From the
The c-kit-encoded tyrosine kinase receptor for stem
cell factor (Kit/SCFR) is crucial for the development of hematopoietic
cells, melanoblasts, and germ cells. Ligand stimulation of Kit/SCFR
leads to receptor dimerization and autophosphorylation on tyrosine
residues. We recently showed, that protein kinase C (PKC) acts in an
SCF-stimulated negative feedback loop, which controls Kit/SCFR tyrosine
kinase activity and modulates the cellular responses to SCF
(Blume-Jensen, P., Siegbahn, A., Stabel, S., Heldin, C.-H., and
Rönnstrand, L.(1993) EMBO J. 12, 4199-4209). We
present here the identification of the major phosphorylation sites for
PKC in Kit/SCFR. Two serine residues in the kinase insert, Ser-741 and
Ser-746, are PKC-dependent phosphorylation sites in vivo and
account for all phosphorylation by PKC in vitro. Together they
comprise more than 60% of the total SCF-stimulated receptor
phosphorylation in living cells and 85-90% of its phosphorylation
in resting cells. Two additional serine residues, Ser-821 close to the
major tyrosine autophosphorylation site in the kinase domain and
Ser-959 in the carboxyl terminus are SCF-stimulated PKC-dependent
phosphorylation sites. However, they are not phosphorylated directly by
PKC-
The receptor for stem cell factor (SCF)
Ligand stimulation of the Kit/SCFR induces its dimerization and
rapid autophosphorylation on tyrosine residues (Blume-Jensen et
al., 1991). Phosphorylated tyrosine residues in receptor tyrosine
kinases mediate the specific binding and subsequent activation of
cytoplasmic signaling molecules containing Src homology 2 (SH2) domains
(Koch et al., 1991; Schlessinger, 1994). By inference, the
phosphorylation of specific amino acid residues in the receptors
specify activation of distinct signaling pathways, why identification
of these autophosphorylation sites is of major importance. Only limited
information is available regarding the tyrosine phosphorylation sites
in Kit/SCFR in vivo, although indirect evidence indicates that
tyrosine 721 becomes phosphorylated in response to SCF and mediates the
binding to phosphatidylinositide 3`-kinase (PI-3`-kinase) (Lev et
al., 1992; Serve et al., 1994). However, most of the
phosphorylation of Kit/SCFR occurs on serine residues in resting as
well as in SCF-stimulated cells, constituting approximately 90% of the
total receptor phosphorylation; a major part of this serine
phosphorylation is mediated by protein kinase C (PKC) (Blume-Jensen
et al., 1993).
PKC is a serine/threonine kinase involved in
the control of cell proliferation, differentiation, and motility
(Nishizuka, 1992). The PKC family presently consists of 12 known
members, of which the classical members, PKC-
In order to further clarify the mechanism
whereby PKC regulates the Kit/SCFR, we report here the identification
of the major PKC-dependent phosphorylation sites in the Kit/SCFR.
Consistent with the notion that PKC is involved in an SCF-stimulated
negative feedback loop, a receptor mutant with the phosphorylation
sites for PKC mutated to alanine residues showed a 2-fold increased
specific receptor tyrosine autophosphorylation and specific
receptor-associated PI-3`-kinase activity compared to the wild type
Kit/SCFR in response to SCF. Furthermore, the kinase activity of the
mutated receptor toward phosphorylation of an exogenous substrate was
increased compared to the wild type Kit/SCFR.
We
further examined the kinase activity of the mutated versus the
wild type receptor in vitro by phosphorylation of the peptide
RDIM(13) in the presence of [
We have previously shown that a majority of the total
phophorylation of Kit/SCFR in unstimulated as well as SCF-stimulated
cells occurs on serine residues (Blume-Jensen et al., 1993).
Most of this serine phosphorylation is mediated by PKC, which acts in
an SCF-stimulated feedback loop, that negatively controls Kit/SCFR
kinase activity and SCF-induced mitogenicity (Blume-Jensen et
al., 1993). The increased Kit/SCFR tyrosine autophosphorylation
and SCF-induced mitogenicity after inhibition of PKC parallels an
increased receptor association with, and specific activation of,
PI-3`-kinase, while activation of the Raf-1/MAPK pathway does not seem
to be increased (Blume-Jensen et al., 1994). In this paper we
have identified all the major phosphorylation sites for PKC in the
human Kit/SCFR in vivo as well as in vitro. The two
major PKC-dependent phosphorylation sites in vivo, serine
residues 741 and 746, are located in the kinase insert region and are
also the two sites that become phosphorylated by PKC in vitro.
These sites are phosphorylated constitutively in resting cells, and
their phosphorylations are increased in response to SCF as well as in
response to PMA. The serine residues 959 and 821 are also SCF- and
PMA-induced PKC-dependent phosphorylation sites. These residues are not
constitutively phosphorylated, nor do they become phosphorylated
directly by PKC-
Numerous observations in this study,
as well as our previous studies, clearly indicate that PKC is activated
in response to SCF in PAE/kit cells. Firstly, the predominant serine
phosphorylation of the Kit/SCFR in resting cells becomes stimulated in
response to both SCF and PMA, and this stimulation is inhibited by
treatment with PKC inhibitors. Secondly, the two major phosphopeptides
in two-dimensional tryptic phosphopeptide maps of Kit/SCFR from intact
cells, which together account for approximately 90-95% of the
basal receptor phosphorylation, become stimulated in response to both
SCF and PMA, and are in both cases inhibited by calphostin C, as shown
in this study. The phosphorylation of each phosphopeptide is accounted
for by a single serine phosphorylation site for PKC. Finally,
inhibition of PKC has profound effects on Kit/SCFR kinase activity and
SCF-induced signal transduction pathways (Blume-Jensen et al.,
1994; Blume-Jensen et al., 1993).
