Immunity, Vol. 4, 515±525, May, 1996, Copyright 1996 by Cell Press
Regulation of Btk Function by a
Major Autophosphorylation Site
Within the SH3 Domain
Hyunsun Park,*# Matthew I. Wahl,*²#
Daniel E. H. Afar,* Christoph W. Turck,³
David J . Rawlings,*Christina Tam,*
Andrew M. Scharenberg,§J ean-Pierre Kinet,§
and Owen N. Witte*?
*Department of Microbiology and Molecular Genetics
University of California, Los Angeles
Los Angeles, California 90095-1662
²University of California, Los Angeles
Cedars Sinai Medical Genetics Training Program
Los Angeles, California 90048
³Howard Hughes Medical Institute
Department of Medicine
Cardiovascular Research Institute
University of California, San Francisco
San Francisco, California 94143-0724
§Laboratory of Allergy and Immunology
Beth Israel Hospital and Harvard Medical School
Boston, MA 02215
?Howard Hughes Medical Institute
Los Angeles, California 90095-1662
Molecular Biology Institute
University of California, Los Angeles
Los Angeles, California 90095-1662
homology domains, SH3, SH2, and SH1 (catalytic) (Tsu-
kada et al., 1993; Vetrie et al., 1993; Rawlings et al.,
1993; Thomas et al., 1993). Mutations in the Btk gene
murine X-linked immunodeficiency (Xid). XLA patients
have a deficit in mature B cells due to a developmental
arrest during pre-B cell expansion (Kinnon et al., 1993).
In Xid, B cell development is selectively impaired and
the number of B cells is reduced by 30%±50% (Wicker
and Scher, 1986). Xid B cells do not respond normally
to a wide variety of activating signals, including thymus-
independent type 2 antigens, surface immunoglobulin
M (sIgM) cross-linking, and interleukin-5 (IL-5), IL-10,
CD38, or CD40 receptor ligation (reviewed by Wicker
and Scher, 1986; Rawlings and Witte, 1995).
Enhancement of catalytic activity in association with
tyrosine phosphorylation is involved in the activation of
many tyrosine kinases (reviewed by Perlmutter et al,
1993). Btk catalytic activity and tyrosine phosphoryla-
tion increase in response to cross-linking orstimulation
of the sIgMcomplex, the IL-5 receptor, orthe IL-6recep-
tor of B cells, or the high affinity IgE receptor of mast
cells (Saouaf et al., 1994; de Weers et al., 1994; Aoki et
al., 1994; Sato et al., 1994; Matsuda et al., 1995; Kawa-
kami et al., 1994). Stimulation of the B cell receptor
(BCR) by sIgM cross-linking increases binding of Src
family tyrosine kinases to the BCR and triggers activa-
tion of Src family tyrosine kinases (reviewed by Cambier
et al., 1994).The activationof Src familytyrosine kinases
precedes Btk activation after sIgM cross-linking, sug-
gesting that Src family tyrosine kinases may be regula-
tors of Btk activation (Saouaf et al., 1994).
Vaccinia virus±driven high level coexpression of Btk
with Lyn results in an increase in both Btk catalytic
activity and Btk tyrosine phosphorylation (Rawlings et
al., 1996). Lyn/Btk coexpression induces Btk phosphor-
ylation at two distinct tyrosine residues by two indepen-
dent steps. PhosphorylationatY551 requires Lyn kinase
activity, indicating that Y551 is a transphosphorylation
site. The sequence surrounding BtkY551 is an excellent
match to the consensus Src family phosphorylationsite
(Hanks, 1988). This transphosphorylation at Y551 is fol-
lowed by phosphorylation at a second site, which is
dependent on Btk catalytic activity.
We had previously identified an activated form of Btk,
which was isolated using a retroviral random mutagene-
sis scheme (Li et al., 1995). Btk*contains a point muta-
tion (E41K) in the PH domain and leads to constitutive
activation of Btk. Btk*can induce transformation of fi-
broblasts and IL-5-independent growth of a pro-B cell
line. The transforming activity of Btk*is associated with
an increase in tyrosine phosphorylation and increased
membrane localizationof Btk. The transforming activity
is dependenton Btk kinaseactivity and phosphorylation
of the consensus phosphorylationsite ofSrc familytyro-
sine kinases (Y551) within the Btk catalytic domain. Ki-
nase-inactive Btk*/K430R lacks tyrosine phosphoryla-
tion but the Btk*/Y551F double mutant accumulates
tyrosine phosphorylation at a low level. These results
Bruton's tyrosine kinase (Btk) plays a crucial role in B
cell development. Overexpression of Btk with a Src
family kinase increases tyrosine phosphorylation and
catalytic activity ofBtk. This occurs by transphosphor-
ylation at Y551 in the Btk catalytic domain and the
enhancement of Btk autophosphorylation at a second
site. A gain-of-function mutant called Btk*containing
E41 to K change within the pleckstrin homology do-
main induces fibroblast transformation. Btk*enhances
the transphosphorylation of Y551 by endogenous Src
familytyrosine kinases andautophosphorylation atthe
second site.We mapped the major Btk autophosphor-
ylation site to Y223 within the SH3 domain. Mutation
of Y223 to F blocks Btk autophosphorylation and dra-
matically potentiates the transforming activity of Btk*
in fibroblasts. The location of Y223 in a potential li-
gand-binding pocket suggests that autophosphoryla-
tion regulates SH3-mediated signaling by Btk.
Bruton's tyrosine kinase (Btk) is a member of the Btk/
Tec subfamily of cytoplasmic tyrosine kinases.Btk con-
tains a pleckstrin homology domain (PH) and proline-
rich sequences at its N terminus, in addition to the Src
#These authors contributed equally to this manuscript.
suggest that Btk*-mediated tyrosine phosphorylation
results from at least two phosphorylation events, which
occur at Y551 and at an unidentified tyrosine residue.
