(Received for publication, October 9, 1996, and in revised form, February 4, 1997)
From the Departments of Microbiology and
§ Biochemistry, Michigan State University,
East Lansing, Michigan 48824-1101
Phosphorylation of CD45, a transmembrane protein-tyrosine phosphatase (PTPase), has been proposed to mediate docking of signaling proteins and to modulate PTPase activity. To study the role of phosphorylation in CD45, in vivo phosphorylation sites of CD45 from 70Z/3.12 cells were identified using 32P labeling, trypsin digestion, two-dimensional peptide mapping, high performance liquid chromatography, phosphoamino acid analysis, matrix-assisted laser desorption/ionization mass spectrometry, and specific enzymatic degradation. Eight phosphopeptides, a through h, were isolated and four phosphorylation sites were identified. All four phosphorylation sites were in the membrane-distal PTPase domain (D2) and the C-terminal tail and none were in the membrane-proximal PTPase domain (D1). One site, Ser(P)939 peptide h, was in the D2 domain and, by comparison to the three-dimensional structure of PTP1B, is predicted to lie at the apex of the substrate binding loop. Ser939 was the only in vitro phosphorylation site for protein kinase C among the phosphorylation sites identified. Four of the C-terminal peptides identified (d, e, f, and g) spanned the same sequence and were derived from the same phosphorylation site in the C-terminal tail, Ser1204. Peptide a was derived from the intact C terminus and comprised a mixture of monophosphorylated peptides containing either Ser(P)1248 or Thr(P)1246. Knowledge of the precise phosphorylation sites of CD45 will lead to the design of experiments to define the role of phosphorylation in PTPase activity and in signaling.
Protein-tyrosine phosphorylation and dephosphorylation play an important role in regulating cellular differentiation, proliferation, and activation. The role of dephosphorylation by CD45 protein-tyrosine phosphatase (PTPase)1 in lymphocyte signaling has been the subject of intense investigation (1-4). CD45 (T200, B220, L-CA) is a transmembrane PTPase of hematopoietic cells of 1268 total amino acids, with a cytoplasmic domain of 702 amino acids containing tandem repeated PTPase homology domains (designated D1 and D2). The membrane-proximal PTPase domain (D1) is considered to be constitutively active, and the second PTPase domain (D2) is usually considered to be inactive. CD45 has been intensely studied because of its well documented role in the antigen-specific activation of B and T cells (1-3). T cells lacking CD45 fail to respond to stimulation of the T cell antigen receptor (5, 6), and the catalytic activity of the CD45 D1 domain is required for T cell receptor activation (7). Chimeric proteins, in which the extracellular domain of CD45 was replaced, restored normal T cell receptor activation (8-10). Similarly, CD45-deficient B cells do not respond to stimulation of the IgM receptor (11). It is believed that CD45 activates the Src family PTKs by dephosphorylating the regulatory Tyr(P) near the C terminus of T cell receptor or B cell receptor-associated Src family kinases (12-15). Since CD45 is abundantly expressed on all nonerythroid hematopoietic cells, it has also been hypothesized that CD45 may be involved in the regulation of other fundamental cell processes such as cell growth and cell cycle (1, 16, 17).
Phosphorylation of the cytoplasmic domain of CD45 has been described, and this phosphorylation has been proposed to play a role in the regulation of biological function by providing docking sites (18) or by altering PTPase activity (18-20). The modulation of CD45 PTPase activity has been found to correlate with phosphorylation of the cytoplasmic domain in several studies. 1) Treatment of T cell clones with a Ca2+ ionophore decreased the Ser phosphorylation of CD45 and simultaneously decreased the PTPase activity of the molecule (20). 2) Phosphorylation of human CD45 (by overexpression of CD45 in Cos cells with p50csk PTK) increased the PTPase activity of CD45 and increased the association of CD45 with a putative substrate, p56lck (18). The p50csk PTK phosphorylation site was identified as Tyr1193. 3) The sequential in vitro phosphorylation of human CD45 by Abl PTK and casein kinase II increased the activity of CD45 for certain substrates (19). 4) The increase in CD45 phosphorylation due to phorbol ester treatment of cells was associated with a decrease in PTPase activity (21). Other reports indicate that the phosphorylation of CD45 increased after stimulation by phorbol esters (22) or interleukin-2 (23) without apparent change in CD45 activity.