The mechanism of
activation of PKC in response to SCF remains to be elucidated. In
previous studies performed in transiently transfected COS cells and
embryonic fibroblasts, as well as in the monocytic cell line MO7e
expressing Kit/SCFR endogenously, it was reported that phospholipase
C-
Ser-959, and to a minor extent Ser-821, are phosphorylated in a
PKC-dependent manner in response to SCF. However, these sites are not
phosphorylated by PKC-
Besides our own previous
findings, accumulating evidence indicates a critical involvement of PKC
in several aspects of Kit/SCFR signal transduction and signal
``cross-talk.'' Thus, PMA-stimulated activation of PKC in
mast cells as well as in SCF-dependent myeloid cell lines leads to
proteolytic release of the extracellular domain of Kit/SCFR (Brizzi
et al., 1994; Yee et al., 1993, 1994). However, PKC-
and SCF-induced down-regulation of Kit/SCFR occurs through independent
mechanisms (Yee et al., 1993). Furthermore, the interleukin-3
receptor, which acts synergistically with Kit/SCFR in initiation of
hematopoietic cell proliferation, becomes phosphorylated in a
PKC-dependent manner on serine and threonine residues in response to
SCF, and this phosphorylation is independent of Kit/SCFR kinase
activity (Liu et al., 1994). Another recent study showed that
treatment of megakaryoblastic Kit/SCFR-expressing tumor cell lines with
PMA led to terminal differentiation concomitant with increased
c-kit mRNA expression (Hu et al., 1994). Finally, the
two transmembrane forms of SCF are proteolytically cleaved to give rise
to the soluble form of SCF in response to PMA, and PKC might be
involved in the tissue-specific regulation of this cleavage (Huang
et al., 1992). The identification of the phosphorylation sites
for PKC in the Kit/SCFR will allow for experiments designed to
determine the role of endogenously activated PKC in the regulation of
Kit/SCFR signaling pathways in intact cells.
Ulla Engström is gratefully acknowledged for
expert production of synthetic peptides and Silvia Stabel is thanked
for the generous gift of purified PKC-
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
in vitro. Both specific receptor tyrosine
autophosphorylation and specific receptor-associated
phosphatidylinositide 3`-kinase activity was increased approximately
2-fold in response to SCF in PAE cells stably expressing
Kit/SCFR(S741A/S746A). Furthermore, the kinase activity of
Kit/SCFR(S741A/S746A) toward an exogenous substrate was increased,
which was reflected as a decreased K
and
an increased V
, in accordance with the negative
regulatory role of PKC on Kit/SCFR signaling.
(
)
is encoded by c-kit, the cellular counterpart of
the oncogene v-kit which was isolated from an acutely
transforming feline retrovirus (Besmer et al., 1986; Qiu
et al., 1988; Yarden et al., 1987). The Kit/SCF
receptor (Kit/SCFR) is a 145-kDa transmembrane receptor tyrosine
kinase, which is allelic with the murine dominant white spotting
(W) locus on chromosome 5 (Chabot et al., 1988; Geissler
et al., 1988), whereas SCF is encoded by the murine steel
(Sl) locus on chromosome 10 (Witte, 1990). The many naturally
occurring mutations in these two loci give rise to defects in the
proliferation, migration, and differentiation of stem cells in the
melanogenic, gametogenic, and hematopoietic lineages (Besmer, 1991).
, -
, and -
,
are Ca
- and phospholipid-dependent. The classical PKC
isoforms become activated by sn-1,2-diacylglycerol produced in
response to, e.g. activation of phospholipase C-
or by
the sequential action of phospholipase D and phosphatidic acid
phosphohydrolase after growth factor stimulation (Nishizuka, 1992).
While most of the known substrates for PKC are cytoskeleton-associated
proteins (Dekker and Parker, 1994), PKC also phosphorylates and
regulates the activity of the receptors for epidermal growth factor,
insulin, and hepatocyte growth factor (Farese et al., 1992;
Gandino et al., 1990; Iwashita and Kobayashi, 1992). Moreover,
PKC acts in an SCF-stimulated negative feedback loop by direct
phosphorylation of the Kit/SCFR. This inhibits SCF-induced kinase
activity and modulates the cellular responses to SCF (Blume-Jensen
et al., 1993). Of several signaling molecules examined, the
increased mitogenicity after inhibition of PKC paralleled an increased
association and specific activation of PI-3`-kinase (Blume-Jensen
et al., 1994).
Site-directed Mutagenesis and Plasmid
Constructs
A 3491-base pair EcoRI-XbaI
fragment of the human c-kit cDNA (nucleotides 704 to 4195
according to Yarden et al.(1987)) was subcloned into the
phagemid vector pALTER-1 (Promega Corp.). Bacterial
cultures were transformed with the construct, infected with helper
phage in the presence of tetracycline, and single-stranded DNA was
prepared. To replace single serine residues with alanine residues in
Kit/SCFR, in vitro mutagenesis reactions were performed using
the following oligonucleotides (mutated bases underlined): Ser-741
Ala, 5`-AAAAGGAGAGCTGTGAGAATAGGC-3` (ON26); Ser-746
Ala,
5`-GAGAATAGGCGCATACATAGAA-3` (ON27); Ser-821
Ala,
5`-AAGAATGATGCTAATTATGTG-3` (ON28); Ser-959
Ala,
5`-CGGATCAATGCTGTCGGCAGC-3` (ON29); Ser-741 and Ser-746
Ala,
5`-GACAAAAGGAGAGCTGTGAGAATAGGCGCATACATAGAAAGA-3` (ON30). To replace the
serine residues 741, 746, 821, and 959 with alanine residues, the
oligonucleotides ON28, ON29, and ON30 were used simultaneously in the
mutagenesis reaction. Mutagenesis reactions were performed according to
the instructions by the manufacturer (Altered Sites
in
vitro mutagenesis system; Promega), and colonies were screened for
mutations by direct sequencing (Sequenase version 2.0 DNA
dideoxynucleotide sequencing kit; U. S. Biochemical Corp.). All
mutations were covered by a SnabI-BstEII fragment
(nucleotide position 1141 to 2970) which was subcloned into the
full-length human c-kit cDNA in the mammalian expression
vector pSV7D (Truett et al., 1985). The c-kit cDNA
was finally cloned into pcDNA1/Neo, a cytomegalovirus-based expression
vector.
