Cell, Vol.

72, 279-290, January 29, 1993, Copyright 0 1993 by Cell Press

Satoshi Tru~kada,‘~~ Douglas C. Saffran,2 Bolen et al., 1991). In addition, several unclassified tyro-
David J. Ba~lings,~ Omella Parolini,3 sine kinases have been reported to be expressed in hema-
R. Cutler Allen,’ lvana Klisak,s Robert S. Sparkes,5 topoietic cells, including csk (Nada et al., 1991) syk (Tani-
Hiromi Kubagawa,6 Thuluvancheri Mohandas,’ guchi et al., 1991) JAKl, JAK2 (Wilks et al., 1991), Tyke
Shirley Duan,2 John W. Belmont,’ Max D. Cooper,6 (Velazquez et al., 1992) and PTK72 (Hutchcroft et al.,
Mary Ellen Conley,3 and Owen N. Whte1p2 1992). While some of these kinases are ubiquitously ex-
‘Howard Hughes Medical Institute pressed (abl and src), others are more restricted and Ick,
2Department of Microbiology and Molecular Genetics hck, and blk are exclusively expressed in hematopoietic
University of California, Los Angeles cells (Marth et al., 1985; Ziegler et al., 1987; Quintrell et
Los Angeles, California 90024 al., 1987; Holtzman et al., 1987; Dymecki et al., 1990).
3Department of Pediatrics The general structure of cytoplasmic tyrosine kinases is
University of Tennessee College of Medicine characterized not only by the tyrosine kinase catalytic do-
and Department of Immunology main, but also by the presence of src homology (SH) do-
St. Jude Children’s Research Hospital mains (reviewed in Koch et al., 1991; Pawson and Gish,
Memphis, Tennessee 38101 1992).
4Howard Hughes Medical Institute Several of the src subfamily tyrosine kinases are cou-
Institute for Molecular Genetics pled to cell surface receptors in lymphocytes and function
Baylor College of Medicine as signal transducers after specific ligand binding. Exam-
Houston, Texas 77030 ples are the associations of CD4 and CD8 with Ick (Veillette
5Department of Medicine et al., 1988), interleukin-2 receptor 8 chain with Ick (Hata-
UCLA School of Medicine keyama et al., 1991) T cell receptor complex with fyn
Los Angeles, California 90024 (Samelson et al., 1990), and the immunogfobulin receptor
6Departments of Medicine, Pediatrics, and Pathology complex with lyn, fyn, and blk (Yamanashi et al., 1991;
Howard Hughes Medical Institute Burkhardt et al., 1991). Alternatively, some cytoplasmic
University of Alabama, Birmingham tyrosine kinases may serve essential regulatory functions.
Birmingham, Alabama 35294 For example, csk was reported to modii the activity of
‘Division of Medical Genetics other tyrosine kinases (i.e., sfc subfamily genes) and
Harbor-UCLA Medical Center down-regulate the signal transduction pathways in which
Torrance, California 90502 they participate (Nada et al., 1991; Okada et al., 1991).
To define genes that regulate B cell growth and differen-
tiation, we have developed in vitro culture models to enrich
Summary for immature B cells and their progenitors (Whitlock and
Witte, 1982; Scherle et al., 1990; Saffran et al., 1992; Faust
We describe a novel cytoplasmic tyrosine kinase, et al., 1993). Enriched populations of murine B cell progen-
termed BPK (B cell progenitor kinase), which is ex- itors were used to prepare cDNA libraries and screened
pressed in aff stages of the B lineage and in myeloid for new members of the cytoplasmic tyrosine kinase fam-
cells. BPK has classic SHl, SH2, and SH3 domains, ily. Here we describe a novel tyrosine kinase named BPK
but lacks myristylation sfgnals and a regulatory phos- (B cell progenitor kinase). BPK has high homology with
phorylation site corresponding to tyrosine 527 of several cytoplasmic tyrosine kinases: Dsrc28C (Gregory
c-src. BPK has a long, basic amino-terminal region up et al., 1987) tecl (fvlano et al., 1990), tech (GenBank and
stream of the SH3 domain. BPK was evaluated as a J. Ihle, personal communication), and itk (Siliciano et al.,
candidate for human X-linked agammagfobulinemia 1992). The high homology and structural similarity among
(XLA), an inherited immu mkfickmy characterized these genes suggests that they form a new subfamily of
by a severe deficit of B and plasma cells and profound cytoplasmic tyrosine kinases. The structure of BPK, its
hypogammaglobulinemia. BPK mrppcd to within 100 restricted expression pattern, and most remarkably its as-
kb of a probe defining the polymorphism most closely sociation with the pathogenesis of X-linked agamma-
linked to XLA at DXS178. Beduction in or the absence globulinemia (XLA), described below, support its role as a
of BPK mRNA, protein expression, and kinase activity key regulator of B cell development.
was observed in XLA preB and B cell lines. t?PK is XLA is the prototypic inherited humoral immunodefi-
llkefy the XLA gene and functions in pathways critical ciency described in 1952 by Bruton (reviewed by Conley,
to B cell expansion. 1992; Cooper et al., 1993). Affected males develop recur-
rent bacterial infections early in life. Circulating immuno-
Introduction globulin levels are profoundly decreased and there is a
severe deficit in B cells and their plasma cell progeny. T
The cytoplasmic protein-tyrosine kinases can be classified cell numbers are normal, and cellular immunity remains
into subfamilies, including the src, the abl, and the fps intact.
groups (Hanks et al., 1988; Eisenman and Bolen, 1990; B cells in female obligate carriers of XLA exhibit nonran-
Cell
200