The elucidation of the role of CD45 phosphorylation in PTPase activity and in signaling has been hampered by the lack of precise knowledge of naturally occurring CD45 phosphorylation sites. The large number of potential phosphorylation sites in the CD45 cytoplasmic domain make identification by sequencing or mutagenesis extremely difficult. To resolve this problem, we have identified the in vivo phosphorylation sites of CD45 in 70Z/3.12 cells (a mouse pre-B cell line) by two-dimensional phosphopeptide mapping, reverse-phase HPLC, phosphoamino acid analysis, enzymatic degradation, and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS).
The mouse pre-B lymphocyte cell line 70Z/3.12 was obtained from the American Type Culture Collection (ATCC) and grown at 37 °C in 90% RPMI 1640 medium (Life Technologies, Inc.) containing 10% heat inactivated fetal bovine serum (FBS) (Life Technologies), 25 mM HEPES, 50 µM 2-mercaptoethanol, 100 units/ml penicillin, 100 units/ml streptomycin (complete RPMI 1640 medium) in 5% CO2. Cells were counted using a Coulter Cell Counter (Coulter Electronics, Hialeah, FL) and maintained in an exponential growth state (0.1-4.0 × 105 cells/ml). Cultures were harvested at 8 × 105 cells/ml with viability in excess of 97%.
32P Labeling70Z/3.12 cells (1 × 107) were washed three times with phosphate-free RPMI 1640 medium (supplemented to 10% with dialyzed FBS; Life Technologies), then resuspended in 1 ml of phosphate-free RPMI 1640 medium, followed by incubation for 1 h at 37 °C. After centrifugation, cells were resuspended in 1 ml of fresh, phosphate-free RPMI 1640 medium. One mCi of [32P]orthophosphate (DuPont NEN Research, Boston, MA) was added and cells were incubated for 4 h at 37 °C.
Immunoprecipitation of CD4570Z/3.12 cells were pelleted, washed twice with ice-cold phosphate-buffered saline (8 mM Na2HPO4, 1 mM KH2PO4, 137 mM NaCl, 3 mM KCl, pH 7.4), and then lysed (2 × 107 cells/ml lysis buffer) in Nonidet P-40 lysis buffer containing 20 mM Tris, pH 8.0, 137 mM NaCl, 1.0% (v/v) Nonidet P-40 (Pierce), 2 mM phenylmethylsulfonyl fluoride, 0.3 µM aprotinin, 1 µM leupeptin, 5 mM EDTA, 1 µM pepstatin A, 50 µg/ml DNase I and RNase A, 1 µM okadaic acid (Life Technologies, Inc.), and 6 mM NaF for 30 min in ice. Lysates were cleared by centrifugation at 12,000 rpm at 4 °C for 20 min and precleared with 25 µl of packed Gammabind Plus Sepharose (Pharmacia Biotech Inc.). Anti-CD45 M1/9.3.4 monoclonal antibody (2 µg, ATCC) was conjugated to 25 µl of Gammabind Plus Sepharose for 1 h at 4 °C in phosphate-buffered saline (pH 7.4), followed by incubation with 0.5 ml of cell lysate (from 107 cells) for 1 h at 4 °C under constant shaking. For SDS-PAGE, immunoprecipitates were washed once with Nonidet P-40 lysis buffer, twice with phosphate-buffered saline (pH 7.4, containing 1 µM okadaic acid, 6 mM NaF), once with 0.5 M LiCl (pH 7.4, containing 1 µM okadaic acid, 6 mM NaF), and twice with 50 mM Tris buffer (pH 8.0, containing 1 µM okadaic acid, 6 mM NaF). Immunoprecipitates were suspended in SDS-PAGE sample buffer and boiled for 3 min.
SDS-PAGE and Western TransferCD45 immunoprecipitate samples were subjected to electrophoresis on 4-15% SDS-PAGE gels for 1.5 h at 120 V. Silver staining was performed to determine the amount of CD45 proteins. CD45 bands were quantitated by optical scanning using an Ambis scanner (Ambis, Inc., San Diego, CA). For 32P-labeled samples, CD45 proteins were transferred to polyvinylidene difluoride (PVDF) membrane (0.2 µm) (Bio-Rad) by electroblotting in 25 mM Tris, 192 mM glycine, 20% (v/v) methanol, pH 8.3, for 4 h at 400 mA. CD45 bands were visualized by autoradiography.