Btk*cannottransform rat-2fibroblasts to agargrowth
unless it is coexpressed with a weakly activated form
of c±src (srcE378G) (D. E. H. A. et al., submitted). Trans-
forming activity correlates with the enhancement of Btk
tyrosine phosphorylation.Btk*-mediated transformation
is suppressed by coexpression of the C-terminal Src
kinase (Csk), which inactivates Src family tyrosine ki-
nases. These results provide genetic evidence for the
role of Src family kinases as upstream regulators of Btk
signaling. The transforming potential of Btk*is further
enhanced by a block deletion of the SH3 domain of Btk
(Li et al., 1995; D. E. H. A. et al., submitted). Analogous
to other cytoplasmic tyrosine kinases, the SH3 domain
of Btk is likely to act as a regulatory domain (Hirai and
Varmus, 1990; Seidel-Dugan et al., 1992; J ackson et al.,
1993; Mayer and Baltimore, 1994; Okada et al., 1993).
Using detailed phosphopeptide analysis, we demon-
strate that the Btk* mutation or Lyn/Btk coexpression
induce indistinguishable patterns of tyrosine phosphor-
ylation on Btk. This suggests that alternative modes
of activation generate common signals leading to Btk
tyrosine phosphorylation. We identified the major Btk
autophosphorylation site and mapped it to Y223 within
the SH3 domain. Mutation of Y223 to phenylalanine
strongly potentiates Btk* transforming activity, sug-
gesting that autophosphorylation serves to regulate Btk
activation. The Y223 residue is highly conserved among
the SH3 domains of a broad array of signaling proteins
and is proposedtolie inthe surfaceofthe ligand-binding
groove (Koyama et al., 1993; Noble et al., 1993; Feng et
al., 1994; Erpel et al., 1995). Thus, Y223 of Btk is likely
to have a criticalrole inSH3 functionofBtk by regulating
The Btk* Mutation Leads to the Phosphorylation
of Two Distinct Tyrosine Residues
by Two Independent Steps
Phosphorylation mapping studies by Rawlings et al.
(1996)haveshownthat theSrc family kinaseLyninduces
Btk tyrosine phosphorylation by transphosphorylation
ofBtk atY551and enhancementofautophosphorylation
at a second site. Phosphorylation at these sites corre-
lates with a greater than 5- to 10-fold increase in Btk
catalytic activity. To determine whether activation by
Btk*correlated with phosphorylation of the same tyro-
sine residues induced by Btk/Lyncoexpression, we ana-
lyzed the phosphorylation patterns induced by Btk*.
Wild-type Btk and Btk*were expressed in NIH 3T3 cells
by retroviral infection. Following in vivo
phate labeling, Btk proteins were immunoprecipitated
from cell extracts and analyzed by two-dimensional
tryptic phosphopeptide mapping (Boyle et al., 1991).
Btk*contained fourmajorphosphopeptides that were
not present in wild-type Btk (Figures 1A and 1B). Phos-
phoamino acid analysis of individual peptides revealed
the presence of phosphotyrosine in the four peptides
(py1, py1?, py2?, and py2??). We definedthe py1 peptides
and py2 peptides as two families of closely related spe-
cies, since they appear to be derived from peptides
containing the same phosphorylation sites (see below).
Inaddition, Btk*exhibited enhanced phosphorylationof
four peptides compared with wild-type Btk. Of these
peptides with enhanced phosphorylation, three con-
tained phosphoserine (ps1, ps2, and ps3) and one con-
tained phosphotyrosine (py2). Analysis of wild-type Btk
showeda prominentphosphothreonine-containing pep-
tide (pt1), which was significantly reduced in Btk*.
To test which phosphopeptides represent autophos-
phorylation or transphosphorylation sites, we analyzed
Figure 1. Comparison of Two-Dimensional
Tryptic Peptide Map of Btk Proteins
(A)Wild-type Btk, (B) Btk*, (C) Btk*/Y551F, (D)
Btk*/K430R Btk proteins were stably ex-
pressed inNIH 3T3cells by retroviralinfection
(seeExperimentalProcedures). Cells (1?107)
were incubated with 1mCi/ml32P-orthophos-
phate in 2 ml of phosphate free DMEM for
3 hr. Btk proteins were immunoprecipitated
using affinity-purified antibodies specific to
the N-terminal region of Btk (N2) (Tsukada et
al., 1993; Li et al., 1995), separated by SDS±
PAGE, blotted onto nitrocellulose, and di-
gested withtrypsin. Thetryptic peptides were
oxidized with performic acid and separated
by thin-layer electrophoresis at pH 1.9 fol-
lowedby chromatography (Boyle etal.,1991).
Phosphopeptides were detected byautoradi-
ography, eluted from the thin-layer cellulose
plate, and subjected to phosphoamino acid
analysis (Boyle et al., 1991). ps, pt, and py
indicate peptides containing phosphoserine,
phosphothreonine, and phosphotyrosine, re-
Regulation of Btk by Autophosphorylation
Figure 2. IgA Protease Cleavage of Btk
The schematic representation indicates the
structural domains of Btk, the possible sites
of IgA protease cleavage, and the N-terminal
and C-terminal fragments generated by the
IgA cleavage.At the bottom ofthe figure, par-
tially purified wild-type protein, expressed by
vaccinia virus coinfection with Lyn (see the
Experimental Procedures) was incubated in
the absence (lanes 1, 3, 5) orpresence (lanes
2, 4, 6) of IgA protease. The digested frag-
ments were separated by SDS±PAGE, and
Western blot analysis was performed using
polyclonal antisera against the N-terminal
portion (N-term, lanes 1, 2) or C-terminal 15
aa of Btk (C-term, lanes 3, 4), or monoclonal
antibodies against phosphotyrosine (anti-PY,
lanes 5, 6). Full-length (80 kDa), N-terminal
(30 kDa), and C-terminal (50 kDa) fragments
the phosphopeptide patterns of a set of Btk* double
mutants.Mutants thatare eitherkinasenegative (K430R)
orthat contain a mutation that abolishes the Src kinase
consensus phosphorylationsite (Y551F)were generated
in conjunction with Btk*. Previous studies by Li et al.