ILKNRNQSLKPQSSIIRKTKKP 195
KEKTRm?NsLVSKmPnFwmDQRwRc py&--------ps~~KKp 170
KYHPKPwADQsYQc KYN---------LFRSSIRKT 165
--PTSLQPQSSLTT pJLL _---- ---- WQQIp 139
KmPcFwIwpTLc 1LENiaoSLKPl3S8HRKTKKP 195

HIBPK PILKKPLPPKPTAAPISTTR 293

ITK R---RSO-QRPEKT----- 257
TKCII A--KRPPPPIPPPE-IWTB ------------------ 235
DgRc2ec A--msgpvK ------- OYIPSNYVOARALLQL 226
ROBPK QILKKPLPPEPTMPISTSKLKK BOYIPSNDVTR-AEDSI 293

LKTATSPW--

MJBPK 491
ITK 457
TRCII 433
MKc2BC 419
HOBPK 442

WBPK RPPTQ- - 589

ITK LFMK-- 555
TKCII EPSRR-- 531
mc2ec TLI(BIYO 519

IQBPK II8 ILDVnm-ES 659

ITK VPS LAmARA-QL 625
TX11 IFT --NLIOS-RF 583
mc2w IFT LALVAQTLTD 590

Figure 1. Comparison of Amino Acid Sequences of Murine BPK, Itk, Tech, Dsrc23C, and Partial Sequence of Human BPK
Stippled areas indicate amino acid residues conserved in all sequences. Significant additional homology between murine BPK (MUBK) and individual
sequences is not indicated. Gaps (indicated by dashes) are introduced to optimize the alignment. The beginnings of the SH3, SH2, and SHl domains
are indicated by arrows. The sequence of tecl is highly homologous with tecll and was not included in the figure. HUBPK, human BPK.