Trypsin Digestion and Two-dimensional Phosphopeptide Mapping32P-Labeled CD45 bands from the PVDF membrane were excised, washed three times with methanol, blocked in 1 ml of 0.5% PVP-360 (Sigma) in 100 mM acetic acid for 30 min at 37 °C, and washed five times with water. Tryptic digestion was performed with 10 µg of trypsin (sequencing grade) (Promega, Madison, WI) for 2 h in 200 µl of 50 mM NH4HCO3, pH 8.3, at 37 °C, and an additional 10 µg of trypsin was added and incubated for another 2 h at 37 °C. Tryptic peptides were recovered in the supernatant and concentrated in a SpeedVac (Savant Instruments, Inc., Farmingdale, NY). The samples were separated by thin layer electrophoresis (TLE) on 20 × 20-cm Kodak cellulose thin layer chromatography (TLC) plates (Eastman Kodak Co.) in the first dimension in pH 1.9 buffer (formic acid (88% w/v)/glacial acetic acid/water, 25:78:897, v/v/v) at 0 °C at 1000 V for 30 min, dried in air, and separated in the second dimension in chromatography buffer (n-butanol/pyridine/glacial acetic acid/water, 15:10:3:12, v/v/v/v) (24, 25). The phosphopeptides were visualized by using a Betascope 603 blot analyzer (Betagen, Waltham, MA).
Phosphoamino Acid Analysis32P-Labeled tryptic phosphopeptides eluted from TLC plates or collected from reverse-phase HPLC were hydrolyzed in 100 µl of 5.7 N HCl for 1 h at 100 °C. The recovered phosphoamino acids were dried in a SpeedVac. Unlabeled phosphoamino acids were added to each sample as standard markers. The samples were analyzed by one-dimensional electrophoresis in pH 2.5 buffer (66.7% pH 3.5 buffer (glacial acetic acid/pyridine/water, 50:5:945, v/v/v, containing 0.5 mM EDTA) and 33.3% pH 1.9 buffer (glacial acetic acid/88% formic acid/water, 78:25:897, v/v/v)) on 20 × 20-cm cellulose TLC plates (Kodak) for 1 h at 0 °C and 500 V. The markers were visualized by ninhydrin and the 32P-labeled phosphoamino acids were visualized by Betascope (Betagen) analysis.
Reverse-phase HPLC Fractionation70Z/3.12 cells (5 × 109 at 8 × 105 cells/ml) were collected, lysed, and immunoprecipitated with anti-CD45 M1/9.3.4 monoclonal antibody to yield approximately 40 µg of CD45 protein. This large scale, unlabeled CD45 immunoprecipitate was then mixed with a smaller amount of a radioactive marker consisting of 32P-labeled CD45 immunoprecipitate from cells grown at the same density as described above. After SDS-PAGE, Western transfer, and tryptic digestion, the digested mixture was fractionated using a microbore reverse-phase HPLC system (Michrom BioResources, Inc.) with a 0-86% gradient of acetonitrile in 0.1% aqueous trifluoroacetic acid and a flow rate of 50 µl/min on a Reliasil C18 column (5 mm, 300 A, 1.0 × 150 mm, Michrom BioResources). The solvent gradient started at 0% solvent B, then increased to 20% solvent B at 15 min, 60% solvent B at 20 min, and 95% solvent B at 21 min, where solvent A was 0.1% trifluoroacetic acid in water and solvent B was a mixture of acetonitrile/0.1% trifluoroacetic acid in water (9:1). The eluant was monitored by a UV absorbance at 214 nm, and fractions were collected every 30 s. A 1-µl sample of each HPLC fraction was spotted on a TLC plate, followed by detection of 32P radioactivity by Betascope (Betagen) analysis.
Mass SpectrometryAll MALDI-MS spectra were obtained on a
Voyager Elite time-of-flight mass spectrometer (PerSeptive Biosystems,
Framingham, MA), used in linear mode, equipped with a nitrogen laser
(337 nm, 3-ns pulse). The accelerating voltage in the ion source was 26 kV. Data were acquired with a transient recorder with 2-ns resolution.
