at both pH 1.9 and 8.9 (data
not shown), consistent with a small peptide containing more than one
basic amino acid residue. Fourth, the first cycle of Edman degradation
altered electrophoretic migration (Fig. 6E, lane 1),
consistent with removal of basic residue. Labeled phosphate was
released at the third cycle (lane 3), suggesting that the
phosphoserine position is three residues from the trypsin and ArgC
cleavage sites.
|
|
Lys49 Is Required for Recognition of 14-3-3 by
SDK1--
In the three-dimensional structure of 14-3-3 , the SDK1
phosphorylation site (Ser58) is localized on helix 3, and
Ser58 participates in dimerization, while Lys49
and other positively charged residues of the helix form part of an
amphipathic groove involved in ligand binding (43). Consistent with the
proposed role of the amphipathic groove in ligand binding, Lys49-Glu mutation disrupted ligand association but
Arg60-Glu had only a small effect (32). To test whether
SDK1 also uses this amphipathic groove of 14-3-3 for substrate
recognition, we examined the effect of the Lys49-Glu and
Arg60-Glu mutations on phosphorylation of 14-3-3 by
activated SDK1. The Lys49-Glu mutation abolished the
ability of 14-3-3 to serve as a SDK1 substrate, while the
Arg60-Glu mutation reduced phosphorylation only by about
50% (Fig. 8A, upper panel).
Equivalent quantities of 14-3-3 were used for each reaction (Fig.
8A, lower panel). These results suggest that the
requirements for SDK1 recognition of 14-3-3 are the same as those for
ligand binding to 14-3-3 (32). It is interesting that the mutation in
Arg60 in position 2+ relative to the phosphorylation site,
had much less effect than the distant Lys49 mutation in
position
9. These findings suggest that direct binding of SDK1 via
the amphipathic groove in 14-3-3 is required for subsequent phosphorylation at Ser58.
|
An SDK Is Associated with 14-3-3 and Is Activated by Exogenous DMS,
Leading to 14-3-3 Phosphorylation in Situ--
14-3-3 proteins were
purified together with SDK1 at the first purification step on
ion-exchange chromatography. To confirm the interaction between 14-3-3 and SDK1, we tested the ability of various 14-3-3 Abs to precipitate
SDK activity from cytosolic extract. The PAN, , and
Abs raised
against different epitopes of 14-3-3 isoforms were utilized. These Abs
detected endogenous 14-3-3
,
, and
found in 3T3 cell lysates
(data not shown). SDK activity was co-immunoprecipitated by PAN Ab or
Ab as indicated by phosphorylation of exogenous 14-3-3
in the
presence of DMS (Fig. 9A). In
contrast,
Ab did not precipitate SDK activity together with 14-3-3, although Western blot analysis revealed a similar level of 14-3-3 proteins in all immunoprecipitates (data not shown). These results
indicate a specific association of an SDK with certain isoforms of
14-3-3. It is reasonable to conclude that SDK1 is probably associated
with 14-3-3
in situ, since the
Ab is
isoform-specific and cross-reacts only with 14-3-3
but not with
14-3-3
,
, or
.
|
![]() |
DISCUSSION |
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---|
We partially purified a sphingosine-dependent protein
kinase, SDK1, that is activated by Sph or DMS but not by other lipids, and specifically phosphorylates certain isoforms of 14-3-3 protein in vitro. Sph-dependent phosphorylation of
endogenous 14-3-3 proteins was previously detected in a crude system,
suggesting that 14-3-3 proteins are major substrates for cellular SDK
activities. Addition of DMS to Balb/c 3T3(A31) fibroblasts stimulates
phosphorylation of 14-3-3 proteins, and SDK activity is associated with
endogenous 14-3-3 ; therefore, SDK1 may also phosphorylate 14-3-3 proteins in intact cells. The failure of SDK1 to phosphorylate PDI and calreticulin, endogenous substrates of other types of SDKs, indicates the existence of several other SDKs with distinct substrate
specificity. Thus, several different kinases, including SDK1, other
SDKs, and certain tyrosine kinases, may mediate signal transduction
induced by Sph and DMS (2, 17, 18, 20).
