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CD45 Negatively Regulates Lyn Activity by
Dephosphorylating Both Positive and
Negative Regulatory Tyrosine Residues in
Immature B Cells
Tatsuo Katagiri, Mami Ogimoto, Kiminori Hasegawa,
Yutaka Arimura, Katsuyuki Mitomo, Masato Okada, Marcus
R. Clark, Kazuya Mizuno and Hidetaka Yakura
J Immunol 1999; 163:1321-1326; ;
http://www.jimmunol.org/content/163/3/1321
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Copyright © 1999 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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References
CD45 Negatively Regulates Lyn Activity by Dephosphorylating
Both Positive and Negative Regulatory Tyrosine Residues in
Immature B Cells1
Tatsuo Katagiri,* Mami Ogimoto,* Kiminori Hasegawa,2* Yutaka Arimura,3*
Katsuyuki Mitomo,* Masato Okada,† Marcus R. Clark,‡ Kazuya Mizuno,* and
Hidetaka Yakura4*
E
ngagement of B cell Ag receptor (BCR)5 by anti-IgM Ab
or multivalent Ags induces a rapid increase in tyrosine
phosphorylation of a number of cellular proteins (1, 2).
Signals are propagated downstream and ultimately control gene
activation leading to activation, cell death, or anergy in B cells,
depending on the differentiation stage, the nature of Ags, and the
presence or absence of costimulation (3, 4). Regardless of the context in which stimulation occurs and the final outcome, BCR signaling is dependent on the proximal activation and recruitment of
protein tyrosine kinases (PTKs): Src-family PTKs (Lyn, Blk, Lck,
and Fyn); and Syk (5–7).
Extensive analyses have demonstrated that one of the crucial
enhancers of BCR signaling is the receptor-type protein tyrosine
*Department of Microbiology and Immunology, Tokyo Metropolitan Institute for
Neuroscience, Tokyo, Japan; †Institute for Protein Research, Osaka University, Suita,
Japan; and ‡University of Chicago School of Medicine, Chicago, IL 60637
Received for publication August 17, 1998. Accepted for publication May 24, 1999.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported in part by grants-in-aid for Scientific Research and for
International Scientific Research from the Ministry of Education, Science, Sports and
Culture.
2
Current address: Howard Hughes Medical Institute, Department of Pathology,
Washington University School of Medicine, St. Louis, MO 63110.
3
Current address: Department of Microbiology and Immunology, Tokyo Women’s
Medical University, School of Medicine, Tokyo 162-8666, Japan.
4
Address correspondence and reprint requests to Dr. Hidetaka Yakura, TMIN, 2-6
Musashidai, Fuchu, Tokyo 183-8526, Japan. E-mail address: [email protected]
5
Abbreviations used in this paper: BCR, B cell Ag receptor; PTK, protein tyrosine
kinase; PY, anti-phosphotyrosine; AP, alkaline phosphatase; PVDF, polyvinylidine
difluoride.
Copyright © 1999 by The American Association of Immunologists
phosphatase, CD45 (8 –12). CD45 has been implicated in both T
and B cell activation based on experiments using CD45-deficient
cells (13–17) and cells from CD45 gene-targeted mice (18, 19). It
has been shown that CD45 in T cells dephosphorylates Lck and
Fyn at the COOH-terminal negative regulatory sites, Tyr505 (20 –
24) and Tyr531 (25–27), respectively. From these findings, a view
has emerged that the binding of the phosphorylated COOH-terminal tyrosine residue of a Src-family PTK to its own SH2 domain
causes the PTK to assume an inactive conformation. CD45 activates the kinase by dephosphorylating the COOH-terminal tyrosine, thereby releasing the inhibitory intramolecular conformation. However, there are also discrepant results in which Lck and
Fyn are hyperphosphorylated and activated in CD45-deficient
YAC-1 T cell clones. In these experiments, the sites of phosphorylation include both the negative COOH-terminal tyrosine (Tyr505)
and the positive autophosphorylation site (Tyr394) of Lck (28, 29).
We have previously demonstrated that in CD45-deficient clones
generated from immature WEHI-231 cells, Lyn is selectively hyperphosphorylated and activated in the absence of BCR ligation.