dom X chromosome inactivation (Conley et al., 1988). This
implies that XLA results from an autonomous B lineage
defect that results in decreased proliferation or decreased
survival of affected cells. The XLA defect may interfere at
several levels in B lineage development. First, pre-B cell
expansion appears abnormal. The ratio of cl-, TdT+ pro-B
cells to pre-B cells has been shown to be inverted to a
predominance of pro-B cells, suggesting retarded pre-B
cell clonal expansion (Nishimoto et al., 1991; Campana et
al., 1990). Second, the maturation of surface immunoglob-
ulin-expressing B cells may also be abnormal. Small num-
bers of peripheral blood B cells (less than 1% of normal)
are present in individuals with XLA. These cells exhibit an
immature phenotype and appear arrested at an early stage
of development (Conley, 1985). This arrest is incomplete
and transition to mature antibody-producing cells does in-
frequently occur.
B cell clonal expansion occurs predominantly through
expansion of the pre-B cell pool (Rolink and Melchers,
1991). In XLA bone marrow cells, however, the prolifera-
tion of cp+ pre-6 cells is significantly reduced (Campana
et al., 1990) and fewer cu+ cells enter S phase (Pearl et
al., 1978). In contrast, the proliferation of TdT+, cu- pro-B
cells is normal. These data suggest that the XLA defect
directly interferes with early B lineage growth and clonal
expansion. Proposed mechanisms for the XLA defect in-
clude abnormal expression of a growth factor receptor, a
defect in a B lineage-specific signal transduction pathway,
or abnormal function of a lineage-specific transcription fac-
tor (Anker et al., 1989).
The XLA gene has been mapped to the midportion of
the long arm of the human X chromosome at Xq22 (Kwan
et al., 1988; Mensink et al., 1988; Malcolm et al., 1987;
Guioli et al., 1989; Kwan et al., 1990; Parolini et al., 1993).
Linkage analysis of over 500 individuals from 80 informa-
tive families has demonstrated no cross over between XLA
and a probe (~212) recognizing a polymorphism on Xq22
at DXS178. No large deletions have been detected in this
region. Recently, yeast artificial chromosome (YAC) and
pulsed-field gel analysis have been used to develop a
physical map of this region (Parolini et al., 1993). B lin-
eage-specific genes located within this region are likely
candidates for the XLA gene.
The BPK gene was independently mapped to Xq22 us-
Deficient Cytoplasmic Tyrosine Kinase in XLA
281
ACTIN-
ing fluorescent in situ hybridization with metaphase
mo8omes and X chromosome somatic cell hybrid analysis.
Multiple lines of evidence support tight linkage of BPK to
the XLA locus. Loss of expression of BPK, only in cell lines
from individuals affected with XLA, strongly supports the
argument that BfK plays a central role in human XLA and
is a critical component of B cell growth and development.
Results
BPK Belongs to a Novel Subfamily of Cytoplasmic
Tyroslne Kinases
A cDNA library prepared from B lymphoid progenitors was
screened with the tyrosine kinase domain sequence of the
human LTK gene under reduced stringency. One clone
contains a homologous but unique sequence in compari-
son with the catalytic domains of several known tyrosine
kinases. We screened a cDNA library of the 702/3 pre-B
cell line using this clone as the probe and obtained a nearly
full-length cDNA. Figure 1 shows the predicted amino acid
sequence of the murine gene. We have designated this
gene BPK. The sequence has a 1977 base open reading
frame, which predicts a protein of 659 amino acid residues
with a calculated molecular weight of 76,449 daltons. The
amino acid sequence between positions 383 and 652 of
BPK has several motifs found in other protein-tyrosine
kinase (SHl) domains (Hanks et al., 1988). The putative
ATP-binding motif, GTGQFG, is at position 409-414, and
the putative autophosphorylation site in the catalytic do-
main is conserved at amino acid position 551. The strong
indicator sequence of tyrosine kinase specificity
main VI corresponds to DLAARN (position 521-526) in
BPK, which is the same as those of abl, fps, and csk, but
different from those of src subfamily genes (DLRAAN).
BPK contains another consensus sequence, PVRWSPPE
(position 560-567) that distinguishes protein-tyrosine ki-
nases from serine/threonine kinases. Amino acid se-
quences corresponding to SH2 (269-382) and SH3 (21&
268) domains are also present. BPK lacks the hydrophobic
amino acid stretch typical of transmembrane regions of
receptor type tyrosine kinases.
An amino-terminal myristylation signal (a glycine at resi-
due 2 and lysine [or arginine] at position 7), essential for
posttranslational myristylation (Kaplan et al., 1988) was
not found in the sequence of BPK. The first methionine is
Figure 2. Northern Analysis of BPK mRNA Ex-
pression
Poly(A) RNA (2 ug) extracted from tissues and
cell lines was electrophoresed in a denatured
1% agarose-formaldehvdeoel and transferred
to nit&cellulose filter. Northern filters were hy-
bridized with 32P-labeled BPK cDNA, followed
by hybridization with 8-actin as the control for
uniform RNA loading. The arrowhead indicates
the BPK transcript (3 kb). The positions of 18s
and 28s rRNAs are indicated. Tissue distribu-
tion of the BPK gene in mice is shown in (A),
and expression of EPK in hematopoietic cell
lines is shown in (B).
chro- followed by alanine, and residue 7 is glutamic acid. The
short carboxyl terminus following the tyrosine kinase cata-
lytic domain of BPK lacks the residue equivalent to tyrosine
527 of c-src. This tyrosine is conserved in all members
of the src subfamily, and its phosphorylation has been
implicated as a mechanism for negatively regulating the
kinase activity of src family genes (Eisenman and Bolen,
1990; Kmiecik and Shalloway, 1987; Piwnica-Worms et
al., 1987). The amino-terminal region of BPK, referred to
as the unique region of cytoplasmic tyrosine kinases, is
unusually long (217 amino acid residues) when compared
with those of the src subfamily (70-100 amino acid resi-
dues). In this region, BPK contains a high number of basic
amino acids (16 basic residues in the first 60 residues).
Comparison of the murine clone and a partial human
BPK cDNA showed an identity of greater than 96% at
the amino acid level, including the amino-terminal unique
region and SH2 and SH3 domains (Figure 1). The deduced
amino acid sequence of BPK was compared with those
of other tyrosine kinases by FASTA search (Lipman and
Pearson, 1985). Amino acid identities of 40%-50% were
observed with Dsrc28C, tech, and itk kinases (Figure 1).
The highest homology was found between BPK, itk, and
tecll. There was significant homology within the unique
amino-terminal segments as well as the catalytic domains.
In addition to the high percentage identity, several fea-
tures of the deduced amino acid sequence of BPK were
shared with Dsrc28C tecll, and itk. These included the
lack of myristylation signals, the lack of carboxy-terminal
regulatory phosphorylation sites, long, basic amino-termi-
in subdo- nal regions, and the presence of SH2 and SH3 domains.
These common features suggest that these genes form a
new subfamily of cytoplasmic tyrosine kinases.
Cell Lineage-Specific Expression
of BPK Transcripts
To determine the expression pattern of BPK transcripts,
poly(A) RNA was extracted from various murine tissues
and analyzed by Northern hybridization using BPK cDNA
as the probe (Figure 2A). A major 3 kb transcript was de-
tected in bone marrow, spleen, lymph node, and faintly in
thymus of adult mice and murine fetal liver (day 14) but
not in nonhematopoietic tissues. Expression of BPK tran-
scripts was further examined in various hematopoietic cell
lines (Figure 28). BPK transcripts were detected in cell
Cdl
282