The matrix used in this work was -cyano-4-hydroxycinnamic acid,
dissolved in water/acetonitrile (1:1, v/v) to give a saturated solution
at room temperature. To prepare the sample for analysis, 1 µl of the
peptide solution (1-10 pmol/µl 0.1% trifluoroacetic acid) was added
to 1 µl of the matrix solution and applied to a stainless steel
sample plate. The mixture was then allowed to air dry on the sample
plate before being introduced into the mass spectrometer. Each spectrum
was produced by accumulating data from 50-256 laser pulses.
Time-to-mass conversion was achieved by external or internal
calibration using bradykinin (MH+ at m/z 1061.2)
and insulin (MH+ at m/z 5734.6). The accuracy of
mass assignments was approximately ± 0.1% (± 1 Da/1000 Da). A
computer program, MSU MassMap (26) was used to calculate the average
masses of all possible peptide and phosphopeptide fragments from CD45,
and the m/z value of the mass spectral peak for the
corresponding MH+ ion. Dephosphorylation of 5 µl of the
HPLC fractions containing phosphopeptide was achieved by incubation of
the peptide with 1~2 units of calf intestine alkaline phosphatase in
50 mM NH4HCO3 buffer (pH 8.0) at
37 °C for 4 h.
Digestion of TLC isolated peptide a with hydroxylamine was performed in 2 M guanidine·HCl, 2 M NH2OH·HCl, 0.2 M K2CO3 (Sigma), pH 9, for 4 h at 45 °C (24). After NH2OH digestion the peptide was purified by HPLC using conditions described above to remove salts (yielding a single radioactive peak at 21 min, retention time) and was analyzed by one-dimensional TLC using the conditions described above. The NH2OH-digested peptide was then digested with 1 µg of endoproteinase Glu-C (Boehringer Mannheim) in 50 mM NH4HCO3, pH 7.8, 18 h at 25 °C. The NH2OH- and endoproteinase Glu-C-digested peptide a was then analyzed by one-dimensional TLC as described above. Radioactivity was detected using a Molecular Dynamics PhosphorImager.
In Vitro Phosphorylation of CD45 by Protein Kinase C (PKC)Purified rat brain PKC was provided by Dr. A. Nairn
(Rockefeller University, New York, NY), and the purified, bacterially expressed cytoplasmic domain of murine CD45 was provided by Dr. P. Johnson (University of British Columbia, Canada) (27). The phosphorylation of 1 µg of cytoplasmic CD45 was carried out with 1 µg of PKC at 30 °C for 30 min in 20 mM Tris·HCl (pH
7.4), 5 mM MgCl2, 1 mM
dithiothreitol, 50 µg/ml phosphatidylserine, 100 nM PMA,
2 mM CaCl2, and 0.01 mCi of
[-32P]ATP (3000 Ci/mmol, DuPont NEN). The reaction was
terminated by the addition of SDS-PAGE sample buffer. The sample was
then boiled and resolved on a 4-15% SDS-PAGE gradient gel and
transferred to a PVDF membrane, followed by autoradiography. The CD45
band was excised, digested with trypsin, and analyzed as described above.
In an effort to map the
in vivo phosphorylation sites of CD45, we first prepared a
two-dimensional map of tryptic phosphopeptides from 70Z/3.12 cells
cultured at high density (8 × 105 cells/ml). Cells
were labeled with [32P]orthophosphate for 4 h and
immunoprecipitated CD45 was separated by SDS-PAGE and transferred to
PVDF for autoradiography (Fig. 1A). The CD45
band was excised, digested with trypsin, and subjected to
two-dimensional phosphopeptide mapping (Fig. 1B). Tryptic
phosphopeptides were designated a-h (Fig. 1B).
Phosphoamino acid analysis of each of the radioactive peptides eluted
from the TLC plate (Fig. 2) revealed that each contained
only Ser phosphorylation, except for phosphopeptide a, which
contained both Ser and Thr phosphorylation in about a 1:1 ratio (Fig.
2, lane 2). No tyrosine phosphorylation of CD45 was
detected. Betascope and phosphorimage analysis was used in this work,
because it was quantitative and it allowed the detection of very low
levels of radioactivity.