(1995) showed that either of these mutations reduces
the tyrosine phosphorylation associated with Btk*and
suppresses the transforming activity infibroblasts. Btk*/
Y551F abolished phosphorylation of py1 but retained
py2, indicating that the py1 peptide was derived from a
peptidephosphorylated onY551(Figure 1C).Incontrast,
the kinase-inactive Btk*/K430R lost phosphorylation of
py2, py2?, and py2?? butretained phosphorylationof py1
(Figure 1D). Thus, while phosphorylation of Y551 does
not require Btk kinase activity, phosphorylation of all
py2-related peptides is dependent on Btk kinase activ-
ity. This indicates that the three py2 peptides (py2, py2?,
and py2??) contain autophosphorylation sites. The py2
peptide migrates at an identical position to the peptide
containing the Btk autophosphorylation site following
Btk/Lyn coexpression. These results suggest that Btk*
and Btk/Lyn coexpression induce highly related phos-
Lyn from Btk. Btk protein was eluted from the column
using high salt (0.5 M NaCl), while Lyn was recovered
from the flow-throughfraction (seeExperimental Proce-
dures). Btk protein was digested with IgA protease,
which cleaves at two adjacent proline-rich motifs C-ter-
minal to the Btk PH domain, resulting in an N-terminal
and a C-terminal fragment of protein. Western analysis
using antibodies specific for phosphotyrosine, Btk N
terminus, and Btk C terminus revealed that Btk tyrosine
phosphorylation sites were all located in the C-terminal
fragment, which includes the SH3, SH2, and SH1 do-
mains (Figure 2).
To test whether the Btk autophosphorylation site re-
sides in the SH3 domain, a Btk*/delSH3 double mutant
(Li et al., 1995) was stably expressed in NIH 3T3 cells.
Tryptic phosphopeptide mapping revealed that deletion
of the SH3 domain abolished all three py2 peptides
induced by Btk*. In contrast, phosphorylation at py1
was unaltered by the SH3 domain deletion (Figure 3A).
In addition, BtkdelSH3 coexpressed withLyn in NIH 3T3
cells retained phosphorylation of py1 (Y551) but lost
phosphorylation of py2. Interestingly, coexpression of
peptide containing phosphotyrosine (py3) (Figure 3B).
The lack of py2 phosphorylation in the absence of the
SH3 domain suggested that the Btk autophosphoryla-
tion site resides within the SH3 domain. Alternatively,
deletion of the SH3 domain may cause a structural
change inthe protein, which prevents autophosphoryla-
tion at a site distal to the SH3 domain. To determine
whetherthe autophosphorylation site resides inthe SH3
The Major Btk Autophosphorylation Site
Resides within the SH3 Domain
To identify the autophosphorylation site in Btk, wild-
type Btk protein was coexpressed with Lyn in NIH 3T3
cells using vaccinia virus. Btk was partially purified by
phosphocellulose chromatography, which separates
Figure 3. Phosphopeptide Map of Btk Containing SH3 Deletion
(A) Btk*/delSH3 stably expressed in NIH 3T3.
(B) BtkdelSH3 coinfected with Lyn in NIH 3T3 cells using vaccinia
(C) In vitro phosphorylated GST±BtkSH3 fusion protein.
Vaccinia virus expressing Btk or Lyn was coinfected into NIH 3T3
cells for 12±15 hr prior to in vivo32P04labeling. For in vivo labeling,
1 ? 107cells were incubated with 1 mCi/ml of32P-orthophosphate
in 2ml ofphosphate-free DMEMfor 3 hr.Btk proteins were immuno-
precipitated by affinity-purified Btk antibodies (N2) and digested
with trypsin as described in the Experimental Procedures. GST±
BtkSH3 fusion proteins were expressed in Escherichia coli and iso-
lated as described in Experimental Procedures. GST±BtkSH3 pro-
tein (4 ?g) was phosphorylated by 50 ng of purified Btk protein in
vitro in 20 mM PIPES (pH 7.0), 20 mM MnCl2, 10 ?M ATP, and 100
?Ci of [?±32P]ATP at 30?C for 1 hr. In vitro phosphorylated GST±
BtkSH3 was resolved by SDS±PAGE, transferred onto nitrocellulose
membrane, and digestedby trypsin.The tryptic peptides were sepa-
rated by thin-layer electrophoresis at pH 1.9, followed by chroma-
tography (Boyle et al., 1991). The locations of py2and py2? peptides
domain, GST±BtkSH3 fusion proteinwas used as a sub-
strate for purified Btk in in vitro kinase assays. Full-
length Btk containing a histidine tag at the C terminus
was purified to near homogeneity by phosphocellulose
chromatography and a Ni2?±NTA affinity column (see
Experimental Procedures). Btk was able to phosphory-
late GST±SH3 but not GST protein alone (data not
shown). Two tryptic phosphopeptides were recovered
from phosphorylatedGST±SH3 proteinwhosemigration
patterns were indistinguishable from py2 and py2? pep-
tides derived from the Btk*protein (Figure 3C). Taken
together, these results strongly suggest that a majorBtk
autophosphorylation site(s)is locatedwithinthe Btk SH3
Figure 4. Physical Mapping of the Btk Autophosphorylation Site
(A) Strategy for mapping the Btk autophosphorylation site. NIH 3T3
cells were coinfected with BTKY551F and Lyn vaccinia virus con-
structs, labeled with32P-orthophosphate, partially purified by phos-
phocellulose chromatography, and then immunoprecipitated by af-
finity-purified Btk antibodies (N2) as described in the Experimental
Procedures. Immunoprecipitates were separated by SDS±PAGE,
blotted onto nitrocellulose, and digested with trypsin. The tryptic
peptides containing phosphotyrosine were isolated using an anti-
phosphotyrosine column, separated by HPLC, and subjected to
automated Edman peptide sequence analysis for 10 cycles as de-
(B) HPLC profile and the amino acid sequences of phosphotyrosine
containing peptides. At the top of the figure, the schematic diagram
of Btk indicates the domain structure of Btk, the locationof tyrosine
223 within the SH3 domain, and the location of tyrosine 551 within
the SH1 domain. A portion of the primary amino acid sequence of
Btk within the SH3 domain is shown. At the bottom of the picture,
the HPLC elutionprofile demonstrates the separationof phosphoty-
rosine containing tryptic peptides. The anti-phosphotyrosine eluate
was separated using a C18 reverse phase HPLC and eluted with a
linear acetonitrile gradient of 5%±27% as described in the Experi-
mental Procedures. Radioactivity in each HPLC fraction was mea-
sured (cpm). Two major peaks containing
and B. Peak fractions containing A or B were pooled, dried, and
Y223 Is the Major Btk Autophosphorylation Site
Rawlings et al. (1996) demonstrated that BtkY551F
coexpressed with Lyn retains the autophosphorylation
site contained in py2 but loses py1 (containing Y551).