Furthermore, receptor stimulation did not significantly enhance
phosphorylation and activation of Lyn (30). BCR-induced Ca21
mobilization, growth arrest, and apoptosis were also negatively
regulated by CD45 in WEHI-231 cells (16). In contrast, CD45
exerted positive, crucial effects on BCR-induced growth arrest and,
to a lesser extent, BCR-induced tyrosine phosphorylation in mature BAL-17 B cells (17). Recent studies on two different B cell
lines revealed somewhat different mode of action of CD45. In
CD45 gene-targeted clones from the chicken DT40 B cell line, Lyn
was shown to be hyperphosphorylated on BCR ligation at the
COOH-terminal negative regulatory residue as well as the positive
regulatory tyrosine. However, the kinase activity of Lyn was
0022-1767/99/$02.00
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Using CD45-deficient clones from the immature B cell line, WEHI-231, we previously demonstrated that CD45 selectively dephosphorylates the Src-family protein tyrosine kinase Lyn and inhibits its kinase activity. To further define the mechanisms of
CD45 action on Lyn, we metabolically labeled Lyn from CD45-positive and -negative WEHI-231 cells and analyzed cyanogen
bromide fragments by SDS-PAGE analysis. Phosphoamino acid analysis confirmed that Lyn is tyrosine phosphorylated with little
serine or threonine phosphorylation. In CD45-negative cells, two bands at 8.2 and 4.1 kDa were phosphorylated in the absence of
B cell Ag receptor (BCR) ligation. The 8.2-kDa band corresponded to a fragment containing the positive regulatory site (Tyr397),
as assessed by its size and its phosphorylation in an in vitro kinase assay. The 4.1-kDa band was phosphorylated by COOHterminal Src kinase, suggesting that it contains the COOH-terminal negative regulatory site (Tyr508). CD45 was also shown to
dephosphorylate autophosphorylated Lyn in vitro. Thus, CD45 dephosphorylates not only the negative but also the positive
regulatory tyrosine residues of Lyn. Furthermore, coimmunoprecipitations using anti-Iga Ab demonstrated that Lyn associated
with the resting BCR was constitutively phosphorylated and activated in CD45-negative cells. In the parental cells, both regulatory
sites were phosphorylated on BCR ligation. Taken collectively, these results suggest that CD45 keeps both BCR-associated and
total cytoplasmic pools of Lyn in an inactive state, and a mechanism by which Lyn is activated by relative reduction of CD45 effect
may be operative on BCR ligation. The Journal of Immunology, 1999, 163: 1321–1326.
1322
Materials and Methods
Cells
The WEHI-231 cell line and its CD45-deficient clone, 10-5, were described
in previous reports (16, 30). These cells were maintained in RPMI 1640
supplemented with 10% FBS, 50 mM 2-ME, 100 mg/ml streptomycin, and
100 U/ml penicillin.
Abs and reagents
Goat (Fab9)2 fragments of anti-mouse IgM Ab and intact anti-mouse IgM
Ab were purchased from Cappel, Organon Teknika (Durham, NC). Polyclonal Ab against Lyn was purchased from Santa Cruz Biotechnology
(Santa Cruz, CA). Anti-phosphotyrosine (PY) mAb (4G10) was purchased
from United Biotechnology (Lake Placid, NY). Anti-Iga Ab was raised by
immunizing rabbits with GST fusion proteins containing the cytoplasmic
tails of the protein. Abs were then purified using the immunogen. Alkaline
phosphatase (AP)-conjugated goat anti-mouse IgG and mouse anti-rabbit
IgG were obtained from Bio-Rad (Richmond, CA), and Jackson Immunoresearch Laboratories (West Grove, PA), respectively.
Enolase was purchased from Sigma (St. Louis, MO).
Cell stimulation, immunoprecipitation, and Western blot analysis
Cells were harvested from log phase cultures, resuspended in fresh prewarmed RPMI 1640 containing 10% FBS supplemented with 20 mM
HEPES, and incubated for 3 h at 37°C. The cells were then stimulated with
25 mg/ml F(ab9)2 fragments of anti-IgM Ab for 1 min, and the reactions
were terminated with ice-cold PBS containing 2 mM Na3VO4 and 2 mM
EDTA (PBS-VE). The cells were centrifuged and solubilized in TNE lysis
buffer (1% Nonidet P-40, 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM
Na3VO4, 2 mM EDTA) or digitonin lysis buffer (1% digitonin, 20 mM
Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM Na3VO4, 2 mM EDTA) supplemented with Protease Inhibitor Mixture (Boehringer Mannheim GmbH,
Mannheim, Germany). The lysates were centrifuged at 10,000 3 g at 4°C
for 30 min, and the supernatants were subjected to further analysis.
Immunoprecipitation and Western blot analyses were performed as previously described (30). Each sample was immunoprecipitated with protein
G-Sepharose (Pharmacia Biotech, Uppsala, Sweden) coupled with Ab
against Lyn or Iga. Anti-Lyn immunoprecipitates were boiled with SDS
sample buffer under reducing conditions and subjected to 10% SDS-PAGE.