Establishment of Stable Kit/SCFR-expressing PAE Cell
Lines
Porcine aortic endothelial (PAE) cells (Miyazono et
al., 1985) were transfected with each of the different c-kit expression constructs. Between 8 and 10 cell clones transfected
with each mutant Kit/SCFR construct were expanded in the presence of
geneticin (G418 sulfate; Life Technologies, Inc.) and analyzed for
receptor expression, receptor synthesis, and tyrosine
autophosphorylation, as described below. Two cell clones for each
construct were finally chosen for further analysis. All cell lines were
routinely grown in Ham's F12 medium supplemented with 10% fetal
calf serum (Life Technologies, Inc.) and 0.25 mg/ml geneticin (G418;
Life Technologies, Inc.), 100 units/ml penicillin, 100 µg/ml
streptomycin, and 2 mML-glutamine.
Antibodies
The Kit/SCFR-specific peptide
antiserum, Kit-C1, recognizing the carboxyl-terminal of the receptor
(Blume-Jensen et al., 1991) was affinity-purified as described
(Blume-Jensen et al., 1993). The mouse anti-phosphotyrosine
monoclonal antibody (PY20) was from Affiniti Research Products Ltd.,
United Kingdom. Affinity-purified rabbit anti-phosphotyrosine
antibodies were prepared according to Rönnstrand et
al.(1987).
Metabolic Labeling, Immunoprecipitation, and Western
Blotting
Serum-starved PAE cells expressing wild type or mutated
Kit/SCFR were labeled for 5 h at 37 °C with 100 µCi/ml each of
[S]methionine and
[
S]cysteine in MCDB 104 medium (Life
Technologies, Inc.) containing 1% fetal calf serum dialyzed against
phosphate-buffered saline. Cells were then stimulated with SCF (150
ng/ml) for 8 min at 37 °C, lysed, and Kit/SCFR immunoprecipitates
by SDS-gel electrophoresis and sequential immunoblotting as described
previously (Blume-Jensen et al., 1994). For quantitation of
specific receptor tyrosine autophosphorylation, densitometric scannings
ensured equal amounts of immunoprecipitated wild type and mutated
receptors. [
P]Orthophosphate Labeling of Cells, Tryptic
Phosphopeptide Mapping, and Two-dimensional Phosphoamino Acid
Analysis-PAE cells expressing wild type or mutated Kit/SCFR
were labeled in phosphate-free Ham's F12 medium supplemented with
0.5% dialyzed fetal calf serum, 15 mM HEPES (pH 7.4), and 2
mCi/ml [
P]orthophosphate for 3 h. Cells were
lysed, and receptors were immunoprecipitated, separated by SDS-gel
electrophoresis, and electrotransferred to nitrocellulose filter
(Hybond-C extra; Amersham) as described (Blume-Jensen et al.,
1993). For tryptic phosphopeptide mapping, protein bands were localized
by exposure on a PhosphorImager, excised from the filter and digested
in situ with trypsin (modified sequencing grade; Promega) for
8-12 h at 37 °C as described (Aebersold et al.,
1987). Two-dimensional phosphopeptide mapping was performed using the
Hunter thin layer electrophoresis apparatus (HTLE-7000; C.B.S.
Scientific Co., Inc., Del Mar, CA) according to Boyle et
al.(1991). First dimension electrophoresis was performed in pH 1.9
buffer (formic acid/glacial acetic acid/double-distilled water
(44:156:1800, v/v/v) for 40 min at 2000 V, and second dimension
ascending thin layer chromatography in isobutyric acid buffer
(isobutyric acid/n-butyl alcohol/pyridine/glacial acetic
acid/double-distilled water (1250:38:96:58:558, v/v/v/v/v). After
exposure on a PhosphorImager, phosphopeptides were eluted from the
plates in pH 1.9 buffer and lyophilized. The fractions were then
subjected to two-dimensional phosphoamino acid analysis and automated
Edman degradation in parallel. For Edman degradation, phosphopeptides
were coupled to Sequelon-AA membranes (Millipore) according to the
manufacturer's instructions and sequenced on an Applied
Biosystems Gas Phase Sequencer Model 470A. The radioactivity in
released phenylthiohydantoin-derivatives from each cycle was
quantitated by exposure on a PhosphorImager.
Immunoprecipitation of Tryptic Phosphopeptides for
Sequencing and Two-dimensional Phosphoamino Acid
Analysis
Tryptic phosphopeptides were dissolved in 200 µl of
50 mM NHHCO
and incubated with 50
µl of a 1:1 slurry of immobilized
-macroglobulin
(Boehringer Mannheim) for 30 min at room temperature to remove the
trypsin. After centrifugation for 1 min in a Microfuge, the supernatant
was incubated with affinity-purified Kit-C1 antibodies for 2 h at 4
°C, and immunocomplexes were collected on Protein A-Sepharose
(Pharmacia). The peptide-depleted supernatant was saved. Beads were
extensively washed, and the immunoprecipitated phosphopeptide eluted by
incubation for 15 min in 30% formic acid. The eluted peptide and the
peptide-depleted supernatant were lyophilized separately and subjected
to two-dimensional phosphopeptide mapping and amino acid sequencing as
described above.
