©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of the Major Phosphorylation Sites for Protein Kinase C in Kit/Stem Cell Factor Receptor in Vitro and in Intact Cells (*)

Peter Blume-Jensen (§) , Christer Wernstedt , Carl-Henrik Heldin , Lars Rönnstrand

From the (1) Ludwig Institute for Cancer Research, Uppsala Branch, Biomedical Center, S-751 24, Uppsala, Sweden

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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- 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.


INTRODUCTION

The receptor for stem cell factor (SCF)() 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).

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-, -, 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).

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.


MATERIALS AND METHODS

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 Sitesin 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- in Vitro

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 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.


RESULTS

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- in Vitro: Identification of Ser-741 and Ser-746 as the Major Phosphorylation Sites for PKC

We further examined the phosphorylation of Kit/SCFR by purified, recombinant 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 NaVO, 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.

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 [-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.


DISCUSSION

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- 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.

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- 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.

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- 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.

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.

  
Table: Characterization of receptor kinase activity and receptor-associated PI-3`-kinase activity of Kit/SCFR(S741A/S746A)



FOOTNOTES

*
This work was supported in part by a research fellowship from The Danish Cancer Society (to P. B.-J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed. Tel.: 46-18-174-291; Fax: 46-18-506-867.

The abbreviations used are: Kit/SCFR, Kit/stem cell factor receptor; PAE, porcine aortic endothelial; PDGF, platelet-derived growth factor; PI-3`-kinase, phosphatidylinositide 3`-kinase; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; SCF, stem cell factor; SH2, Src homology 2.

P. Blume-Jensen, unpublished results.

P. Blume-Jensen and L. Rönnstrand, unpublished results.

P. Blume-Jensen, L. Rönnstrand, and C. Wernstedt, unpublished results.

O. Kozawa, unpublished results.

P. Blume-Jensen, unpublished results. Raf-1 and MAPK (kindly provided by Dr. Silvia Stabel) were immobilized on protein A-Sepharose beads using specific antibodies. Kit/SCFR, specifically eluted from either immunoprecipitates or from glycoprotein-enriched fractions of PAE/kit cell lysates, was added, and kinase assays were performed in the presence of [-P]ATP.


ACKNOWLEDGEMENTS

Ulla Engström is gratefully acknowledged for expert production of synthetic peptides and Silvia Stabel is thanked for the generous gift of purified PKC-. Dr. Osamu Kozawa has provided helpful comments on the manuscript.


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