Separated proteins were blotted onto nitrocellulose membranes, and the
membranes were incubated overnight with anti-PY mAb and anti-Lyn Ab,
followed by AP-conjugated goat anti-mouse IgG and mouse anti-rabbit
IgG, respectively. The blots were visualized by developing them with an
AP Conjugate Substrate kit (Bio-Rad, Hercules, CA). Anti-Iga immunoprecipitates were washed with 0.5% digitonin lysis buffer, and dissolved in
1 ml of 1% Nonidet P-40, 0.1%SDS lysis buffer. The supernatants were
immunoprecipitated with anti-Lyn Ab- or normal rabbit IgG-coated protein
G beads. The precipitates were washed with TNE and then subjected to
SDS-PAGE or to in vitro kinase assays. The intensity of each band was
measured with a Bio-Rad densitometer.
In vitro kinase assay
In vitro kinase assay was performed as previously described (30). Cells
were solubilized in TNE or digitonin lysis buffer, and the supernatants were
immunoprecipitated with anti-Lyn Ab or anti-Iga and anti-Lyn Abs. The
immunoprecipitates were washed with lysis buffer and then with kinase
buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 10 mM magnesium acetate,
20 mM MnCl2). For in vitro kinase assays, 0.35 MBq [g-33P]ATP (37–110
TBq/mmol, Amersham, Arlington Heights, IL) in kinase buffer containing
10 mM cold ATP was added to the anti-Lyn immunoprecipitates. For antiIga and anti-Lyn immunoprecipitates from digitonin lysates, reactions
were performed in the same buffer without cold ATP together with exogenous substrate enolase. To assess phosphorylation of Tyr508 of Lyn, 1 ml
of recombinant COOH-terminal Src kinase (Csk) (33) was added to the
anti-Lyn immunoprecipitates during the in vitro kinase assay. The reactions
were terminated by adding SDS sample buffer, and the samples were subjected to 10% SDS-PAGE analysis. The resulting gels were treated with 1
N KOH at 60°C for 90 min to hydrolyze phosphoserine and phosphothreonine, dried, and analyzed with a BAS 2000 Bio-Imaging Analyzer (Fuji
Photo Film, Tokyo, Japan).
Metabolic labeling and CNBr digestion
Cells were washed with phosphate-free RPMI 1640 and cultured at 4 3 106
cells/ml for 3 h at 37°C in phosphate-free medium supplemented with 10%
dialyzed FBS. The cells were harvested and labeled with 370 MBq of
[32P]orthophosphate in 2 ml of medium for 1 h at room temperature. After
addition of 8 ml of phosphate-free medium to the culture, cells were incubated for 3 h at 37°C. Labeling was stopped by adding ice-cold PBS-VE,
and the labeled cells were lysed and immunoprecipitated with anti-Lyn Ab
as described above. Bound proteins were eluted in SDS sample buffer,
applied to 10% SDS-PAGE, and transferred onto a nitrocellulose membrane. The Lyn bands (p53 and p56), which were identified by autoradiography and subsequent immunoblotting, were excised and eluted with 250
ml of 150 mg/ml CNBr in 70% formic acid at room temperature for 2 h. The
eluted proteins were dried in a vacuum concentrator (Tomy Seiko, Tokyo,
Japan) to remove formic acid, and the final pellets were dissolved in SDS
sample buffer and applied to SDS-PAGE in 15–25% gradient gel. The
separated proteins were transferred to a polyvinylidine difluoride (PVDF)
membrane and subjected to BAS 2000 analysis.
Phosphoamino acid analysis
Phosphoamino acid analysis was performed as described earlier (34). 32Plabeled proteins were excised from a PVDF membrane and hydrolyzed
with 6 N HCl at 110°C for 2 h. The samples were dried, dissolved in 5 ml
of distilled water, and spotted onto TLC plates. Electrophoresis was conducted in pH 1.9 buffer for 20 min at 1.5 kV. After drying, the plates were
subjected to second dimension electrophoresis in pH 3.5 buffer for 16 min
at 1.3 kV. Separated phosphoamino acids were analyzed with a BAS 2000.
In vitro dephosphorylation assay
WEHI-231 cells stimulated with or without anti-IgM Ab were lysed with
TNE, and Lyn was immunoprecipitated and subjected to in vitro kinase
assays to phosphorylate the autophosphorylation site. CD45 was immunoprecipitated from WEHI-231 cells by using anti-CD45 mAb. The immunoprecipitates were washed with TNE, and CD45 was eluted with 50 ml
0.17 M glycine-HCl buffer, pH 2.0. The eluted supernatants were neutralized immediately by adding 0.45 ml 1 M Tris-HCl buffer, pH 9.0, and
dialyzed to PBS. A phosphatase assay was performed by incubating autophosphorylated Lyn with 0.5 ml purified CD45 for 60 min at 37°C. After
incubation, the precipitates were washed with TNE and subjected to SDSPAGE. The results were visualized by autoradiography.