To identify the tyrosine autophosphorylation site by
physical mapping, phosphorylated py2 peptide was en-
riched by coexpression of BtkY551F with Lyn by vac-
cinia virus inNIH3T3cells.Following invivo labeling with
32P-orthophosphate, purified Btk protein was digested
with trypsin and phosphotyrosine-containing peptides
were isolated using an anti-phosphotyrosine antibody
column. The peptides were separated by reverse phase
high pressure liquid chromatography (HPLC) and radio-
activity in each fraction was measured (Figure 4A). One
major peak (B) and one minor peak (A) (Figure 4B) were
independently subjected toautomated Edmandegrada-
tion analysis for 10 cycles. Sequence analysis revealed
that peptides A and B contain overlapping sequences
32P were indicated as A
Regulation of Btk by Autophosphorylation
within the SH3 domain. Peptide B originated from a
predicted tryptic digestionsite. However, peptideA was
derived from a cleavage at L222, which probably was
generatedby contaminating protease activityinthe tryp-
sinpreparation. Independent microsequencing analysis
of these two fractions showed preferential loss of posi-
tionY223, indicating itis the onlyphosphotyrosine-mod-
ified residue in either peptide (Figure 4B).
Since the predicted tryptic peptide containing the Btk
autophosphorylation site has another tyrosine residue,
Y225, it is possible that generation of multiple py2 pep-
tides arose from differentialphosphorylation ontwo dif-
ferent tyrosine residues in the same tryptic peptide. To
test this possibility, the tryptic peptides derived from
in vitro phosphorylated GST±SH3 protein by Btk were
sequenceddirectly.Following tryptic digestion ofinvitro
phosphorylated GST±BtkSH3 fusion protein, phospho-
tyrosine containing peptides were isolated on an anti-
phosphotyrosine antibody column, separated by HPLC
fractionation, and subjected to 12 cycles of automated
Edmansequence analysis. The results showed that only
Y223 was phosphorylated in each peptide. Thus, the
different migration properties of py2 peptides do not
result from differential phosphorylation of the same
tryptic peptide, but are likely due to the generation of
heterogeneous products by contaminating proteases.
Conceivably, they could arise from additional protein
modifications that occur within the same phospho-
Mutation of Y223 Potentiates Btk*
Btk*is a weak transforming allele inrat fibroblasts com-
pared with NIH 3T3 cells. However, Btk*-transforming
activity is dramatically potentiatedby coexpressionwith
a weakly activated form of c±src (srcE378G) in rat-2
cells. srcE378G contains an activating mutation in the
catalytic domain (Levy et al., 1986) but does not trans-
formrat-2cells (D. E. H.A. etal., submitted).Thesynergy
betweenBtk*and srcE378G correlates with asignificant
increase in Y551 phosphorylation and enhanced Btk
autophosphorylation. Btk*-transforming activity is fur-
ther enhanced by a block deletion of the SH3 domain
when coexpressed with srcE378G. This supports the
role of the Btk SH3 domain in regulating Btk activity
(D. E. H. A. et al., submitted).
We tested whetherthe point mutation of Y223 to phe-
nylalanine, which cannot serve as a phosphate ac-
ceptor, stimulates or inactivates Btk*-mediated trans-
formation. If Btk autophosphorylation inactivates SH3
function similar to a deletion of the SH3 domain, muta-
tion of Y223 would activate Btk signaling leading to
enhanced Btk*transformation. Alternatively, ifBtk auto-
phosphorylation serves as an activation mechanism,
then mutation of Y223 would reduce Btk*transforma-
tion. To test these possibilities, the Y223F mutation was
generated in the context of wild-type Btk and Btk*.
Figure 5. Effect of Y223F Mutation in Btk*Transforming Activity
Parental rat-2 cells and cells expressing SrcE378G were infected
with retroviruses encoding wild-type Btk, BtkY223F, Btk*, or Btk*/
Y223F and were plated into soft agar in medium containing 20%
fetal calf serum at 1 ? 104cells/plate. Colonies equal to and larger
than 0.5 mm in diameter were counted after 3 weeks in soft agar.
The approximate locations of point mutations in the Btk subdomain
are indicated by arrows in the schematic representations of the
different Btk forms. The numberof colonies are indicated and repre-
sent averages fromtwo different independentexperiments. The pic-
tures were taken after colonies were grown for 3 weeks in agar.
These Btk forms were introduced into either rat-2 cells
or rat-2 cells coexpressing srcE378G. Tryptic phospho-
peptide mapping of in vivo
showed that the Y223F mutation abolished phosphory-
lation of py2 peptides (data not shown).
Rat-2 cells expressing wild-type Btk or Btk* formed
only a low number (<10) of small colonies (<0.5 mm
diameter) per 104cells plated in soft agar (Figure 5). As
previously shown, coexpression of Btk*with SrcE378G
in rat-2 cells resulted in the growth of numerous (>350),
medium- to large-sized colonies (0.5±1.5 mm in diame-
ter) in agar after 3 weeks in culture. Mutation of Y223
32P-labeled Btk molecules
subjected to 10 cycles of automated Edman degradation sequence
analysis. PH, pleckstrin homology domain; SH, src homology do-
mains; pro, proline-rich sequences. Locations of Y223 and Y551 are
Table 1. Differences in Transforming Activity of Btk*and Btk*/Y223F
Number of Colonies in Soft Agar
5% Serum Number of Cells Plates (? 103) 20% Serum Number of Cells Plates (? 103)
25 1025 10
Rat-2 cells expressing SrcE378G were infected with retroviruses encoding Btk*or Btk*/Y223F. Cells (2 ? 103, 5 ? 103, or 1 ? 104) were plated
in soft agar in either 5% or 20% fetal calf serum. The growth of colonies in soft agar was allowed to proceed for 3 weeks, at which time the
colonies larger than 0.5 mm in diameter were counted. The transforming strength was discernible between Btk*and Btk*/Y223F within 10
days after plating in soft agar.
in Btk*resulted in a dramatic increase in number(>800)
and sizeofcolonies (1.0±1.5mmindiameter)whencoex-
pressed with SrcE378G (Figure 5). Btk*/Y223F induced
more rapidly the appearance of colonies and the acidifi-
cation of the media (within2 weeks)thanBtk*did (within
To quantify betterthe difference intransforming activ-
ity between Btk*and Btk*/Y223F, rat-2 cells coexpress-
ing these Btk mutants with SrcE378G were plated in
soft agar at three different cell concentrations in either
5% or20% serum(Table 1).Cells expressing Btk*/Y223F
formed at least three times more colonies than cells
expressing Btk*under all conditions (Table 1).