6 C D E Figure 3. Detection of the Protein Product of

BPK and Its Kinase Activity
(A) Characterization of anti-BPK antiserum.
(kd) 1 P 2 P P12 P P Murine BPK cDNA cloned into pBluescript was
transcribed in vitro under the control of the T7
promoter. Transcribed RNA was then trans-
lated in rabbit reticulocyte lysate in the pres-
ence of [“Slmethionine (lane 1). The in vitro
translation product was then immunoprecipi-
tated by anti-BPK antiserum (lane 2). (In each
experiment, the preimmune serum was used in
lane Pas a control.) The positions of molecular
weight markers are indicated at left. The arrow-
head indicates the BPK protein.
(B) Detection of BPK protein in vivo. 70213 cells
were metabolically labeled with 100 uCi of
[%]methionine for 4 hr, and the cell lysate was
immunoprecipitated by anti-BPK antiserum.
(C) Kinase activity of BPK protein in vitro. BPK
protein immunoprecipitated from 7Oz/3 cells by
anti-BPK antiserum was assayed for kinase ac-
tivity by adding [Y-‘~P]ATP in 20 mM PIPES (pH 7.0) containing 20 mM MnCl at 30°C, 15 min (lane 1). In lane 2, acid-denatured enolase was added
as a substrate (the asterisk indicates enolase).
(D) Tyrosine specificity of autophosphorylation of BPK. BPK protein immunoprecipitated from 70x/3 cells by anti-BPK antiserum was subjected to
in vitro kinase reaction by adding cold ATP (10 f&l), followed by immunoblotting with an anti-P-Tyr antibody. (Dark regions on the bottom are the
cross-binding of the second antibody [goat anti-mouse immunoglobulin G] to rabbit immunoglobulin G.)
(E) Anti-P-Tyr immunoblotting of BPK immunoprecipitated from 70213 cell lysate (without in vitro kinase reaction).