Results
CD45 dephosphorylates and inactivates Lyn tyrosine kinase
In the immature B cell line WEHI-231, absence of CD45 led to
constitutive hyperphosphorylation and activation of Lyn, but not
other PTKs including Lck, Blk, Btk, and Syk (30) (T.K. and H.Y.,
unpublished observations). Representative results from anti-PY
blot analyses and in vitro kinase assays of Lyn in WEHI-231 and
its CD45-deficient clone (10-5) are shown in Fig. 1A. Anti-IgM
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reduced as compared with the activity of wild-type cells (31). In
CD45-negative, BCR-transfected J558Lmm3 plasmacytoma cells,
only the COOH-terminal tyrosine of Lyn was constitutively hyperphosphorylated, and the enzymatic activity of Lyn was significantly lower than its counterpart isolated from CD45-positive
cells (32). The reasons for these differences have not yet been well
defined.
This study was initiated to elucidate the mechanisms whereby
CD45 regulates Lyn tyrosine kinase in immature B cells. Studies
on Lyn in immunoprecipitates from 32P-labeled WEHI-231 CD45deficient cells with anti-Iga Ab demonstrated that Lyn, total cellular as well as BCR-associated, was constitutively tyrosine phosphorylated and activated. Cyanogen bromide (CNBr) cleavage
mapping of Lyn clearly demonstrated that both the COOH-terminal negative regulatory residue (Tyr508) and the positive regulatory
residue (Tyr397) were phosphorylated before BCR stimulation in
CD45-deficient cells. In the parental cells, by contrast, phosphorylation of not only Tyr397 but also Tyr508 was induced by BCR
ligation. Thus, in WEHI-231 cells, the main function of CD45 is to
dephosphorylate two major regulatory residues, Tyr508 and Tyr397,
and inactivate Lyn kinase in both the receptor-associated and total
cellular pools before BCR ligation. These results also suggest a
mechanism by which BCR ligation reduces the negative effect of
CD45, thereby inducing the phosphorylation and activation of Lyn
kinase.
NEGATIVE REGULATION OF Lyn KINASE BY CD45
The Journal of Immunology
1323
sively on tyrosine residues irrespective of BCR ligation, and in
10-5 cells, tyrosine phosphorylation of Lyn was enhanced 6.4-fold
even in the absence of BCR ligation (Fig. 1B). These results suggest that CD45 selectively dephosphorylates and inactivates Lyn in
WEHI-231 cells. Negative regulation of a Src-family kinase by
CD45 is in apparent contrast to the prevailing view that CD45
dephosphorylates the COOH-terminal negative regulatory tyrosine
residue, thereby activating the kinase activity (8, 11).
Both negative and positive regulatory tyrosine residues are
dephosphorylated by CD45
stimulation of the parent cells induced a 7.7-fold increase in tyrosine phosphorylation, but in the CD45-deficient clone, tyrosine
phosphorylation was constitutively elevated 3.6-fold and was not
enhanced further by BCR ligation. The kinase activity of Lyn was
induced only after BCR ligation in the parent, whereas Lyn was
constitutively activated (3.8-fold) in CD45-deficient cells, and the
activity was slightly increased (to 5.5-fold) on BCR ligation.