Ligand Binding Analysis
Recombinant human
SCF was labeled with
I (Amersham) according
to the chloramine-T method (Hunter and Greenwood, 1962), and Scatchard
analyses were performed as described (Blume-Jensen et al.,
1993).
Phosphorylation of Wild Type and Mutated Kit/SCF Receptor
by Purified PKC-
Serum-starved PAE cells
expressing wild type or mutated Kit/SCFR were lysed and Kit/SCFR
immunoprecipitated in the presence of 2 mMN-ethylmaleimide to irreversibly inhibit the Kit/SCFR kinase
activity before phosphorylation by PKC- in Vitro
in the presence of
[
-
P]ATP, as described (Blume-Jensen et
al., 1993).
Phosphatidylinositide 3`-Kinase Assays
PAE cells
expressing wild type or mutated Kit/SCFR were serum-starved overnight,
and cells were lysed before and after SCF stimulation. Aliquots of the
lysates were incubated with anti-Kit-C1 antibodies, and receptor
immunoprecipitates were subjected to PI-3`-kinase assays using
phosphatidylinositol as a substrate exactly as described (Blume-Jensen
et al., 1994). Receptors immunoprecipitated in parallel from
other aliquots were used to quantitate receptor numbers by
densitometric scannings of sequential immunoblottings as described
above. PI-3`-kinase activity was normalized to receptor number.
Lineweaver-Burk Plots
Wild type or mutated
Kit/SCFR were immunoprecipitated from cell lysates with an excess of
Kit-C1 antibodies, and stimulated with 0.5 µg/ml SCF in
vitro. Lysates were aliquoted, and half of the aliquots were
phosphorylated by PKC- in a mixed micelles assay in the presence
of 100 µM nonradioactive ATP. Phosphorylation of the
peptide RDIM(13) in the presence of [
-
P]ATP
was then performed exactly as described (Blume-Jensen et al.,
1993). To normalize the receptor number, parallel aliquots of the
receptor immunoprecipitates were subjected to quantitative
immunoblotting using
I-labeled protein A as secondary
reagent and quantitation using a Fuji Image Analyzer.
A Subset of SCF- and PMA-stimulated Tryptic
Phosphopeptides from Kit/SCFR Are Phosphorylated Mainly on Serine
Residues in Vivo and Their Phosphorylation Is Inhibited by Calphostin
C
We earlier reported that most of the phosphorylation of
Kit/SCFR occurs on serine residues and is mediated by PKC, which acts
in an SCF-stimulated feedback loop (Blume-Jensen et al.,
1993). In order to to identify the phosphorylation sites for PKC in the
Kit/SCFR, cells were labeled with
[P]orthophosphate and stimulated with SCF or PMA
before and after inhibition of PKC with calphostin C (Kobayashi et
al., 1989). After lysis, receptors were immunoprecipitated,
separated by SDS-gel electrophoresis, and subjected to tryptic
digestion and two-dimensional phosphopeptide mapping (Boyle et
al., 1991). As shown in Fig. 1A, the
phosphorylation of the two major phosphopeptides (denoted 1 and 2) is
stimulated by both SCF and PMA. Besides, several additional
phosphopeptides, including numbers 3 and 4 which were not seen in
unstimulated cells, appeared. Treatment of cells with calphostin C led
to a decreased relative intensity of phosphopeptides 1 and 2, while 3
and 4 were no longer detectable. The phosphopeptides from both
unstimulated cells and cells stimulated with SCF or PMA were scraped
off the thin layer chromatography (TLC) plates and subjected to
two-dimensional phosphoamino acid analysis. Phosphopeptides 1, 2, and 3
were phosphorylated entirely on serine residues
(Fig. 1B; after SCF stimulation). Phosphopeptide 4 in
Fig. 1A was more difficult to analyze as it co-migrated
with (an)other phosphopeptide(s) (Fig. 1A and data not
shown
), (
)and varying phosphorylation on serine
and tyrosine residues was observed.
These initial
experiments thus indicated that a subset of at least 3 peptides,
denoted 1, 2, and 3 in
Fig. 1A, were phosphorylated entirely on serine residues
in response to SCF as well as PMA in a PKC-dependent fashion.
Phosphopeptide 4 was also phosphorylated in a PKC-dependent manner, but
its apparent co-migration with other peptide(s) excluded any further
conclusions regarding its regulation.
Figure 1:
Two-dimensional tryptic phosphopeptide
maps of Kit/SCFR from [P]orthophosphate-labeled
PAE/kit cells and phosphoamino acid analyses of phosphopeptides.
A, serum-starved, orthophosphate-labeled cells were left
untreated or stimulated with SCF or PMA in the presence or absence of
calphostin C as indicated. Kit/SCFR was immunoprecipitated from cell
extracts, separated by SDS-gel electrophoresis, electrotransferred to
Hybond-C extra nitrocellulose membrane, and digested in situ with trypsin. Phosphopeptides were separated on cellulose thin
layer chromatography (TLC) glass plates by high voltage
electrophoresis, followed by ascending chromatography. The plates were
exposed on a PhosphorImager as well as to film (exposure time 20 h at
-70 °C). The indicated electrophoresis direction is from the
anode to the cathode. B, phosphopeptides were eluted from the
phosphopeptide maps and subjected to two-dimensional phosphoamino acid
analysis. The numbers indicate the peptide spots in A from the SCF-stimulated sample. Identical results were obtained
from PMA-stimulated samples. The stippled circles indicate the
locations of the phosphoamino acid markers, and S, T,
and Y to the right indicate the relative positions of
phosphorylated serine, threonine, and tyrosine, respectively. The
plates were exposed on a PhosphorImager.