In separate experiments, NIH 3T3 cells were infected
with retroviruses encoding wild-type Btk, BtkY223F,
Btk*, or Btk*/Y223F and plated in soft agar at 1 ? 104
cells/plate in 5% or 20% serum. Btk wild-type or
BtkY233 did not induce agar colony formation. Cells
expressing Btk*/Y223F formed numerous (>800), large-
after plating. Cells expressing Btk*gave rise to only a
few (50±100), medium-sizedcolonies underthese condi-
tions (data not shown).
3).Btk*/Y223F exhibited asmall increaseinkinase activ-
ity (<2-fold) compared with Btk*in both autophosphor-
ylation and transphosphorylation reactions (Figure 6,
We also compared the transphosphorylation activity
between Btk*and Btk*/Y223F using a synthetic peptide
of 12 aa containing sequences surrounding Y223 as
a substrate. The transphosphorylation activity of Btk*/
Y223F was slightly elevated compared with Btk* (<2-
fold),which was consistentwiththe enolase phosphory-
lation results (data not shown). This demonstrates that
the Y223F mutation does not dramatically enhance Btk
kinase activity. Thus, increase in transforming activity
Y223F Mutation Does Not Significantly Affect
Btk Kinase Activity
The increase in biological activity of Btk*/Y223F may be
explained by a dramatic increase in Btk kinase activity,
which is illustrated in the cases of transforming mutants
of abl and src oncogenes (Coussens et al., 1985; Iba et
al., 1985; Konopka and Witte, 1985; Lugo et al., 1990).
To test whether the Y223F mutation enhances tyrosine
kinase activity, we compared the in vitro kinase activity
of Btk in the presence or absence of Y223F mutation.
Wild-type Btk, BtkY223F, Btk*, Btk*/Y223F, and kinase-
inactive Btk*/K430R were coexpressed with SrcE378G
inrat-2 cells. Btk proteins were immunoprecipitated and
analyzed forautokinaseand transphosphorylationactiv-
ity using enolase as a substrate. Btk*/K430R exhibited
no autokinase and very low transphosphorylation activ-
ity (Figure 6, lane 5). This indicates that the immunopre-
cipitates were largely free of contaminating kinases.
When the extent of phosphorylation is normalized to
the amount of protein present in each kinase assay,
BtkY223F had similarautokinase andtransphosphoryla-
tion activities compared with wild-type Btk (Figure 6,
lanes 1 and 2). As previously shown by Li et al. (1995),
the in vitro autokinase activity of Btk*protein was not
enhanced compared with wild-type Btk (Figure 6, lane
Figure 6. Tyrosine Kinase Activities of Btk* or Btk*/Y223F Coex-
pressed with SrcE378G
Rat-2 cells expressing SrcE378G were infected with retroviruses
encoding wild-type Btk (lane 1), BtkY223F (lane 2), Btk* (lane 3),
Btk*/Y223F (lane 4), and kinase-inactive Btk*/K430R (lane 5). Btk
proteins were immunoprecipitated from 1 ?107cells using antibod-
ies directed against Btk (N2). Btk autokinase as well as transphos-
phorylationactivity using enolase as a substrate (2?g/reaction) was
measured in 20 mM PIPES (pH 7.0), 20 mM MnCl2, 20 ?M ATP, 10
?Ci of [?±32P]ATP at 30?C for 10 min. Phosphorylated proteins were
resolved by SDS±PAGE and detected by autoradiography. The level
of Btk proteins was analyzed by Western blot analysis of the immu-
noprecipitates using anti-Btk antibodies (N2) (?Btk). The positions
of Btk and enolase are indicated by arrows.
Regulation of Btk by Autophosphorylation
of Btk*/Y223F is most likely due to an alteration in signal
transduction mediated in some manner by the SH3
Kinetic studies by Saouafetal.(1994)showedsequen-
tial activationof cytoplasmic tyrosine kinases uponsIgM
cross-linking of B cells. Btk activation follows that of
Lyn and Blk, while Syk becomes activated gradually
following Btk activation. This suggested that Btk tyro-
sine phosphorylation in B cells may be dependent on
activation of Src family tyrosine kinases. Genetic analy-
sis of animals carrying null alleles of Fyn orLyn demon-
strate the specific role of these src family tyrosine ki-
nases in Btk signaling. Although Fyn knockout animals
do not display any severe abnormality in B cell develop-
ment, B cells derived from these animals are defective
in IL-5 signaling (Appleby et al., 1995). B cells from Lyn
knockouts show impaired response to sIgM stimulation
and lack of B-1 B cell production (Hibbs et al., 1995).
Btk knockout animals display an Xid phenotype with
impaired responses in IL-5 and sIgM signaling (Khan et
al., 1995; Kerner et al., 1995). Thus, Lyn and Fyn appear
to participate in distinct Btk-mediated signaling during
B cell development.
A Two-Step Mechanism for Btk
In this report, we show that two different modes of acti-
vation lead to phosphorylationof the same two tyrosine
residues of Btk. The same tyrosine phosphorylations
can be induced upon sIgM cross-linking of a human B
cell line (Rawlings et al., 1996), which correlates with
enhancement of Btk autokinase activity. While phos-
phorylationofBtk atY551by Src family kinases is crucial
for activating Btk function, it is not yet clear how phos-
phorylation at Y223 regulates Btk signaling in B cells.