lines representing all stages of B cell development, myelo-
monocytic cell lines, and a macrophage cell line. Tran-
scripts of BPK were not detected in T lineage cell lines.
A similar pattern of B cell and myeloid expression was
observed when a panel of human hematopoietic cell lines
were examined (data not shown).
Detection of BPK Protein and Its Tyrosine
Kinase Activity
The expression of BPK gene products was investigated
using an antiserum prepared against the unique amino-
terminal region (see Experimental Procedures). The speci-
ficity of this antiserum was demonstrated by immunopre-
cipitating in vitro translated BPK protein labeled with
[%]methionine (Figure 3A). In vitro translated BPK pro-
teins exhibited molecular masses of 77 (corresponding to
the full-length amino acid sequence), 66, 56, and 50 kd
(corresponding to translational initiations at internal methi-
onines). Antiserum against the amino-terminal region of
BPK efficiently precipitated the 77 and 66 kd translation
products. We subsequently used this antiserum for the
detection of BPK protein in vivo. Several cell lines were
metabolically labeled with [%)methionine and immuno-
precipitated with anti-BPK antiserum. The major protein
species of 77 kd was detected in B lineage cells (Figure
38) and myelomonocytic cells (data not shown). The ap-
pearance of the 77 kd protein correlated with the expres-
sion pattern of BPK transcripts among B lineage cells
tested (data not shown).
Cell lysates of 7Oz/3 were immunoprecipitated by anti-
BPK antiserum and assayed for autokinase activity. The
results (Figure 3C, lane 1) demonstrate that BPK protein
is able to catalyze the autophosphorylation reaction. Fur-
thermore, BPK had transphosphorylation activity for eno-
lase (lane 2; whereas histone Hl, casein, and immuno-
globulin heavy chain were not phosphorylated [data not
shown]). In the presence of the exogenous substrate, auto-
phosphorylation of BPK became weak, which may reflect
the effect of substrate competition or preference for trans-
phosphorylation by the BPK kinase activity. BPK protein
immunoprecipitated from 7Oz/3 cell lysate was immu-
noblotted with anti-P-Tyr antibody after an in vitro kinase
reaction (Figure 3D). The appearance of the 77 kd band
showed that BPK protein was autophosphorylated on tyro-
sine residues in vitro. Phosphoamino acid analysis of in
vitro labeled BPK protein revealed that only tyrosine resi-
dues were autophosphorylated (data not shown). How-
ever, we could not detect tyrosine phosphorylation of BPK
in vivo by anti-P-Tyr blotting of BPK protein precipitated
from 7Oz/3 cell lysate (Figure 3E). The anti-murine BPK
serum also recognized the human BPK protein (see
below).
l3PK Maps to the Long Arm of the X Chromosome
(Xq22) by Fluorescence In Situ Hybridization
and Somatic Cell Hybrid Analysis
Murine BPK cDNA was used to screen a human DNA cos-
mid library to isolate human BPK clones (Experimental
Procedures). Cosmid C3-1 was selected on the basis of
hybridization to a murine unique region probe after sec-
ondary screening. A more restricted unique region sub-
clone (U4X3) was derived from C3-1 (see Experimental
Procedures). The U4X3 unique region and murine BPK
cDNA probes revealed restriction fragments of identical
size in Southern blot analysis of human genomic DNA
(Figure 4). The location of the U4X3 subclone was estab-
lished by hybridization to specific restriction fragments
mapped within the cosmid clone C3-1 (Figure 48). Both
the full-length cosmid clone (C3-1) and the unique region
subclone U4X3 were used as human-specific BPKprobes.
Fluorescence in situ hybridization was used to deter-
mine the chromosomal location of human BPK (data not
Deficient Cytoplasmic Tyrosine Kinase in XLA
203