Phosphorylation of Kit/SCFR by PKC-
We further examined the phosphorylation of
Kit/SCFR by purified, recombinant PKC- in Vitro:
Identification of Ser-741 and Ser-746 as the Major Phosphorylation
Sites for PKC
in vitro. Kit/SCFR
was immunoprecipitated from serum-starved PAE/kit cells and
kinase-inactivated by incubation with the sulfhydryl-reagent
N-ethylmaleimide. The receptors were then subjected to
phosphorylation by PKC-
in the presence of
[
-
P]ATP in a mixed micelles kinase assay
with phosphatidylserine and PMA as stimulating agents (Sözeri
et al., 1992). Phosphorylated receptors were separated by
SDS-polyacrylamide gel electrophoresis, electrotransferred to
nitrocellulose membrane before tryptic digestion in situ and
analysis by two-dimensional phosphopeptide mapping. As shown in
Fig. 2A, two major phosphopeptides, which were
phosphorylated entirely on serine residues (Blume-Jensen et
al., 1993),
were located at positions corresponding to
those of 1 and 2 in Fig. 1A. Furthermore, mixing tryptic
digests of Kit/SCFR from
[
P]orthophosphate-labeled PAE/kit cells after
PMA or SCF stimulation with the tryptic digest of the Kit/SCFR
phosphorylated by PKC-
in vitro showed that the peptides
co-migrated.
(
)
Figure 2:
Identification of the major
phosphorylation sites for PKC in Kit/SCFR. A, Kit/SCFR
immunoprecipitated from detergent extracts of PAE/kit cells was
kinase-inactivated by treatment with N-ethylmaleimide and
phosphorylated by purified PKC- in a mixed micelles assay in
vitro in the presence of [
-
P]ATP. The
receptor was analyzed by two-dimensional tryptic phosphopeptide mapping
performed exactly as described in the legend to Fig. 1A. The
picture is from a PhosphorImager exposure. B, the Kit/SCFR
phosphorylated in vitro by PKC-
was chemically cleaved
with cyanogen bromide and immunoprecipitated with an affinity-purified
peptide antiserum against the kinase insert region. The fragment was
subjected to amino acid sequencing, and the radioactivity released in
each cycle was measured by spotting onto TLC plates and exposure on a
Fuji Image Analyzer. Identical results were obtained from Kit/SCFR
labeled in vivo with [
P]orthophosphate
before and after stimulation with either SCF or PMA. The two
phosphorylated serine residues 741 and 746 are indicated in the
sequence of the putative fragment of the
Kit/SCFR.
In order to identify these
peptides and their sites of phosphorylation, phosphopeptides 1 and 2
from both in vivo and in vitro phosphorylation
experiments were coupled to a Sequelon-AA membrane (Millipore) for
amino acid sequencing. In both cases, a significant radioactivity
release was found in cycle 1 of peptide 1 and cycle 3 of peptide 2.
Compiling all the theoretical tryptic peptides derived from a complete
digestion of the Kit/SCFR revealed that 2 peptides have a serine
residue at position 1 and 7 peptides have a serine residue at position
3. Based on results obtained by sequencing of all the major spots from
two-dimensional phosphopeptide maps of Kit/SCFR from SCF-stimulated
PAE/kit cells, as well as secondary proteolytic cleavages using
different proteases, we concluded that two peptides from
the kinase insert of the receptor, containing Ser-741 and Ser-746, were
likely to be the phosphopeptides 1 and 2, respectively. To obtain
further evidence for this notion, Kit/SCFR from both SCF- and
PMA-stimulated [
P]orthophosphate-labeled PAE/kit
cells, as well as in vitro PKC-phosphorylated Kit/SCFR, was
cleaved with cyanogen bromide and precipitated with an
affinity-purified peptide antibody raised against a peptide from the
kinase insert of the Kit/SCFR (see ``Materials and Methods''
for details). In all three cases, amino acid sequencing of the
CNBr-cleaved peptide revealed significant release of radioactivity in
cycles 17 and 22, which corresponds to serine residues 741 and 746 in
the kinase insert (Fig. 2B). These findings thus
indicated that the major in vivo and in vitro phosphorylation sites for PKC in the Kit/SCFR are Ser-741 and
Ser-746.
Ser-959, and Possibly Ser-821, in the Kit/SCFR Are
PKC-dependent SCF- and PMA-stimulated in Vivo Phosphorylation
Sites
The phosphopeptides 3 and 4 in the receptor were clearly
dependent on PKC (Fig. 1A), but not phophorylated by
PKC- in vitro (Fig. 2A). Sequencing of the
serine-phosphorylated peptide 3 gave a significant radioactivity
release in position 3.
(
)
Of the candidate tryptic
peptides with a serine residue in position 3, the most
carboxyl-terminal peptide of Kit/SCFR (amino acids 959-976)
contains Ser-959 in a typical PKC phosphorylation consensus recognition
sequence (RIN
S
. . . ). To
examine if this peptide corresponds to phosphopeptide 3 in
Fig. 1A, we sequenced the phosphopeptide obtained by
immunoprecipitation with Kit-C1 of tryptic Kit/SCFR digests obtained
from either SCF- or PMA-stimulated
[
P]orthophosphate-labeled PAE/kit cells. In both
cases, radioactivity release was observed in cycle 3, as
expected.
Furthermore, the phosphopeptide
immunoprecipitated from PMA-stimulated PAE/kit cells was analyzed by
two-dimensional phosphopeptide mapping. As shown in Fig. 3, it
migrated as phosphopeptide 3 from Fig. 1. In addition, in the
tryptic digest that was depleted of the immunoprecipitated
phosphopeptide, spot 3 disappeared (Fig. 3). Identical results
were obtained from SCF-stimulated cells.