Phosphoamino acid analysis was also performed on Lyn in
WEHI-231 and 10-5 cells. Cells metabolically labeled with
[32P]orthophosphate were cultured with or without 25 mg/ml antiIgM Ab for 1 min, immunoprecipitated with anti-Lyn Ab, and
subjected to SDS-PAGE analysis. Phosphoamino acid analysis on
32
P-labeled Lyn demonstrated that Lyn was phosphorylated exclu-
Lyn in BCR complex is constitutively phosphorylated and
activated in a CD45-deficient clone
Previous reports suggested that CD45 exerts its regulatory effects
differently on the total pool and the TCR- or CD4-associated pools
of Src-family PTKs (27, 35). It is therefore possible that the observed regulation of CD45 in WEHI-231 applied only to the total
pool of Lyn and not to the more physiologically relevant subpool
of Lyn associated with the BCR. To exclude this possibility, we
examined the phosphorylation state and the kinase activity of Lyn
in the BCR complex. WEHI-231 and its CD45-deficient clone 10-5
were labeled with [32P]orthophosphate. After incubating with or
without 25 mg/ml anti-IgM Ab for 1 min, cells were lysed with
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FIGURE 1. Lyn is hyperphosphorylated and activated in a CD45-deficient clone from WEHI-231 cells. A, WEHI-231 and its CD45-deficient
clone (10-5) were either stimulated with 25 mg/ml anti-IgM Ab for 1 min
(1) or unstimulated (2), solubilized, and immunoprecipitated with protein
G-Sepharose coated with anti-Lyn Ab. The immunoprecipitates were subjected to SDS-PAGE analysis and Western blotting with anti-PY mAb
(top) or anti-Lyn Ab (bottom). Middle, results from an in vitro kinase assay
performed on Lyn immunoprecipitates for 3 min. The intensity of two
bands was normalized to the quantity of total Lyn present, and the relative
levels of tyrosine phosphorylation and kinase activity were expressed as
multiples of the levels observed in unstimulated WEHI-231. B, WEHI-231
and 10-5 cells were metabolically labeled with [32P]orthophosphate and
stimulated with (1) or without (2) 25 mg/ml anti-IgM Ab for 1 min. Cells
were immunoprecipitated with anti-Lyn Ab and then subjected to SDSPAGE analysis. The 32P-labeled Lyn bands were cut out from the PVDF
membrane, hydrolyzed, and then subjected to phosphoamino acid analysis
as described in Materials and Methods. The numbers at the bottom are the
relative levels of tyrosine phosphorylation. Phosphorylation levels in unstimulated WEHI-231 cells were assigned as 1. The results are representative of five separate experiments.
To address the mechanisms by which CD45 regulates Lyn activity,
the dephosphorylation sites were determined by the CNBr cleavage method. CD45-positive and -negative WEHI-231 cells were
metabolically labeled with [32P]orthophosphate, and Lyn immunoprecipitations were treated with CNBr. The resulting fragments
were resolved by SDS-PAGE. CNBr cleavage of Lyn is expected
to yield a fragment of 4.1 kDa containing the COOH-terminal
negative regulatory tyrosine residue (Tyr508) and a fragment of 8.2
kDa containing the autophosphorylation site (Tyr397), among others (Fig. 2A). In the parental cells, BCR ligation induced phosphorylation of 8.2- and 4.1-kDa fragments by 2.7- and 3.5-fold,
respectively (Fig. 2B). In CD45-deficient cells, however, phosphorylation of the 8.2-kDa fragment was ;2-fold greater than control even in the absence of anti-IgM stimulation, and the 4.1-kDa
fragment was even more strongly phosphorylated (4.1-fold). The
phosphorylation levels of the respective fragments were not significantly enhanced by BCR stimulation in the CD45-deficient
clone, 10-5. The 8.2-kDa fragment corresponded to a band containing the positive regulatory tyrosine residue Tyr397, as assessed
by its size and phosphorylation in an in vitro kinase assay (Fig.
2C). The 4.1-kDa fragments from both WEHI-231 and 10-5 were
phosphorylated by Csk (Fig. 2D), suggesting that it contained the
negative regulatory residue Tyr508. These results suggest that BCR
ligation induces phosphorylation of Lyn not only at the autophosphorylation site but also at the negative regulatory tyrosine residue.
The net result of phosphorylation at both sites is an activation of
the kinase. Furthermore, in the absence of CD45, both the positive
and negative regulatory tyrosine residues of Lyn were constitutively phosphorylated; nevertheless, the kinase activity was
increased.
To firmly establish that CD45 dephosphorylates the positive
regulatory tyrosine residue of Lyn, we examined whether CD45
directly dephosphorylates Tyr397 of Lyn in vitro. Lyn was immunoprecipitated from WEHI-231 cells and phosphorylated by in
vitro kinase assays. As shown in Fig. 2C, Lyn prepared by this
method was phosphorylated predominantly at Tyr397 but not at
Tyr508. When incubated with autophosphorylated Lyn for 60 min,
CD45 strongly dephosphorylated Lyn (Fig. 3, lane 2 vs lane 4).
Thus, CD45 dephosphorylates not only the negative but also the
positive regulatory tyrosine residues both in vitro and in vivo.