normal or reduced human X chromosomes were evalu-

ated. The WK human cosmid subclone, U4X3, containing
exonic sequence from the unique region of the gene was
used for hybridization with somatic cell hybrid DNA in
Southern blot analysis (Figure 5). The BPK probe hybrid-
ized specifically with DNA from somatic hybrids containing
the entire human X chromosome and to DNAfrom reduced
X chromosome cell lines containing regions overlapping
Xq21-22. There was no hybridization with X chromosomal
regions telomeric to Xq22. These results were further con-
firmed by Southern blot analysis using DNA specimens
from individuals containing X chromosomal deletions re-
sulting in the eye disorder, choroideremia (Cremers et al.,
Probe: 1989). The U4X3 probe hybridized with DNA specimens
Human BPK BPK
Cosmid Subclone cDNA with large X chromosome deletions spanning Xq21.1-
Xq21.3 (data not shown). These results and those ob-
tained by fluorescence in situ hybridization independently
localized the human BPK gene to Xq22.
B
The BPK Gene Is Tightly Linked to the Region
of Xq22 Identified as the XLA Region Locus
F-i
The gene for XLA has been mapped to the midportion of
the long arm of the X chromosome at Xq22. In linkage
studies of more than 500 individuals in over 80 informative
B B B
families, no recombination has been detected between
B : BamHl XLA and a probe (~212) with a LOD score greater than 11,
Cosmid Subclone Probe H : Hindlll
recognizing a polymorphism at DXS178 (Kwan et al., 1988;
Figure 4. Characterization of the Human BPK Cosmid Clone C3-1
Mensink et al., 1988; Malcolm et al., 1987; Guioli et al.,
(A) Autoradiograph of restriction enzymedigested human genomic
1989; Kwan et al., 1990; Parolini et al., 1993). The closest
DNA. The human cosmid subclone U4X3 or murine BPK cDNA was reported flanking markers, at DXS442 and DX94, are ap-
used as a probe on Southern blots of genomic DNA digested with proximately 3 CM apart (Parolini et al., 1993). To localize
EcoRl or Hindlll. Sizes were estimated from a Hindllldigested 5 phage the gene for BPK more precisely, the U4X3 human cosmid
standard.
unique sequence probe was used to screen a panel of
(6) A partial restriction map was constructed by restriction digestion
of cosmid DNA as described (Experimental Procedures). The region YAC clones that formed a partial contig map in the region
containing the human BPK cosmic subclone sequence was deter- defined by the flanking markers. The BfK probe hybrid-
mined by hybridization of specific BamHl and Hindlll fragments to the ized with a 200 kb YAC, designated A2. This YAC had been
U4X3 probe.
previously isolated using the probe most closely linked to
XLA, ~212 (Figure 8). The A2 YAC also hybridized to mu-
rine BPK cDNA probes (data not shown). All five YAC
shown). The 28 kb BPK-specific cosmid clone C3-1 was clones isolated with a probe, A2R, defining one of the ends
hybridized to metaphase spreads. Fluorescent micros- of the human insert (Parolini et al., 1993) in the A2 YAC,
copy revealed twin fluorescent dots on sister chromatids were also positive with the BPK cosmid probe (Figure 8).
of the X chromosome in female metaphase spreads. This None of the 21 additional YACs, including a YAC con-
was confirmed by simultaneous hybridization with an a taining human sequence from the opposite end of the A2
satellite probe, specific for repeat sequences on the X YAC, were recognized by the BPK cosmid probe. Further
centromere, and the C3-1 probe. SPK-specific twin dots restriction mapping demonstrated that the BPKgene was
were present on both X chromosomes, which were posi- within 80 kb of the end of the A2 YAC and within 100 kb
tively identified by the intensely staining satellite probe. of the XLA-linked ~212 probe (Figure 8; additional data not
When staining male chromosome spreads, fluorescent shown). These results demonstrate that the BPK gene is
twin dots were only present on the single X chromosome very tightly linked to the region of Xq22 identified as the
(data not shown). XLA region locus.
To determine the specific region of hybridization on X, In an attempt to identify deletions in the BPKgene, geno-
ratios of centromere-to-probecentromere-to-telomere dis- mic DNAs from multiple patients with XLA were evaluated
tances were calculated from photomicrographs and plot- by Southern analysis. Using a panel of multiple restriction
ted against an X chromosome band map archetype (Harn- enzymes and several probes, including the human BPK
den and Klinger, 1985). From 10 independent metaphase cosmid unique region and full-length or subfragment mu-
spreads, 8 fluorescent signals were localized to Xq22, 2 rine and human cDNA sequences, no unique restriction
to Xq21.3, and 2 to Xq23. fragment length polymorphisms were identified. This indi-
To confirm independently the X localization of W’K, a cated that a common, large deletion in the coding region
panel of rodent-human somatic cell hybrids containing of BPK was unlikely. Small deletions, point mutations, or
A

Human X retained
+ + + + + - -
Human X only
f
=r
I
21.:

p :

11 iin

1 qll.1
11 ;
12
13

21 : 1
q 22;
23
24
1

’
’ q22 1 q21-22

q26

I t

Figure 5. X Chromosome Mapping of BfK Using Somatic Cell Hybrid Analysis

(A) Hybridization pattern of DNA obtained from X chromosome somatic hybrids using the WK cosmid unique probe: plus sign indicates detectable
signal; minus sign indicates no signal. The portion of X chromosome retained in each somatic hybrid is indicated (see Experimental Procedures).
BPK was localized to Xq21-22 as indicated.
(6)Autoradiograph of Southern analysis, using SPKcosmid unique region probe, of genomic DNAfrom an individual with five copies of X chromosome
(penta X), a normal male and somatic cell hybrids retaining portions of the X chromosome as indicated.

alterations in distant upstream exons of BPK, not covered formed pre-B and B cell lines from normal individuals, obli-
by the available probes, however, remained possible. gate carriers of XLA, and patients with XLA was examined
for potential alteration in expression or function of BPK.
Lines were derived from normal individuals, obligate carri-
Deficient Expression of BPK In Epstein-Barr ers of XLA, and patients with XLA (Table 1). The lines
Virus-Transformed B Cell Lines from XLA Patients represented early (slgM-) and late (slgM+) stages of B cell
To address directly whether BPK expression was altered development derived from both bone marrow and periph-
in XLA, B cell lines derived from affected individuals were eral blood lymphocytes and represented patients with both
evaluated. A panel of Epstein-Barr virus (EBV)-trans- typical and milder disease phenotypes.
Deficient Cytoplasmic Tyrosine Kinase in XLA
285