Contrary to a recent report by Mahajan et al. (1995),
we demonstrate that Y551 is a transphosphorylation
site.They arguethatY551 is amajorBtk autophosphory-
lation site, since a Y551F mutation abolishes Btk tyro-
sine phosphorylation in their experiments. However, we
have observed that the Y551F mutant immunoprecipi-
tated from NIH 3T3 cells is still kinase active and be-
comes autophosphorylated atothersites (Lietal., 1995).
The discrepancy may be due to different expression
levels of catalytically active Btk proteins in two indepen-
dent experimental settings. Our mapping studies and
mutational analyses clearly illustrate that the major Btk
autophosphorylation occurs at Y223, which resides in
a distinct peptide from that containing Y551. Mahajan
et al. (1995) showed that CNBr cleavage of autophos-
phorylated Btk yielded a single-sized phosphopeptide
that they thought was phosphorylated only at Y551.
However, CNBr cleavage of Btk protein can generate
two peptides of about 7 kDa containing either Y551 (61
aa from D510±M570) or Y223 (63 aa from G164±M226).
Since these two peptides could not be resolved by one-
dimensional SDS±PAGE, the peptides derived from au-
tophosphorylated Btk by CNBr cleavage in their report
likely containspecies phosphorylated atY551and Y223.
SH3 Domain in Btk Signaling
Autophosphorylations of Src family and receptor tyro-
sine kinases occur in their catalytic domains and en-
hance their enzymatic activities (reviewed by Cooper
and Howell, 1993; Kazlauskas, 1994). In contrast, the
major Btk autophosphorylation site is mapped to Y223
in the SH3 domain. The precise role of the Btk SH3
domain in signaling and its ligand specificity is not
known. Mutations in the SH3 domain of c±src or c±abl
increase their transforming potentials, suggesting that
their SH3 domains have a role in down-regulation of
their kinaseactivity (J acksonand Baltimore, 1989; Franz
et al., 1989;Hiraiand Varmus, 1990; J acksonet al., 1993;
Mayer and Baltimore, 1994; Okada etal., 1993). Deletion
of the Btk SH3 domain enhances Btk*-mediated trans-
formation of fibroblasts upon stimulation by SrcE378G
(D. E. H. A. et al., submitted). Since deletion ormutation
of the Btk SH3 domain does not appear to affect Btk
kinase activity directly, itis likely to regulate Btk function
by binding to regulatory molecules.
SH3 domains bind to peptides containing proline-rich
sequences. The SH3 domain core is composed of two
perpendicularand antiparallel three-stranded ?-pleated
sheets. The hydrophobic surface contains shallow
grooves formed by highly conserved aromatic amino
acids that contact proline-rich peptides. Recent struc-
tural and mutational analyses of SH3 domains from a
wide range of signaling molecules identified important
residues involved in these protein±protein interactions
(Knudsen et al., 1995; Maignan et al., 1995; Goudreau
et al., 1994; Terasawa et al., 1994; Yu et al., 1994; Koy-
ama et al., 1993; Kohda et al., 1993; Lim et al., 1994;
Feng et al., 1994). Based on structural comparison with
the Fyn SH3 domain, Btk Y223 represents one of the
highly conserved aromatic residues forming the surface
of the peptide-binding groove (Noble et al., 1993). Muta-
tional analysis of the c±src SH3 domain demonstrated
that Y90 of c±src (equivalent to Btk Y223) is critical for
SH3-mediated protein±protein interactions (Erpel et al.,
1995). Thus, Y223 of Btk is highly likely to play a crucial
role in ligand binding.
Role of Src Family Tyrosine Kinases
in Btk Activation
The Btk*mutation increases membrane localization of
Btk (Li et al., 1995). A recent structural study shows that
PIP2 binds to the PH domain at the site of the Btk*
mutation (E41K) (Ferguson et al., 1995). This suggests
that Btk* may alter the binding affinity of the Btk PH
domain to specific phosphoinositol lipids that may aid
Btk association with membranes. We have demon-
strated that activation of Src family tyrosine kinases
potentiate Btk*transforming activity, while suppression
of Src family tyrosine kinases by Csk diminishes Btk*
activation (D. E. H. A. et al, submitted). This data sup-
ports the idea that Src family tyrosine kinases act as
upstream regulators of Btk. In this assay, Src kinases
show synergy only withBtk*not with wild-type Btk. Btk*
is presumably more accessibleto activatedSrc kinases,
which results inenhanced tyrosine phosphorylationand
prepared by transient transfection of 293T cells (Muller et al., 1991;
Pear et al, 1993) and were used to infect NIH 3T3 cells. Stable cell
lines expressing Btkwere generated by selectioninG418.Recombi-
nant vaccinia viral constructs expressing Lyn or BtkdelSH3 were
produced as described previously (Rawlings et al., 1996).
Function of Phosphorylation at Y223
in the SH3 Domain
Phosphorylationof a tyrosine residue inthe SH3 domain
of c±src has been recently implicated as a mechanism
of regulating SH3-mediated signaling. The Y138 residue
of the c±src SH3 domain (equivalent to Y263 of Btk)has
beenshownto be phosphorylatedby the PDGF receptor
(M. Broome and T. Hunter, personal communication).
The Y138F mutation acts as a dominantnegative inhibi-
tor of PDGF-stimulated mitogenesis without inhibiting
Src catalytic activity (M. A. Broome and T. Hunter, sub-
mitted). Interestingly, phosphorylation at Y138 signifi-
cantly diminishes SH3 binding affinity for peptide li-
gands containing proline-rich sequences, suggesting a
role for this phosphorylation in SH3-mediated ligand
We have shown that mutation of Y223 of Btk potenti-
ates Btk*transforming activity. A block deletion of the
SH3 domain of Btk*induces a similar phenotype, sug-
gesting that mutation of Y223 is equivalent to inactiva-
tion of SH3 function. This leaves us with two alternative
interpretations. First, activation of Btk by phosphory-
lation at Y551 leads to autophosphorylation at Y223,
which would down-regulate Btk activity. In this case,
mutation of Y223 would prevent down-regulation of Btk
by autophosphorylation and result in activation of Btk
function. Alternatively, both mutation of Y223 or phos-
phorylation at Y223 may alter SH3 function and lead to
activation of Btk signaling. Since the Y223F mutation
does not significantly change Btk catalytic activity, it is
likely that in either case Y223 in the SH3 domain regu-
lates Btk activity by modulating binding to cellularregu-
latory molecules. One must certainly include the possi-
bility that phosphorylation of Y223 creates a docking
site for a SH2 containing protein or proteins.