Separation of Tryptic Peptides of CD45 by Reverse-phase HPLC
Reverse-phase HPLC was performed to isolate larger
quantities of the tryptic phosphopeptides for detailed analysis of the phosphorylation sites of CD45. Approximately 40 µg of CD45 (about 200 pmol) was purified from 5 × 109 70Z/3.12 cells
(8 × 105 cells/ml) by immunoprecipitation with
M1/9.3.4 monoclonal antibody. A radioactive tracer was prepared by
immunoprecipitating CD45 from 4 × 107 70Z/3.12 cells
(8 × 105 cells/ml) labeled with 4 mCi of
32Pi for 4 h, and added to the
nonradioactive preparation. After tryptic digestion, the CD45 peptide
mixture was subjected to fractionation by HPLC. Fractions were
collected every 30 s and numbered according to HPLC retention time
(Fig. 3A). The radioactive fractions were identified by application of 1 µl of each fraction to a TLC plate, followed by Betascope detection (Fig. 3B). Fractions at the
following times were found to be radioactive and are hereafter
designated by their retention time: 4, 18, 18.5, 20.5, 21, 21.5, 22, and 23.
Each radioactive HPLC fraction was aligned with each phosphopeptide
from two-dimensional phosphopeptide map by subjecting each HPLC
fraction to one-dimensional TLE and one-dimensional TLC (Fig.
4, A and B). The unique mobility
in one dimension of each spot allowed us to correlate the HPLC fraction
with the two-dimensional phosphopeptide map. TLE analysis indicated
that each HPLC fraction contained primarily one radioactive peptide,
except for fraction 4, which contained two peptides (designated peptide
b/c on the two-dimensional phosphopeptide map). TLC analysis
indicated that each HPLC fraction contained primarily one radioactive
peptide, except for fraction 21.5 which was separated into two
phosphopeptides on TLC (designated peptide e,f). The TLE and
TLC results matched the HPLC results in which both fraction 4 and 21.5 eluted as doublets. The HPLC chromatographic pattern was consistent
with TLE (each consecutive fraction (except part of 4 and 18.5)
generally became less charged; Fig. 4A) and TLC (each
consecutive fraction became more hydrophobic; Fig. 4B). The
results from one dimensional analysis allowed the correlation of each
radioactive HPLC peptide fraction with each two-dimensional
phosphopeptide spot (peptide designation shown at the bottom of Fig.
4). The analysis of fractions 18 and 20.5 is not shown since these were
found to be the same as peptides 18.5 and 21, respectively.
Phosphoamino acid analysis of each radioactive HPLC fraction indicated
the presence of Ser(P), except for fraction 18.5, which also contained
Thr(P) (data not shown), thus confirming that this fraction was the
two-dimensional phosphopeptide spot a.
MALDI-MS Identification of Peptides
MALDI-MS analysis was
performed on each radioactive HPLC fraction to determine the identity
of the CD45 phosphopeptides. The observed mass values obtained for each
radioactive HPLC fraction were compared with a table of predicted
masses of all possible tryptic peptides of CD45 cytoplasmic domain
below 3000 Da (including partially digested, unphosphorylated, and
phosphorylated peptides). Each sample was then treated with alkaline
phosphatase and subjected to re-analysis by MALDI-MS to observe loss of
phosphate (80 Da or multiples of
80 Da shifts in the spectra). The
results of MALDI-MS analysis for each radioactive HPLC fraction are
described below.
The MALDI-MS spectrum of
HPLC fraction 18.5 contained a single peak at m/z 2960.2 (Fig. 5A). Comparison with a table of
calculated mass values of all possible tryptic peptides of CD45
cytoplasmic domain (724 possible peptides) indicated a close match to
the predicted peak at 2958.9, corresponding to the monophosphorylated peptide 1239-1268 (Tables I and II).
Dephosphorylation of the sample followed by MALDI-MS clearly showed a
loss of only one phosphate (80 Da) to m/z 2880.0. Because
phosphoamino acid analysis showed peptide a was
phosphorylated equally on Ser and Thr, it must therefore consist of a
mixture of phosphopeptides with the same sequence: one with a single
Ser(P) and one with a single Thr(P). The Ser(P) and Thr(P) sites were
localized using the following strategy. Digestion of peptide
a with NH2OH at Asn-Gly resulted in a single
32P-phosphopeptide, which eluted at 21 min from HPLC,
compared with undigested peptide a, which eluted at 18.5 min
(data not shown). This HPLC-purified peptide contained both Ser(P) and
Thr(P) (data not shown) and had a slightly slower mobility by TLC than peptide a (Fig. 5B, lanes 2 and
3). (HPLC purification was required to remove salts before
TLC or enzymatic digestion.). Thorough digestion of the HPLC purified
radioactive peptide with endoproteinase Glu-C resulted in only one
32P-phosphopeptide with altered TLC mobility (Fig.