123458 123456 123456 Figure 6. Localization of the BPK Gene within

a Panel of YACs in the Region Most Closely
Linked to XLA
(Top) Analysis of YACs by pulsed-field gel elec-
200 kb -
trophoresis. Lane 1, YAC A2, isolated with the
60 kb- anonymous probe most closely linked to XLA
(~212) at DXS178 after pulsed-field gel electro-
phoresis without restriction digest. Lanes 2-6,
A. Probe at DXS178 B. Probe within BPK C. Probe derived
from end of YAC YACs isolated with a probe derived from one of
gene
in lane 1 the ends of the A2 YAC, A2R, digested by Mlul
and separated by pulsed-field gel electrophore-
sis. The blot was probed with: ~212, recogniz-
’ 100 kb
___________________ 6 ing polymorphism at DXS178, in (A); U4X3,
5 BPK unique sequence human cosmid probe, in
4 (B); and A2R, A2 YAC end probe, in (C). The
3 BPK cosmid and A2R probes identify the same
-7 2 DNA fragments. The BPK cosmid probe did not
1 hybridize with 21 additional YACs including
i ic one, YAC 7, containing DNA from the opposite
end of YAC A2 (data not shown).
(Bottom) A restriction map showing Mlul sites
M M M M (M) in genomic DNA (bold line), with alignment
of YACs A2 (1) and 2-6 and probes (A-C) used
DXS178 Locus above. YAC 6 is chimeric (indicated by dotted
line). YAC 7, isolated with the probe from the
oppositeendof A2, did not hybridize with ~212,
BPK, or A2R allowing placement of ~212 on the
map.

It is possible that XLA B cell lines represent immortaliza- able or deficient in all XLA patient-derived cell lines. Lines
tion of an unusual subset of 6 lineage cells. However, the from normal individuals and obligate carriers expressed
data presented in Table 1, the spectrum of cell phenotypes kinase activity similar to the control B cell line RAJI (Figure
and patients evaluated, and previous analysis of similar 7). Notably, one XLA B cell line (D. C.) produced a dimin-
XLA-derived lines and peripheral lymphocytes would ar- ished but detectable level of kinase activity. This line was
gue against this suggestion (Fu et al., 1980; Tsuchiya et derived from an individual with late onset of recurrent bac-
al., 1980; Lau et al., 1989; Levitt et al., 1984; Anker et al., terial infection and possibly a milder disease phenotype.
1989). The decrease in kinase activity of BPK in the XLA lines
Using the polyclonal antiserum recognizing human did not appear to be due to the presence of a nonspecific
BPK, lysates from normal and XLA lines were immunopre- trans-acting phosphatase or inhibitor. The autokinase ac-
cipitated and tested for kinase activity in an in vitro autoki- tivity of an unrelated kinase, c-abl, was evaluated using
nase assay. Strikingly, BPK kinase activity was undetect- protein extracted from each of the cell lines. All lines dem-
onstrated equivalent levels of c-abl autokinase activity
(data not shown).
Table 1, EBV-Transformed B Cell Lines Several explanations could be considered for the ab-
- sence of kinase activity in XLA cell lines. These included
Tissue
Lines Genotype Source B Cell Phenotype mutations in the kinase domain or deficient production of
- BPK protein. To examine the latter possibility, lysates from
Ill-6 Normal BM Pro-B (lg.)
III-7 Normal BM Pro-B (lg.) [%]methionine-labeled control and XLA cell lines were
ELB-5 Carrier BM B (IgMK > IgM?$ used for immunoprecipitation with polyclonal antibody rec-
ELB-10 Carrier BM B (IgMx > ISMA)@ ognizing the BPK unique region. All tested XLA lines had
EKG-4.59 Carrier PBL B WV absent or decreased protein expression (data not shown)
BT-8 XLA” BM B WW
BDB-BM XLA” BM Pre-B (u only)
which paralleled the level of autokinase activity. This result
BDB-30 XLA” PBL B NW
argued against a mutation in the kinase domain, but led
D. C. XLA” PBL B (IgW to the possibility of deficient BPK mRNA expression in the
c. c. XLA” PBL Pre-B (Z?ZZiZ) XLA cell lines.
EBV-transformed B cell lines were derived as described in Experimen- To evaluate BPKmRNA expression, total RNA prepared
tal Procedures and Kubagawa et al. (1988) and Conley et al. (1986). from EBV cell lines was analyzed by Northern blotting us-
a Represent typical cases of XLA with B cell levels <l% of normal. ing human BPKcDNA as a probe (see Experimental Proce-
D D. C. was derived from an atypical patient with <I% the normal
dures). Normal B cell lines expressed the 3.0 kb BPK tran-
level of B cells and low immunoglobulin G levels (<200 mg/dl) having
unusually late onset of recurrent infection. script, while XLA lines expressed little to no detectable
c C. C. was derived from an atypical patient with <I% the normal level level of the BPK transcript (Figure 8). The reduction or
of B cells but with immunoglobulin G levels greater than 200 mgldl. absence of mRNA transcripts in patient cell lines explains
d TheELBQand-lOcell lineswerederivedfrom thesamepatient. Both the lack of immunoreactive protein and kinase activity.
lines are oligoclonal and the degree of K to h expression is indicated.
Low but detectable amounts of BPK mRNA were present
Cell
266