1324
NEGATIVE REGULATION OF Lyn KINASE BY CD45
FIGURE 2. Both the autophosphorylation and COOH-terminal regulatory tyrosine residues of Lyn are dephosphorylated by CD45. A, Schematic
representation of CNBr cleavage sites of Lyn. The autophosphorylation site
(Tyr397) and the COOH-terminal regulatory tyrosine residue (Tyr508) are
expected to be in fragments of 8.2 and 4.1 kDa, respectively. p, A tyrosine
residue. B, WEHI-231 and 10-5 cells were metabolically labeled with
[32P]orthophosphate, and stimulated with (1) or without (2) 25 mg/ml
anti-IgM Ab for 1 min. Cells were immunoprecipitated with anti-Lyn Ab
and subjected to SDS-PAGE analysis. The 32P-labeled Lyn bands were
excised from the nitrocellulose membrane and eluted with CNBr in formic
acid. The eluted proteins were subjected to SDS-PAGE in 15–25% gradient
gel, transferred to a PVDF membrane, and analyzed with a BAS 2000. The
Lyn protein applied was also assessed by Western blotting with anti-Lyn
Ab. The numbers in the middle indicate the relative increases in phosphorylation at Tyr397 and Tyr508 expressed as multiples of the phosphorylation
level seen in unstimulated WEHI-231 cells, based on the intensities normalized to the amount of Lyn assayed. C, Cells were stimulated with (1)
or without (2) 25 mg/ml anti-IgM Ab for 1 min and immunoprecipitated
with anti-Lyn Ab. An immune complex in vitro kinase assay was performed in the presence of [g-33P]ATP and subjected to SDS-PAGE. The
Lyn bands were processed as in B. The relative increases in phosphorylation at Tyr397 were calculated as in B. D, An immune complex kinase assay
was performed on Lyn from unstimulated WEHI-231 and 10-5 cells in the
presence of [g-33P]ATP and Csk, and CNBr cleavage analysis was conducted as in B. The results are representative of three experiments.
digitonin and immunoprecipitated with anti-Iga Ab. The immunoprecipitates were dissolved in 1% Nonidet P-40, immunoprecipitated with anti-Lyn Ab or control IgG, and subjected to SDSPAGE analysis. The results revealed that on BCR ligation, p53Lyn
and p56Lyn were phosphorylated in the parental cells (Fig. 4A, lane
2) but that in the CD45-deficient cells, Iga-associated Lyn was
hyperphosphorylated even in the absence of BCR stimulation (Fig.
4A, lane 3). Control IgG did not precipitate Lyn (Fig. 4A, lanes 5
and 6). Similar results were obtained with anti-Igb immunoprecipitation (data not shown).
The enzymatic activity of Lyn in BCR complexes was examined
after digitonin lysis and immunoprecipitation with anti-Iga Ab.
Compared with the parental cells, Lyn activity, both autophosphorylation and phosphorylation of enolase was significantly increased
even before BCR ligation in the CD45-deficient clone 10-5 (Fig.
5A, lanes 1, 2 vs lanes 3, 4). These results suggest that both receptor-associated and not associated Lyn pools were constitutively
hyperphosphorylated and activated in the absence of CD45. Thus,
CD45 dephosphorylates both the positive and negative regulatory
tyrosine residues of Lyn, and the net effect is inactivation of the
kinase.
Discussion
Our previous studies showed that CD45 inhibits Lyn kinase activity in WEHI-231 cells and negatively regulates BCR-initiated effector phenomena such as growth arrest and apoptosis (16, 30). To
further characterize the mechanisms by which CD45 regulates a
Src-family PTK, we examined the relationship between the phosphorylation state of regulatory tyrosine residues of Lyn and the
enzymatic activity. The results demonstrate that in the absence of
FIGURE 4. Lyn in BCR complex is hyperphosphorylated in the CD45deficient clone. A, WEHI-231 (lanes 1, 2, and 5) and 10-5 (lanes 3, 4, and
6) cells were labeled with [32P]orthophosphate and stimulated with (lanes
2 and 4) or without (lanes 1, 3, 5, and 6) 25 mg/ml anti-IgM Ab for 1 min.
Cells were lysed with digitonin and immunoprecipitated with anti-Iga Ab.
The immunoprecipitated BCR complex was dissolved with 1% Nonidet
P-40 buffer, immunoprecipitated with anti-Lyn Ab (lanes 1– 4) or control
normal rabbit IgG (lanes 5 and 6) and subjected to SDS-PAGE analysis.
The 32P radioactivity was visualized by autoradiography. B, Lyn proteins
were detected by Western blotting with anti-Lyn Ab. The results are
representative of three separate experiments. Arrows indicate p56Lyn
and p53Lyn.
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FIGURE 3. CD45 dephosphorylates the positive regulatory tyrosine in
vitro. A, Lyn was immunoprecipitated with anti-Lyn-coated protein
G-Sepharose from TNE lysates of unstimulated (lane 1) or antiIgM-stimulated (lane 2) WEHI-231 cells and autophosphorylated in vitro
with [g-33P]ATP. CD45 was purified from WEHI-231 as described in Materials and Methods. CD45 was then incubated at 37°C for 60 min with the
autophosphorylated Lyn from unstimulated (lane 3) or anti-IgM-stimulated
(lane 4) cells. After incubation, the beads were washed and eluted in SDSPAGE sample buffer, and the aliquots were subjected to 10% SDS-PAGE.