5B, lane 4), which contained both Ser(P) and
Thr(P) (data not shown). Thus we conclude that the phosphorylation
sites of peptide a were Thr1246 and
Ser1248. This conclusion is based on the observation that
only the N-terminal portion of peptide a, after digestion at
Asn-Gly, contains Glu. Furthermore, extensive digestion of this peptide
with endoproteinase Glu-C results in only one product with a single Ser
and a single Thr.
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Fraction 4 contained two extremely hydrophilic Ser(P)-containing phosphopeptides, b and c, which were found in the non-retained fraction from the HPLC column. Mass spectral signals for this fraction were not obtained. Their signals were probably suppressed by co-eluting salt contaminants, and efforts to remove the peptides from these contaminants were not successful.
Peptide d, Fraction 21MALDI-MS analysis of
fraction 21, peptide d (Fig. 6A),
exhibited one phosphorylated peak and one non-phosphorylated CD45
peptide. The mass spectral peak at m/z 2127.4 shifted to m/z 2048.3 (80 Da) after treatment with alkaline
phosphatase (Table I). Ser1204 was identified as the
phosphorylation site, since there is only one Ser in this peptide and
Thr(P) was not detected by phosphoamino acid analysis. The other major
peak at m/z 1548.0 did not move after phosphatase treatment
and was identified as an unphosphorylated CD45 peptide (573-585) from
the membrane-proximal region of the cytoplasmic domain.
Peptide e/f, Fraction 21.5
MALDI-MS analysis of
fraction 21.5 showed three major peaks that correspond to CD45 peptides
in the fraction (Fig. 6B). One at m/z 1548.0 represented the same unphosphorylated fragment identified in the
previous fraction. The other two peaks, m/z 1886.2 and 1999.6, were found to shift by 80 Da after dephosphorylation: m/z 1999.6 to m/z 1921.6 and m/z
1886.2 to m/z 1807.5 (Fig. 6B). Two
phosphopeptides were also found in this fraction by one-dimensional TLC
(Fig. 4B). The peak at m/z 1999.6 was compared
with the calculated mass values and identified as peptide e
with a calculated m/z of 1999.0 (Table I). The only possible
phosphorylation site, Ser1204, was the same phosphorylation
site of peptide d, and the difference between phosphopeptide
d and e is one N-terminal Lys.
The observed mass of peptide f, m/z 1886.2 (Fig. 6B), did not match any tryptic phosphopeptide of CD45 but instead matched a product of peptide d involving loss of the C-terminal GGK from peptide d (loss of 242.4 Da (calculated)). Instability of the Asp-X (D-X) peptide bond at low pH is well known and likely resulted in backbone cleavage of the D-G bond in our peptides (28-31), and the procedure used involved dissolving the peptides in pH 1.9 buffer before electrophoresis or HPLC. Peptide f was phosphorylated since 80 Da was lost after phosphatase treatment. Peptide f, resulting from loss of GGK, was found in the appropriate position in HPLC (more hydrophobic), TLC (more hydrophobic) and TLE (more negatively charged) compared with peptide d. The TLC and TLE positions of peptides e and f were distinguished because a portion of peptide e was found in the preceding HPLC fraction 21 (Fig. 4B), which resulted in a weak signal of m/z 1999.6 in the mass spectra of fraction 21. Despite the fact that these peptides resulted from chemical cleavage, the peptide maps were remarkably reproducible.
Peptide g, Fraction 22The MALDI-MS spectrum of
fraction 22 displayed a number of signals. Only one peptide,
m/z 1757.4 (Fig. 6C, Table I), shifted by 80 Da
to m/z 1677.1 after phosphatase treatment. No putative tryptic phosphopeptide has a m/z value close to 1757.4, but
this number matched that expected from the loss of GGK from peptide e. Thus the four peptides, d, e,
f, and g, were all derived from the same sequence
region and therefore represent a single phosphorylation site,
Ser1204.