SDK1 phosphorylates the ,
, and
, isoforms of 14-3-3 at Ser
adjacent to Trp in the sequence Arg-Arg-Ser-Ser-Trp-Arg. 14-3-3 proteins have consensus sequences for phosphorylation by several protein kinases (41). The site phosphorylated by SDK1 fits the consensus motif for phosphorylation by PKA and PKC (41); however, PKA
fails to phosphorylate 14-3-3 (40), and PKC phosphorylates a different
site that is conserved in all isoforms of 14-3-3 (
,
,
,
,
and
) (40, 42). 14-3-3
and
isolated from sheep brain are
phosphorylated on Ser184 in the site Ser-Pro-Glu-Lys (44),
a consensus sequence motif of proline-dependent protein
kinase (41). Casein kinase I phosphorylates 14-3-3
and
on
residue 233 (45, 46). A breakpoint cluster region protein (Bcr)
phosphorylates Ser, and Bcr-Abl phosphorylates Ser and Tyr, on 14-3-3
in vitro, but the phosphorylated residues have not been
mapped (47).
The crystal structure of 14-3-3 at 2.9-Å resolution indicates a dimer,
and each monomer contains nine helices (Fig.
10A) (43). The longest
helix, helix 3, interacts with helix 1 of the opposite monomer and
contributes to the dimer interface as well as the ligand binding
groove. Many residues located in this helix are invariant among
vertebrates, yeast, and plants (43). Mutagenesis and structural studies
indicate that the surface of helix 3 that faces helix 5 forms the
pocket in 14-3-3 that binds to phosphopeptide ligands of the sequence
Arg-Ser-X-pSer-X-Pro (32, 43, 48, 49). However,
the residue (Ser58 of 14-3-3 ) that is phosphorylated by
SDK1 in helix 3 of one monomer faces away from the ligand-binding
pocket and toward the other monomer in the 14-3-3 dimer (Fig.
10B; Ser shown in red). This Ser is buried in the
dimer interface, lying just 5.8 Å from a conserved Arg
(Arg18 in 14-3-3
) of the other monomer. After
phosphorylation, a negative charge on phosphoserine 58 may interact
with the positive charge on the side chain of Arg18,
resulting in dimer stabilization. Thus, the newly phosphorylated 14-3-3 monomer may sequester other 14-3-3 monomers, preventing monomer
functions such as interaction with inactive Raf (50). The possibility
described here for 14-3-3
may also apply to 14-3-3
and
,
because the key residues of the dimer interface are conserved across
the 14-3-3 family (43, 49).
|
The location of the SDK1 phosphorylation site in the dimer interface of 14-3-3 also raises the question of how SDK1 phosphorylates this buried residue. Although the recombinant 14-3-3 used during SDK1 purification may be partly monomeric, the phosphorylation of endogenous 14-3-3 proteins suggests that SDK1 or Sph can promote either dimer dissociation or conformational changes that expose the phosphorylation site. PKC may not induce such conformational changes, because PKC fails to phosphorylate the SDK1 site in native 14-3-3 but does phosphorylate this site in a peptide substrate (40).
All protein kinases interact via their catalytic pocket with the phosphoaccepting hydroxyamino acid, as well as with several specificity conferring residues that flank the phosphoacceptor site (51). Substrate recognition by SDK1 was investigated using mutants Lys49-Glu (Fig. 10B, yellow) and Arg60-Glu (green). Lys49, which is located nine amino acids (2.5 helix turns) from the phosphorylation site, facing the opposite side of helix 3 (43), had a surprisingly large effect on phosphorylation by SDK1. On the other hand, Arg60-Glu mutation, located 2 amino acids C-terminal to the phosphorylation site, reduced phosphorylation by only 50%, suggesting that the positive charge of Arg60 has little effect on SDK1 activity. Lys49 faces the proposed 14-3-3 ligand binding groove (32). The requirement for a functional binding groove in 14-3-3, and the lack of a strong requirement for a residue close to the phosphorylation site, suggest that the molecular interactions mediating binding of SDK1 to 14-3-3 are not typical of most protein kinases and their substrates.
Sph is generated from sphingolipid degradation in the plasma membrane,
and can be transferred to other intracellular membrane compartments.