This, as well as
experiments in which the immunoprecipitated phosphopeptide was mixed
with total tryptic digests and analyzed by phosphopeptide
mapping,
clearly showed that phosphopeptide 3 corresponds
to the carboxyl-terminal Kit/SCFR tryptic peptide and is phosphorylated
at Ser-959 after SCF and PMA stimulation. In order to exclude the
possibility that the lack of phosphorylation of Ser-959 in the in
vitro phosphorylation experiments with PKC-
was due to steric
hindrance from the Kit-C1 antibody, we also performed these experiments
with affinity-purified Kit-KI antibody recognizing a sequence in the
kinase insert. Using this antibody, no phosphorylation of Kit/SCFR was
observed at all, despite the fact that the receptor was detectable by
immunoblotting.
This result shows that Ser-959 is not a
direct phosphorylation site for PKC-
in vitro.
Furthermore, the lack of detectable receptor phosphorylation is
consistent with the conclusion that the Ser-741 and Ser-746 in the
kinase insert of Kit/SCFR are the major phosphorylation sites for PKC
(see above), as these two sites are protected from phosphorylation by
the Kit-KI antibody.
Figure 3:
Identification of Ser-959 in the Kit/SCFR
as a PKC-mediated phosphorylation site. Kit/SCFR was immunoprecipitated
from cell-extracts of [P]orthophosphate-labeled
cells and digested in situ with trypsin as described in the
legend to Fig. 1A. The tryptic phosphopeptides were
immunoprecipitated using affinity-purified Kit-C1 antibodies against
the carboxyl terminus of Kit/SCFR, and the immunoprecipitated peptide,
as well as the peptide-depleted tryptic digest, was analyzed by
two-dimensional phosphopeptide mapping. The number indicates
the corresponding phosphopeptide 3 in Fig. 1A. The experiment
shown is from PMA-stimulated cells, but identical results were obtained
from SCF-stimulated cells (exposure at -70 °C for 40
h).
Sequencing of the serine- and
tyrosine-phosphorylated phosphopeptide 4 was difficult due to
co-migration with other phosphopeptides in the two-dimensional maps,
complicating its isolation. However, of several amino acid sequencings
of the phosphopeptide, reproducible release of radioactivity was
observed in cycles 3 and 5. Of the theoretical tryptic
peptides from Kit/SCFR intracellular domain with a serine residue at
position 3, only one has a tyrosine residue at position 5, while no
peptides have a tyrosine residue at position 3. This peptide
(K-NDSNY
. . . ) covers Tyr-823 which
corresponds to the tyrosine kinase domain autophosphorylation site
Tyr-857 in the structurally related platelet-derived growth factor
(PDGF) receptor-
. Furthermore, our results from sequencing of
phosphopeptides derived from in vitro autophosphorylated
Kit/SCFR had shown this site to be an autophosphorylation site.
It was therefore plausible that the PKC-dependent phosphopeptide
4 contained phosphorylated Ser-821, and this residue was therefore
included among those subjected to site-directed mutagenesis (see
below).
Analysis of PAE Cells Stably Transfected with Mutated
Kit/SCFR
In order to finally verify that the serine residues
741, 746, 821, and 959 are PKC-dependent phosphorylation sites, we
changed these residues in the Kit/SCFR to alanine residues singly, as
well as in combinations, by site-directed mutagenesis. The mutated
c-kit cDNAs were used to establish stably expressing
Kit/SCFR-mutant PAE cell lines. Serum-starved cells were labeled with
[S]methionine and
[
S]cysteine and stimulated with SCF for 7 min at
37 °C before cell lysis. Mutated Kit/SCFRs were then
immunoprecipitated from cell lysates, separated by SDS-gel
electrophoresis, and electrotransferred to polyvinylidene difluoride
membrane. After exposure on a PhosphorImager, filters were sequentially
immunoblotted first with anti-phosphotyrosine antibodies and then,
after stripping, with anti-Kit-C1 antibody. One cell clone of each
receptor-construct was chosen, and, in all cases, the mutated Kit/SCFRs
were found to be processed properly and to become
tyrosine-phosphorylated in response to stimulation with SCF
(Fig. 4). Scatchard analyses of several clones of each of the
different Kit/SCFR-mutants revealed ligand binding affinities between
0.13 and 0.68 nM, comparable to that of the wild type Kit/SCFR
expressed in PAE cells (Blume-Jensen et al., 1993).
Furthermore, all receptors were able to transduce specific
SCF-stimulated signals leading to biological responses, such as
mitogenicity and actin reorganization.
Figure 4:
Analysis
of PAE cell lines stably transfected with mutated Kit/SCFRs.
Serum-starved PAE/kit mutant cell lines were labeled with
[S]methionine and
[
S]cysteine, stimulated with SCF in the presence
of Na
VO
, and mutated Kit/SCFRs were
immunoprecipitated from cell extracts. After separation by SDS-gel
electrophoresis and electrotransfer to polyvinylidene difluoride
membrane, filters were exposed on film (72-h exposure time). The
different cell mutants are indicated in the top of the figure
(A). The filter was sequentially probed with affinity-purified
anti-phosphotyrosine antibodies (B), stripped, and reprobed
with affinity-purified Kit-C1 antibodies (C). In all
panels, the arrow indicates p145
.
Please note that the figure does not allow for comparison of specific
receptor tyrosine phosphorylation, as different ECL exposures are used
for the different receptor mutants in both B and
C.