MALDI-mass spectrometry of
HPLC fraction 23, peptide h, showed two mass spectral peaks
at m/z 1325.6 and m/z 1676.1 (Fig.
7, Table I). After dephosphorylation with alkaline
phosphatase, the peak at m/z 1325.6 remained unchanged
(m/z 1324.4) and the peak at m/z 1676.1 shifted
to m/z 1595.4 (-80 Da, loss of one phosphate). This mass
matched a tryptic phosphopeptide with a calculated mass of 1675.7 designated h (Table I). Since there is only one Ser residue
in this peptide, the phosphorylation site is Ser939. This
phosphopeptide includes one site (Arg937) that is not
cleaved by trypsin when the sequence -RXpS- is present (24).
The second peak in this fraction, at m/z 1325.6, matched unphosphorylated form of the same peptide with a slightly shorter N
terminus. Thus the unphosphorylated and phosphorylated
Ser939 peptides were present in the same mass spectrum.
In Vitro PKC Phosphorylation of CD45
Since CD45
phosphorylation increases after PMA treatment of cells (21, 22), we
wished to determine if any of the sites that we identified could serve
as substrates for PKC phosphorylation. The phosphorylation sites at
Ser939 (h) and Ser1204
(d-g) conform to the consensus sequence described for PKC
(see "Discussion"). Activation of 70Z/3.12 cells with PMA resulted
in increased phosphorylation on both Ser939 and
Ser1204 (data not shown), but it is difficult to conclude
that this phosphorylation was a direct result of PKC activity. To
resolve this question, bacterially expressed, purified murine CD45
cytoplasmic domain was phosphorylated by a purified preparation of rat
brain PKC (Fig. 8). Phosphorylated CD45 was gel-purified
to separate it from the lightly autophosphorylated PKC present after
the in vitro kinase reaction (Fig. 8A). Tryptic
phosphopeptide mapping of CD45 indicated that there were several CD45
peptides phosphorylated. However, only one peptide matched the mobility
of an in vivo phosphorylated peptide, i.e.
peptide h. That peptide h is an in vitro target of PKC was confirmed by mixing the eluted PKC peptide labeled h (from Fig. 8B) with in vivo
labeled CD45 peptides (Fig. 8D). PKC-labeled peptide
h contained only Ser(P) (data not shown), and its position
was precisely coincident with in vivo labeled peptide
h.
The identification of CD45 in vivo phosphorylation
sites is essential to determine which of the many possible sites may be functionally relevant to the modulation of activity and to the docking
of CD45 associated proteins. The current study was designed to
characterize the in vivo phosphorylation sites of CD45. Four CD45 in vivo phosphorylation sites were identified using a
combination of two-dimensional phosphopeptide mapping, reverse-phase
HPLC, phosphoamino acid analysis, MALDI-MS, and specific enzymatic
degradation. The method used is advantageous because phosphopeptides
can be detected in the picomole range, because phosphorylation can be confirmed by observation of a loss of 80 Da after dephosphorylation, and because the target phosphopeptide does not need to be absolutely pure (26). The major type of phosphorylated amino acid observed in CD45
was Ser(P) with a minor amount of Thr(P) in peptide a. This
is the first time that Thr(P) has been observed in CD45. Three
phosphorylation sites were identified in the C-terminal tail,
Ser1204, Thr1246, and Ser1248
(summarized in Fig. 9) and one phosphorylation site,
Ser939, was in the D2 domain (peptide h). No
phosphorylation sites were observed in the D1 domain or in the
connector between PTPase domains.
The phosphorylation site at Ser939 (peptide h)
is conserved in mouse, rat, and human CD45; instead of
Ser939, chicken and shark CD45 have a Ser in an adjacent
residue. In addition, the sequence around the Ser939 is
highly conserved among PTPases. The three-dimensional structure of the
phosphatases has been found to be remarkably conserved (32), and
comparison of CD45 and PTP1B predicts that Ser939 is
located at the apex of the substrate binding loop at a position homologous to Arg47 in PTP1B (33). This suggests that
phosphorylation of Ser939 may be important in the
regulation of binding of substrates in the D2 domain or in the binding
of signaling proteins which may interact with the D2 active site.
Ser939 also falls in the homologous interaction region
resulting in dimer formation of RPTPD1, which blocks the active site
(34). Potential dimerization of CD45 (D1-D1 or D1-D2) may modulate
activity since the PTPase activity of a CD45-epidermal growth factor
receptor chimeric molecule was functionally inactivated upon induced
dimerization with epidermal growth factor (9).