The 33P radioactivity was visualized by autoradiography. B, Lyn proteins
were detected by Western blotting with anti-Lyn Ab (B). The data are
representative of three independent experiments. Arrows indicate p56Lyn
and p53Lyn.
The Journal of Immunology
FIGURE 5. BCR-associated Lyn is constitutively activated without
CD45. A, WEHI-231 (lanes 1 and 2) and 10-5 (lanes 3 and 4) cells were
stimulated with (lanes 2 and 4) or without (lanes 1 and 3) 25 mg/ml antiIgM Ab for 1 min. The cells were lysed with 1% digitonin lysis buffer and
immunoprecipitated with anti-Iga coated-protein G-Sepharose. The immunoprecipitated BCR complex was dissolved with 1% Nonidet P-40 buffer
and immunoprecipitated again with anti-Lyn Ab. The immunoprecipitated
Lyn was subjected to in vitro kinase assays with exogenous substrate enolase. B, Lyn proteins were detected by Western blotting with anti-Lyn Ab.
The results are representative of three experiments.
ulatory tyrosine, activating their enzymatic activity. This was
based on the initial descriptions of CD45-negative T cell lines in
which the Src-family PTKs were found to be hyperphosphorylated
at their COOH-terminal tyrosines and inactive (20 –27, 37). However, subsequent studies revealed that the function of CD45 might
be more complex. In CD45-negative human HPB-ALL T cells,
anti-CD4-induced activation and tyrosine phosphorylation of Lck,
particularly the CD4-associated pool, was higher than those of
CD45-positive cells (35). Studies on three T cell lines, YAC-1,
SAKRTLS, and HPB-ALL, demonstrated that Lck and Fyn are
constitutively hyperphosphorylated and paradoxically activated in
the CD45-negative clones and that in vitro exposure of CD45 to
Lck leads to decreases, rather than increases, in the kinase activity
(28). CNBr cleavage mapping showed that Lck in the CD45-negative clones is hyperphosphorylated at both the negative regulatory
tyrosine residue (Tyr505) and the autophosphorylation site (Tyr394)
(28). Phosphoamino acid analysis confirmed that the increased
phosphorylation in CD45-negative YAC-1 cells is restricted to tyrosine residues, Tyr505 and, to a lesser extent, Tyr394 (29). Further
mutational analysis revealed that mutation of Tyr505 to phenylalanine results in increased kinase activity whereas mutation of
Tyr394 to phenylalanine decreases enzymatic activity. The double
mutation, Tyr505-Phe and Tyr394-Phe, led to inactivation of the
kinase (29). These results suggest that without CD45, phosphorylation at Tyr394 induces activation of Lck despite hyperphosphorylation at Tyr505 and that the phosphorylation state of Tyr394 may
have a dominant role in the regulation of Lck.
In B cells, there have been discrepant reports as well. Lyn from
CD45-negative, BCR-reconstituted J558Lmm3 plasmacytoma
cells was shown to be hyperphosphorylated at the COOH-terminal
tyrosine residue. The kinase was neither activated nor recruited to
the BCR complex (32). A study on the chicken DT40 B cell line
showed that CD45 dephosphorylates both the positive and the negative regulatory tyrosine residues of Lyn but the activity of Lyn is
enhanced in the presence of CD45 and that BCR-induced tyrosine
phosphorylation and Ca21 mobilization are severely impaired in
the absence of CD45 (31). Although the sites dephosphorylated by
CD45 are different, both studies underscore the importance of dephosphorylation of the COOH-terminal tyrosine of Lyn in certain
processes of BCR-mediated activation. In contrast, our previous
reports (16, 30), together with the present study, propose a different mode of CD45 action, in which CD45 dephosphorylates both
Tyr508 and Tyr397 of Lyn and keeps the kinase in an inactive state,
exerting negative modulatory effects on BCR-induced Ca21 mobilization, growth arrest, and apoptosis. It is also important that
BCR ligation induces phosphorylation of not only Tyr397 but also
Tyr508, yet activates the kinase. All these results suggest that there
must be a mechanism whereby BCR ligation negates the effect of
CD45, thus inducing phosphorylation of two regulatory sites and
activation of the kinase.
What accounts for all the phenotypic differences is not clearly
elucidated at present. Recent crystal structural and functional analyses on the inactive form of Src-family PTKs, c-Src and Hck (38 –
40), may give us an important hint as to the role of CD45 in the
regulation of this family of PTKs. These studies demonstrated that
although the SH2 domain binds to the phosphorylated COOHterminal tail, this interaction does not block the catalytic site.