To verify that
Ser-741 and Ser-746 are phosphorylation sites for PKC, and that Ser-959
and Ser-821 are PKC-dependent phosphorylation sites, we analyzed all
the stably expressing Kit/SCFR-mutants by two-dimensional
phosphopeptide mapping. Serum-starved receptor-mutant cells were
labeled with [P]orthophosphate, stimulated with
SCF or not; the cells were then lysed in nondenaturing lysis buffer;
and Kit/SCFR was immunoprecipitated. In parallel experiments, mutated
Kit/SCFRs were immunoprecipitated from unlabeled cells and treated with
N-ethylmaleimide before they were phosphorylated by PKC-
in vitro in the presence of
[
-
P]ATP. Both the in vivo and
in vitro phosphorylated receptors were then subjected to
two-dimensional tryptic phosphopeptide mapping as described under
``Materials and Methods.'' As shown in Fig. 5, the
S741A and S746A single mutations led to complete disappearence of
phosphopeptides 1 and 2, respectively, indicating that phosphorylation
on these serine residues are solely responsible for these two major
phosphopeptides. Furthermore, in the double mutant
Kit/SCFR(S741A/S746A) both phosphopeptides 1 and 2 were completely
absent from in vivo-phosphorylated cells, and the
Kit/SCFR(S741A/S746A) did not become phosphorylated at all by PKC
in vitro. The S959A and S821A single mutations led to the
disappearance of the tryptic phosphopeptides 3 and 4, respectively,
from both control and SCF-stimulated cells. Phosphopeptide 3 was of
varying intensity in the PAE/kitS821A mutant of unknown reasons. The
in vitro phosphorylation by PKC-
of each of these two
mutants was identical with that of the wild type receptor, in
accordance with the results obtained above, that Ser-741 and Ser-746
are the phosphorylation sites for PKC-
in vitro. Finally,
in the mutant where all 4 serine phosphorylation sites were replaced
with alanine residues, PAE/kit(S741A/S746A/S821A/S959A),
phosphopeptides 1, 2, 3, and 4 were absent in the in vivo maps, as well as both the major in vitro phosphorylated
peptides 1 and 2. These results thereby verified our data obtained from
the wild type Kit/SCFR-expressing PAE cells, that Ser-741 and Ser-746
in the kinase insert region of Kit/SCFR are the major in vivo and in vitro phosphorylation sites for PKC, while Ser-821
and Ser-959 are minor, PKC-dependent in vivo phosphorylation
sites (Fig. 6).
Figure 5:
Two-dimensional tryptic phosphopeptide
mapping of wild type and mutated Kit/SCFRs from stably expressing PAE
cell lines. Wild type as well as mutated Kit/SCFR were
immunoprecipitated from cell extracts of
[P]orthophosphate-labeled cells before and after
SCF stimulation or from unlabeled cells, before phosphorylation by PKC
in vitro in the presence of
[
-
P]ATP, as indicated. The receptors were
analyzed and processed for tryptic phosphopeptide mapping exactly as
described in the legends to Figs. 1 and 2. The left side of
the panels indicates the receptor analyzed in each row, and
the numbers in all the mutant phosphopeptide maps indicate the
missing phosphopeptide(s) as compared to the similarly treated wild
type Kit/SCFR. Films were exposed for variable lengths of time (from 24
h up to 168 h at -70 °C) to partially correct for different
levels of receptor expression.
Figure 6:
Schematic illustration of the relative
positions of the PKC-mediated serine phosphorylation sites in Kit/SCFR.
The figure illustrates the cytoplasmic part of the Kit/SCFR with the
two parts of the tyrosine kinase domain shown as boxes. The
solid arrowheads indicate the two serine residues in the
kinase insert, which are the major phosphorylation sites for PKC both
in vivo and in vitro. The open arrowheads indicate the PKC-dependent phosphorylation sites, which do not
become phosphorylated by PKC in
vitro.
Kit/SCFR(S741A/S746A) Exhibits Increased Kinase Activity
in Vivo and in Vitro
We earlier reported that inhibition of PKC
leads to an increased SCF-stimulated Kit/SCFR kinase activity reflected
as an increased tyrosine autophosphorylation in intact cells as well as
an increased kinase activity toward an exogenous peptide in vitro (Blume-Jensen et al., 1993). In order to examine if the
regulation of the receptor kinase activity by PKC can be ascribed to
the phosphorylation by PKC of the serine residues in the kinase insert,
we compared the kinase activity of the Kit/SCFR(S741A/S746A) with that
of the wild type Kit/SCFR. Cells were stimulated with SCF or left
untreated, lysed, and similar amounts of receptors were
immunoprecipitated from either wild type or mutant PAE/kit cells. After
separation by SDS-gel electrophoresis, proteins were electrotransferred
to polyvinylidene difluoride membrane, and the filters were probed
sequentially first with anti-Kit-C1 and then, after stripping, with
anti-phosphotyrosine antibodies. By densitometric scannings, the
specific receptor tyrosine phosphorylation was examined ().
The specific SCF-induced tyrosine phosphorylation of
Kit/SCFR(S741A/S746A) was 2.5-fold that of the wild type receptor. Even
the basal specific tyrosine phosphorylation of the mutated receptor was
increased almost 4-fold compared to the wild type receptor.
-
P]ATP. Wild
type and mutated receptors were immunoprecipitated, incubated with SCF
in vitro, and half of each of the samples were phosphorylated
by PKC-
in the presence of cold ATP. Aliquots of the samples were
then incubated for 2 min at 30 °C with the peptide RDIM(13) at
different concentrations in the presence of
[
-
P]ATP, and the samples were analyzed by
SDS-gel electrophoresis and quantification using a Fuji Image Analyzer.
The presence of equal receptor amounts in the phosphorylation reactions
was ensured by quantitative immunoblotting of samples processed in
parallel. From the resulting Lineweaver-Burk plots,
K
and V
of the
reactions were deduced. PKC phosphorylation of the wild type Kit/SCFR
prior to the kinase assay increased the K
of the reaction from 0.3 mM to 0.6 mM and
reduced the V
of the reaction, consistent with
our earlier findings ( and Blume-Jensen et al. (1993)). The K
of the mutated
receptor was 0.3 mM both before and after phosphorylation by
PKC, and the phosphorylation kinetics was largely unaffected by
phosphorylation by PKC (). Thus, substituting the two
serine residues in Kit/SCFR which are phosphorylated by PKC with
alanine residues led to an increased ligand-induced receptor kinase
activity.