However, Sph traffic into the soluble phase of the cytoplasm has not
been investigated. The majority of SDK1 activity is in the cytosol, but
it is not clear whether the Sph that activates SDK1 is in the membrane
or cytosol. SDK1 may be activated by translocation to the membrane,
since 14-3-3 is associated with SDK1 in situ, and
partially purified membrane SDKs phosphorylate 14-3-3 (21). In
addition, 14-3-3 proteins, which are found in cytosol, are also
associated with the plasma membrane (52), and can promote translocation
of kinases to different cellular compartments upon stimulation with
agonists. For example, serum stimulation translocates KSR-14-3-3
complex from cytosol to membrane, resulting in association with Raf-1
and activation (29). In contrast, the
and
isoforms promote
translocation from cytoskeleton to cytosol of A20 and PKC
,
respectively (53, 54). The
isoform inhibits PKC
by preventing
its translocation to membrane (54). Because SDK1 is associated with
14-3-3, association of 14-3-3 with membrane could also provide a means
for activation of SDK1 by membrane-bound Sph.
We used DMS to demonstrate Sph-dependent phosphorylation of
14-3-3 in situ, since in fibroblasts this Sph derivative
cannot be phosphorylated or modified to create other potential kinase activators. DMS and Sph are found naturally in cells, and enzymes that
convert Sph to DMS have been detected in tissues, suggesting that DMS
is a physiological activator (15, 16, 55). A relatively low
concentration of exogenous DMS (2 µM) induced rapid
phosphorylation of 14-3-3 in intact cells. The rapid penetration of Sph
or DMS into platelets (56) and the fact that 80% of DMS is
incorporated in A31 cells within 5 min (data not shown) suggest an
intracellular activation of SDK1 by Sph or DMS. Extracellular agonists
such as platelet-derived growth factor and insulin-like growth factor, which increase Sph levels within the cell, may also stimulate 14-3-3 phosphorylation. Further studies are needed to correlate phosphorylation of ,
, and
isoforms of 14-3-3 (SDK1
substrates) with the increase of intracellular Sph levels induced by
growth factors. Such studies would shed light on the regulatory role of
Sph and SDK1 activity in intracellular signaling via modulation of the
function of specific 14-3-3 isoforms.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Kimie Murayama and Tsutomu Fujimura for preparation of hexadecapeptide, Dr. Edwin Krebs for the gift of anti-CKII Ab, Dr. Ari Helenius for the gift of calreticulin, Dr. Yasuyuki Igarashi for helpful discussions, Melissa Jurica and Dr. Barry Stoddard for computer assistance, Dr. R. Liddington for coordinates, and Dr. Stephen Anderson for scientific editing and preparation of the manuscript.
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FOOTNOTES |
---|
* This work was supported in part by National Cancer Institute Outstanding Investigator Grant CA42505 (to S. H.) and National Institutes of Health Grants CA54786 (to J. C.) and GM53165 (to H. F.).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.
Burroughs Wellcome Fund New Investigator.
** To whom correspondence should be addressed. E-mail: hakomori{at}u.washington.edu.
The abbreviations used are:
Sph, sphingosine; Ab, antibody; AEBSF, 4-(2-aminoethyl)-benzenesulfonyl fluoride; CKII, casein kinase II; DAG, diacylglycerol; DMS, N,N'-dimethylsphingosineDTT, dithiothreitolErk, extracellular signal-regulated mitogene-activated protein kinaseFBS, fetal bovine serumFT, flow-throughKSR, kinase suppressor of
rasNC, nitrocellulosePDI, protein disulfide isomerasePKA, protein kinase APKC, protein kinase CPKM, catalytic domain of
protein kinase CPS, L--phosphatidyl-L-serineSDK, sphingosine-dependent protein kinaseSph-1-P, sphingosine
1-phosphateTPA, 12-O-tetradecanoylphorbol-13-acetatePAGE, polyacrylamide gel electrophoresisDMEM, Dulbecco's modified
Eagle's mediumMOPS, 4-morpholinepropanesulfonic acid.
2 R. R. Subrumanian, S. C. Masters, and H. Fu, manuscript in preparation.
3 T. Megidish, K. Takio, K. Titani, Y. Igarashi, A. Helenius, and S. Hakomori, manuscript in preparation.
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REFERENCES |
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