Peptide h is also of interest because it contains the target for Abl kinase, which is proposed to modulate the PTPase activity of CD45-D2 (19). Phosphotyrosine was not detected in our in vivo study, even when the PTPase inhibitor (phenylarsine oxide) was included in the isolation. It is possible that CD45 tyrosine phosphorylation occurs transiently or at very low stoichiometry. Peptide h also overlaps with a fodrin-binding site (E930ENKKKNRNS939) of CD45, and fodrin binding increases the PTPase activity of CD45 (35, 36). Phosphorylation at Ser939 may serve to regulate the binding of CD45 to the cytoskeleton. Other studies have suggested that CD45 interacts with the cytoskeleton and has a role in the coordination of cytoskeletal remodeling (37, 38).
In the current report, peptide h (Ser939) was not the major phosphorylated peptide observed. However, it is unlikely that phosphorylation reached equilibration during the labeling since CD45 is expressed at high levels and the turnover of CD45 protein is very slow (39). Thus, the phosphorylation of CD45 in our study probably resulted primarily from turnover of phosphate at individual phosphorylation sites. It is difficult to reach conclusions about stoichiometry from our 32P incorporation experiments, since, for example, a very stable phosphorylation site would only become weakly phosphorylated. In the current study the stoichiometry of phosphorylation at Ser939 was more directly estimated by comparison of the MALDI-MS signals for peptide h and an almost identical non-phosphorylated peptide containing Ser939 in the same HPLC fraction (Table I and Fig. 7). Assuming comparable trypsin cleavage, the intensity of the MALDI-MS signal of the dephosphorylated form of peptide h was of the same magnitude as the equivalent non-phosphorylated peptide in the same mass spectrum, suggesting equal abundance.
The phosphorylation site at Ser1204 is conserved in mouse and rat but not in human, chicken, or shark CD45. The other sites in the C-terminal tail, Thr1246 and Ser1248, are not precisely conserved among these species, but this region (within 1-2 residues) contains multiple Ser and/or Thr residues suggesting that this may also be a phosphorylation site common to all species. The MALDI-MS signals of peptides b and c (HPLC fraction 4) were not observed because of the presence of salts in this fraction. It is possible that phosphopeptides in this fraction are homologous to a peptide obtained from a tryptic digest of in vivo 32P-labeled, human Jurkat cell CD45 (40). This sequence, which is highly conserved in mouse CD45, contains casein kinase II phosphorylation sites and in vitro phosphorylation of the cytoplasmic domain of CD45 by casein kinase II revealed one major phosphopeptide which migrated very close to peptides b and c in two-dimensional mapping (data not shown).
The phosphorylation sites Ser939 and Ser1204
conform to the consensus sequence described for PKC (41). We have
confirmed that, of these potential in vivo phosphorylation
sites, only Ser939 is a target for in vitro PKC
phosphorylation. Several other peptides were also labeled in the
in vitro PKC reaction, which did not appear in in
vivo labeled CD45. Increased phosphorylation at Ser939
and Ser1204 was observed after PMA treatment of 70Z/3.12
cells (data not shown), and other studies have indicated that CD45 was
phosphorylated after PMA treatment (21, 22, 42). It has been reported
that other PTPases such as PTP1B, RPTP, and PTP-PEST are also
substrates of PKC (43-45), and in vitro PKC phosphorylation
of PTP-PEST resulted in decreased PTPase activity (45).
Our results suggest that Ser939 may be an important phosphorylation site, which is phosphorylated in vivo by PKC. This phosphorylation may play a role in the regulation of D2 domain activity, in potential dimerization involving the D2 domain, or in the interactions of other molecules with the active site of the D2 domain. The C-terminal phosphorylation sites may also have a regulatory role in CD45 activity. Direct confirmation of the role of these phosphorylation sites in the function of CD45 is currently under investigation by site-directed mutagenesis.
We are grateful for the generous contributions of Dr. A. Nairn, who provided purified PKC, and Dr. P. Johnson, who provided purified cytoplasmic domain of CD45. The MSU Mass Spectrometry Facility is supported in part by Grant RR00480 from the National Center for Research Resources (NCRR) of the National Institutes of Health.