A genetic study in C. elegans implicates the SLI-1
gene product, a homolog of mammalian c±cbl, as a
negative regulatorof a receptortyrosine kinase pathway
(Yoon et al., 1995). This suggests a similarrole forc±cbl
in down-regulating tyrosine kinase signaling pathways
inmammalian cells. Recently, it was reported thata GST
fusion protein containing the SH3 domain of Btk can
bind to the c±cbl protooncogene product from B cell
extracts (Cory et al., 1995). It is not known whetherfull-
lengthBtk is ableto interactwithc±cbl. Autophosphory-
lation at Y223 is predicted to increase negative electro-
static potential and perturb the hydrophobic nature of
the ligand-binding surface of the SH3 domain. This is
likely to attenuate binding of proline-rich peptides to
the SH3 domain. Btk autophosphorylation substantially
increases uponB cell activationbut the role ofBtk auto-
phosphorylation in B cell signaling is not known. A fur-
ther search for B cell proteins that interact with subdo-
mains of Btk should provide candidate regulators of Btk
Construction of GST±BtkSH3 Fusion Protein
Polymerase chain reaction was performed to amplify the region
encoding the SH3 domain (nucleotides 787±951) from murine Btk
cDNA using the primers SH3-5? (5?-GTGGATCCAAGGTCGTGGCCC
TTTAT-3?) and SH3-3? (5?-CCGAATTCTGTAGCCTTCCTGCCCAT).
The polymerase chain reaction product was cloned into pGEX-2T
(Pharmacia Biotech). The fusion protein was induced and purified
as described previously (Frangioni and Neal, 1993).
An EcoRI fragment of Btk (nucleotides 136±1457) containing the
Btk*mutationwas clonedinto M13mp18vector and single-stranded
DNA was generated. Site-directed mutagenesis was performed on
single-stranded DNA template using an oligomer (5?-ATGTAATCGA
AAAGGGCCA-3?)containing phenylalanineattheY223 position(Am-
ersham). DNA sequencing was performed to verify the presence of
the Y223F mutation. Constructs containing BtkY223F orBtk*/Y223F
were generated in the pSR?MSVtk±neo vector (Muller et al., 1991)
and were transfected into 293T cells to generate helper-free retro-
In Vivo32P04-Labeling, Two-Dimensional Tryptic
Phosphopeptide Mapping, and Phosphoamino
Cells (1 ? 107) were used for32P-orthophosphate labeling. To allow
sufficient protein expression, vaccinia viruses encoding Btk were
infectedinto 1?107NIH3T3cells 12±15hrpriortoinvivo32P-labeling
(Rawlings et al., 1996). Cells were washed with phosphate-free
DMEM and labeled with 1 mCi/ml of32P-orthophosphate (New En-
glandNuclear)in2 mlofphosphate-free DMEMfor3 hr.Cellextracts
were prepared in lysis buffer (20mM HEPES [pH7.4], 100 mMNaCl,
3% Triton X-100, 10% glycerol, 2 mM EDTA, 1 mM Na3V04, 10 mM
NaF, and protease inhibitors including 1 mM PMSF, 5 ?g/ml aproti-
nin, and 5 ?g/ml leupeptin). Btk was immunoprecipitated with 20
?g of affinity-purified antibody (N2), which specifically recognizes
the N-terminal portion of Btk (Tsukada et al., 1993; Li et al, 1995).
Immunoprecipitates were separated by SDS±PAGE (6% polyacryl-
amide), blotted onto nitrocellulose membrane, and visualized by
autoradiography. Btk protein was excised from the membrane and
digested with 15 ?g TPCK±trypsin (Sigma) twice for 3 hr at 37?C in
50 mM NH4HCO3, lyophilized, and treated with performic acid at
4?C for 1 hr (Luo et al., 1991). Two-dimensional analysis of tryptic
peptides was performed by thin-layer electrophoresis at pH 1.9,
followed by chromatography in phosphochromatography buffer
(Boyle etal., 1991). For phosphoamino acid analysis, peptides were
extracted fromthin-layercellulose plates using pH1.9electrophore-
sis buffer and hydrolyzed for 1 hr at 110?C in 6 Na±HCl.32P-labeled
amino acids were mixed with phosphoamino acid standards and
separated bythin layerelectrophoresis at pH 3.5(Boyle etal., 1991).
Standards were developed by ninhydrin staining and
amino acids were identified by autoradiography.
Large Scale Purification of Btk from Vaccinia
NIH 3T3 cells (2 ? 109) were coinfected with vaccinia viruses ex-
pressing BtkY551F and Lyn. After a 12±18 hrinfection, 5 ? 107cells
were washed with phosphate-free DMEMand incubated with 10±20
mCiof32P-orthophosphate in5±10 ml ofphosphate-free DMEM plus
0.1% dialyzed fetal calf serum for an additional 4±6 hr. Cells were
treated with 200 ?M of Na3VO41 hr prior to harvesting and lysed in
the lysis buffer as described in the previous section. Cell extracts
were applied to a phosphocellulose column (Whatman)and washed
with buffers containing low salt and detergent (50 mM Tris [pH 7.4],
150 mM NaCl, 0.1% Triton X-100, 10% glycerol, 1 mM Na3V04),
mediumsalt anddetergent(300mM NaCl),low saltwith nodetergent
(150 mM NaCl), and then eluted with high salt buffer without deter-
gent (500 mM NaCl). By immunoblot analysis, Btk was observed to
Cell Culture and Retrovirus or Vaccinia
NIH 3T3 cells were grown in Dulbecco's modified Eagle's medium
(DMEM) supplemented with 10% calf serum. Recombinant Btk was
cloned into the retroviral expression vector, pSR?MSVtk±neo, as
described by Li et al., (1995). Helper-free retroviral stocks were
Regulation of Btk by Autophosphorylation
elute with 500 mM NaCl, while Lyn was recovered in the initial flow-
through fraction. The phosphocellulose eluate was concentrated
through a Centricon C-30 filter (Amicon) and stored at ?20?C with
25% glycerol. Btk was immunoprecipitated with affinity-purified an-
tibodies (N2) from the phosphocellulose eluate, separated by SDS±
PAGE, blotted onto nitrocellulose, and visualized by autoradiogra-
phy. Btk protein was digested with 15±25 ?g of TPCK±trypsin twice
for 90 min at 37?C, dried, and resuspended in ddH2O. The tryptic
peptides were denatured at 90?C for 10 min and then applied to
an anti-phosphotyrosine column (1G2-Sepharose). The column was
washed with 50 mM ammonium acetate (pH 7.0), and then eluted
with 50 mM ammonium acetate and 500 mM acetic acid (pH 3.7).