Rather, the kinase is probably inactive because the linker sequence
between the catalytic and SH2 domains binds the SH3 domain (38,
39, 41). Furthermore, the PTK that is phosphorylated at the
COOH-terminal tyrosine was shown to be activated by disturbing
these intramolecular interactions with exogenous SH2 and SH3
ligands (40). Thus, regulation of Src-family PTKs seems to be very
complex such that the state of phosphorylation at the two major
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CD45, Lyn is activated and hyperphosphorylated exclusively at
tyrosine residues (Fig. 1). Phosphorylation was observed in the
absence of BCR stimulation and occurred on both the negative
regulatory Tyr508 and the positive regulatory Tyr397 (Fig. 2). This
negative regulation was observed not only in the total cellular Lyn
pool but also in the more physiologically important pool of kinases
associated with the BCR (Figs. 4 and 5). Thus, CD45 dephosphorylates the two major regulatory tyrosine residues and inactivates
Lyn kinase in WEHI-231 cells.
Accumulating evidence suggests that Src-family PTKs are regulated in part by tyrosine phosphorylation of the inhibitory site in
the COOH-terminal tail and the stimulatory site in the kinase domain (36). The different phosphorylation states stabilize a repressed or an activated conformation respectively. Csk phosphorylates the COOH-terminal tyrosine, keeping Src-family PTKs in a
repressed conformation (33). Therefore, one way to activate Srcfamily PTKs is by dephosphorylating the COOH-terminal tyrosine. Mutations in the kinase and SH2 and SH3 domains led to
activation of Src, suggesting that conformational changes in these
regions are also important factors for activating Src-family PTKs
(36). Our results showing that a Src-family PTK can be active even
if its negative regulatory COOH-terminal tyrosine is phosphorylated appear to contradict the conventional model of regulation.
However, there are several explanations for our findings. One is
that the phosphorylation state of the positive regulatory tyrosine is
dominant over that of the negative regulatory tyrosine as has been
suggested previously (29). An alternative possibility is that in Srcfamily PTKs that have phosphorylated COOH-terminal tyrosines,
activation can occur by a phosphorylation-independent mechanism. It is also possible that the observed phosphorylation at both
sites could be caused by the presence of two populations of Lyn,
each phosphorylated at different residues. In this case, the enhanced kinase activity is a reflection of the dominant presence of
Lyn phosphorylated at the stimulatory Tyr397. This possibility has
not been examined in this or any other studies. Given that phosphorylation at the inhibitory Tyr508 was 2.2-fold higher than that at
the stimulatory Tyr397 in activated Lyn (Fig. 2B), we think this
possibility is unlikely.
It has been argued that the main function of CD45 is to activate
Src-family PTKs. A consensus model is that Csk phosphorylates
the COOH-terminal negative regulatory tyrosine residue of Srcfamily PTKs, keeping PTKs in an inactive conformation, and that
on Ag receptor ligation CD45 dephosphorylates the negative reg-
1325
1326
Acknowledgments
We thank Drs. James Clements and Gary Koretzky (University of Iowa,
Iowa City, IA) for advice on phosphoamino acid analysis and Dr. Philip
Cohen (University of Dundee, Dundee, U.K.) for critical comments on
CNBr mapping data.
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regulatory tyrosine residues is but one determiner of activation.
Various factors, including unknown SH2 and SH3 ligands, contribute to the net enzymatic activity. It may also be possible that
tyrosine phosphorylation at the two sites is a consequence of activation or inactivation induced by conformational alterations that
dictate the accessibility of Src-family PTK phosphorylation sites to
other PTKs and protein tyrosine phosphatases (36). In light of
these structure-function relationships, our findings are not necessarily idiosyncratic but reflect a common regulatory activity of
CD45. This is supported by observations in other CD45-negative
B cell clones in which BCR-induced tyrosine phosphorylation of
total cellular proteins is slightly reduced, but phosphorylation of a
few species of proteins is almost completely defective (17). Differences observed in the PTK activity in a variety of CD45-deficient cells may be explained partly by the different availability of
molecules within the cell capable of affecting the conformation of
the Src-family PTKs (42).
In summary, CD45 has an inhibitory effect on Lyn by dephosphorylating the autophosphorylation site as well as the COOHterminal regulatory tyrosine in immature WEHI-231 cells. This
regulation exerted on not only total cellular Lyn but also the BCRassociated pool of Lyn. Furthermore, the fact that both regulatory
tyrosines are phosphorylated and activated by BCR ligation suggests that BCR signaling machinery may have a mechanism by
which the negative effect of CD45 is somehow inhibited.
NEGATIVE REGULATION OF Lyn KINASE BY CD45