Increased Specific Kit/SCFR(S741A/S746A)-associated
PI-3`-kinase Activity
We earlier showed that the SCF-stimulated
Kit/SCFR-associated PI-3`-kinase activity was significantly increased
after inhibition of PKC (Blume-Jensen et al., 1994). We
therefore further examined the PI-3`-kinase activity associated with
the Kit/SCFR(S741A/S746A) and compared it to that associated with the
wild type Kit/SCFR. Equal amounts of wild type and mutated receptors
were immunoprecipitated from lysates of unstimulated or SCF-stimulated
cells. The immunocomplexes were then incubated with
phosphatidylinositol in the presence of
[-
P]ATP, and phospholipids were extracted
and separated by ascending chromatography. The phosphorylated products
were quantified using a Fuji Image Analyzer, and the phosphorylation
relative to the receptor amount was estimated based on semiquantitative
immunoblottings of parallel samples. The SCF-stimulated specific
receptor-associated PI-3`-kinase activity was increased 2.2-fold in the
mutated compared to the wild type receptor (). Also the
basal receptor-associated PI-3`-kinase activity was increased in the
mutated compared to the wild type receptor, being 3-fold higher in the
mutated receptor. In conclusion, these data are consistent with our
earlier findings, indicating that the phosphorylation by PKC of serine
residues in Kit/SCFR negatively affects the receptor-associated
PI-3`-kinase activity.
in vitro. Substitution of the two direct
phosphorylation sites for PKC in Kit/SCFR, Ser-741 and Ser-746, with
alanine residues led to a 2-fold increase in SCF-stimulated specific
receptor tyrosine autophosphorylation and a 2.2-fold increased specific
receptor-associated PI-3`-kinase activity compared to that of the wild
type receptor. Furthermore, the Kit/SCFR exhibited a decreased
K
and an increased V
in an in vitro kinase assay toward an exogenous
substrate. These findings thus support the notion that the inhibition
mediated by PKC on Kit/SCFR signaling is, at least partially, due to
the phosphorylation of the two serine residues, Ser-741 and Ser-746, in
the kinase insert of Kit/SCFR.
associated with, and became tyrosine-phosphorylated by, the
ligand-stimulated Kit/SCFR (Hallek et al., 1992; Herbst et
al., 1991; Reith et al., 1991). In contrast, Lev et
al.(1991) found only negligible tyrosine phosphorylation of
phospholipase C-
and no increased inositol 1,4,5-trisphosphate
production in response to SCF in Kit/SCFR-transfected fibroblasts.
Accordingly, when tyrosine phosphorylation of phospholipase C-
in
PAE cells stably transfected with the Kit/SCFR was compared with that
observed in PAE cells stably transfected with the PDGF
-receptor,
SCF induced only marginal tyrosine phosphorylation of phospholipase
C-
, while PDGF-BB induced a marked tyrosine phosphorylation of
phospholipase C-
(Blume-Jensen et al., 1994). More recent
studies have clearly shown that phospholipase D, but not
phosphatidylinositide- or phosphatidylcholine-specific phospholipase C,
becomes activated in response to SCF and is the main pathway
responsible for production of diacylglycerol in rat peritoneal mast
cells and in PAE/kit cells (Koike et al., 1993).
(
)
The SCF-induced activation of phospholipase D is dependent
on Kit/SCFR tyrosine kinase activity and the action of phosphatidic
acid phosphohydrolase, as shown by the use of the inhibitors genistein
and propranolol, respectively. Thus, SCF-induced activation of
phospholipase D does not seem to be dependent on activation of
phospholipase C-
, in contrast to what was recently reported to be
the case for the PDGF-stimulated activation of phospholipase D in
canine kidney epithelial cells (Yeo et al., 1994). It cannot
be excluded, however, that some of the recently discovered,
nonclassical PKC isoforms become activated in other ways in response to
SCF. Thus, the ubiquitously expressed, Ca
- and
phorbol ester-insensitive PKC-
becomes activated directly by
phosphatidylinositol 3,4,5-trisphosphate, the product of PI-3`-kinase
(Nakanishi et al., 1993). In this study, we observed an
increased SCF-stimulated specific receptor-associated PI-3`-kinase
activity in PAE/kit(S741A/S746A) cells compared to that observed in
PAE/kit cells paralleling an increased kinase activity of the mutated
receptor (). This is in accordance with our earlier
observations of an increased SCF-stimulated Kit/SCFR association with
PI-3`-kinase activity after inhibition of PKC-
and -
in
PAE/kit cells, which parallels an increased SCF-stimulated mitogenicity
(Blume-Jensen et al., 1994, 1993). It is an interesting
possibility that the increased mitogenicity is partially mediated via
phosphatidylinositol 3,4,5-trisphosphate-activated PKC-
, which is
mitogenic for fibroblasts (Berra et al., 1993). This question,
as well as the mechanism for the increased receptor association with,
and activation of, PI-3`-kinase are the subject for future studies.
in vitro. Of other
serine/threonine kinases we have tested so far, neither purified MAPK
nor Raf-1 phosphorylates the Kit/SCFR in vitro.
(
)
Furthermore, cyclic AMP-dependent protein kinase does not
phosphorylate Kit/SCFR after forskolin and isobutyl methylxanthine
treatment of intact cells.
Most likely, Ser-959 and Ser-821
are phosphorylated by serine/threonine kinases that are downstream in
the signaling cascade from PKC, and/or their dephosphorylation is
prevented by PKC-mediated inhibition of a normally constitutively
active serine/threonine phosphatase.
Table:
Characterization of receptor kinase activity and
receptor-associated PI-3`-kinase activity of
Kit/SCFR(S741A/S746A)
-
P]ATP.
. Dr. Osamu Kozawa has
provided helpful comments on the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.