samples were plated in duplicate in medium containing either 5%
or 20% fetal calf serum. Colonies equal to or larger than 0.5 mm in
diameter were counted 2±4 weeks after plating.
In Vitro Btk Kinase Assay
Rat-2 cells expressing SrcE378G were infected with retroviruses
encoding different forms of Btk. Cell extracts were prepared in lysis
buffer (20 mM Tris [pH 7.4], 150 mM NaCl, 1% Triton X-100, 2 mM
EDTA, 1mM Na3V04, 10 mM NaF, and protease inhibitors including
1 mM PMSF, 5 ?g/ml aprotinin, and 5 ?g/ml leupeptin) and Btk was
immunoprecipitated by antibodies recognizing Btk (N2). Immuno-
precipitates were washed four times in lysis buffer and the kinase
activity of Btk proteins was measured inkinasebuffer(20 mMPIPES
[pH 7.0], 20 mM MnCl2, 20 ?M ATP, 10 ?Ci of [?±32P]ATP) including
2 ?g of enolase as an exogenous substrate for 15 min at 30?C. The
reaction mixtures were boiled in SDS sample loading buffer and
separated by SDS±PAGE (8%). Phosphorylated proteins were de-
tected by autoradiography. The level of Btk protein was measured
by Western blot analysis using antibodies against Btk (N2).
HPLC Purification of Tryptic Peptides
and Peptide Sequencing
The eluate from the anti-phosphotyrosine columnwas concentrated
to 250 ?l and applied to a Betasil C18 250 mm ? 4.6 mm reverse
phase HPLC column (Keystone Scientific), using a flow rate of 1.25
ml/min.Aftera 10minwash at5% acetonitrile, the phosphopeptides
were eluted with a linearacetonitrile gradientof 5%±27%. Fractions
were obtained at 1 min intervals and radioactivity in each fraction
was measured. Peak fractions containing
and subjected to ten cycles of automated Edman degradation se-
quence analysis. Blank cycles at the appropriate position indicated
the presence of a phosphotyrosine residue.
32P were pooled, dried,
Correspondence should be addressed to O. N. W. We thank D.
Saffran, and A. Satterthwaite for their helpful comments, and J .
Shimaoka and J . White for assistance in preparation of the manu-
script and figures. We are grateful to Dr. K. Faull for provision of
HPLC facilities. We are grateful to Drs. L. Zipursky, S. Smale, and
D. Black for their critical comments on the manuscript. We thank T.
Li for providing us with Btk retroviruses and R. M. Kato for excellent
technical assistance. This work is supported by a fellowship from
the CancerResearch Institute(H. P.),Human Genetics Training grant
GM08243 (M. W.), Special Fellowship of the Leukemia Society of
America (D.E. H. A.),NationalInstitutes of HealthPhysicianScientist
Award AR01912 (D. J . R.), Pediatric Scientist Training Grant fel-
lowship (A. S.), and National Cancer Institute Grants (O. N. W.).
O. N. W. is an Investigatorof the Howard Hughes Medical Institute.
IgA Protease Cleavage
Btk proteins expressed by vaccinia virus in 1 ? 107NIH 3T3 cells
were partially purified by phosphocellulose chromatography as de-
scribed above. The Btk proteins were incubated with 1 ?g of IgA
protease (Boehringer Mannheim) in 50 mM Tris (pH 7.4) at 16?C for
16 hr, lyophilized, separated by SDS±PAGE (15%), and blotted onto
nitrocellulose. Western blot analysis was performed using poly-
clonal antibodies that specifically recognize either the N-terminal
(N2)orthe C-terminal15aa ofBtk,oramonoclonalantibody directed
against phosphotyrosine (4G10, Upstate Biotechnology, Incorpo-
rated). Secondary antibody was either goat anti-rabbit or goat anti-
mouse conjugatedto horseradish peroxidase (Bio-Rad).Theprotein
was detected using chemiluminescent signal (ECL, Amersham).
Received November 5, 1995; revised April 2, 1996.
Purification of Histidine-Tagged Btk Protein
Wild-type Btk containing a six histidine tag at the C terminus was
constructed in the pCD-SR?296 expression vector (Takee et al.,
1988). Btk was expressed in 293T cells by transient transfection.
Cells were lysed in lysis buffer 2 days after transfection (see the
previous sections). Cellextracts were applied to aphosphocellulose
column (Whatman) in 50 mM Tris (pH 7.4), 150 mM NaCl, 5 mM
EDTA, 1% Triton-X 100, 10% glycerol, and protein inhibitors (1 mM
PMSF, 5 ?g/ml aprotinin, and 5 ?g/ml leupeptin). The column was
washed with 50 mM Tris (pH 7.4), containing 250 mM NaCl and Btk
protein was eluted with 300±500 mM NaCl in 50 mM Tris (pH 7.4).
The eluted proteins were applied to a Ni2?±NTA agarose column
(Qiagen). The column was washed with 5 mM imidazole (pH 7.0)
and the Btk protein was eluted with 25 mM imidazole (pH 7.0) in
15% glycerol. Btk protein was concentrated using Centricon C-30
filters (Amicon) and stored at ?20?C.
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Note Added in Proof
The data reported in the text as D.E.H.A. et al., submitted, and M.A.
Broome and T. Hunter, submitted, are now in press:
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Witte, O.N. (1996). Regulation of Btk by Src family tyrosine kinases.
Mol. Cell. Biol. 15, in press.
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