Normal XLA dicted lack of a myristylation signal. Several tyrosine ki-

nases that are located in the cytosolic fraction have been
reported, including c-fps (Young and Martin, 1984) csk
(Nada et al., 1991), and FAK (Schaller et al., 1992). For
most of the tyrosine kinases localized in the cytosol, their
biological functions are unknown. However, csk has been
EPK reported to phosphorylate specifically the regulatory tyro-
p77 -
sine residues (corresponding to tyrosine 527 of c-src) of
Genotype src subfamily proteins and diminish their tyrosine kinase
YYYt! tt +-P+vv
activities (Okada et al., 1991). It is possible that BPK may
Sex FFF,M M M M t-l,
,F F be another example of a modulator type tyrosine kinase.
Several cytoplasmic tyrosine kinases, including lyn, fyn,
Figure 7. Absent or Reduced Expression of BPK Kinase Activity in blk, and PTK72 (Yamanashi et al., 1991; Burkhardt et al.,
EBV-Transformed B Cell Lines from XLA Patients
1991; Hutchcroft et al., 1992) are thought to be associated
BPK protein was immunoprecipitated from lysates of a panel of EBV- with the immunoglobulin receptor complex and possibly
transformed B cell lines with polyclonal anti-BPK antiserum. In vitro
autokinase activity was assayed in the presence of [T-“P]ATP. Phos-
function in B cell activation pathways. Association of BPK
phorylation of the 77 kd BPK protein is indicated by a bar. Sex and with the immunoglobulin receptor complex remains to be
genotype of each individual are indicated. tested.
Several lines of evidence suggest that defective expres-
sion of BPK is involved in the pathogenesis of XLA: First,
in two affected individuals upon extended exposure of the BPKis tightly linked to theXLA locus. Second, BPKexpres-
autoradiographs. In one XLA line, D. C., this expression sion is reduced in B cell lines derived from patients with
was consistent with the low levels of detectable protein XLA, but not in obligate carriers or normal individuals. The
and kinase activity observed. Interestingly, in the XLA cell detectable, but markedly reduced, levels of BPK mRNA in
line, BT-8, there was detectable mRNA expression, yet no some XLA patients may correlate with the variable pheno-
detectable kinase activity. It is likely that these XLA lines type of the disease. Third, BPK is a B lineage-specific
represent distinct mutations producing transcripts with dif- cytoplasmic tyrosine kinase representing an appropriate
fering effects on mRNA stability, protein expression, or candidate gene for a disorder manifested by abnormal
function. pre-B cell proliferation. It remains possible that deficient
BPK expression in XLA is the result of a genetic change
Discussion in an as yet undefined tightly linked gene or regulatory
sequence controlling the expression of BPK. A closely
We have identified and characterized a novel cytoplasmic
tyrosine kinase, BPK, which is exclusively expressed in
cells of hematopoietic origin. Sequence analysis of BPK
demonstrates homologies to DsrQBC, tecl, tecll, and itk, Normal XLA

suggesting that BPK belongs to a new subfamily of cyto-

I I I
plasmic tyrosine kinases with these genes. The features
of the gene organization of this subfamily include lack of
myristylation signals, lack of a carboxy-terminal regulatory
phosphorylation site, long, basic amino-terminal regions,
and the presence of SH2 and SH3 domains.
The product of the Dsrc28C gene is expressed during
embryogenesis in the central nervous system of Drosoph-
O-ACTIN _
ila and may be involved in segmentation and neuronal ,i,;&
,:.
I_
differentiation (Gregory et al., 1987; Vincent et al., 1989).
Tecl is predominantly expressed in liver and human hepa-
tocellular carcinoma (Mano et al., 1990). Itk is found in T Genotype
Y rfxka++=P
cells (Siliciano et al., 1992). Tecll is expressed in hemato- Sex F M F M t-l M
poietic cells, predominantly in myeloid lineage and T lin-
eage cells (J. N. Ihle, personal communication). BPKtran-
scripts are detected in B lineage and myelomonocytic Figure 8. Absent or Reduced Expression of BPK mRNA in EBV-
Transformed B Cell Lines from XLA Patients
cells, but not in T lineage cells. This expression pattern is
Autoradiograph of Northern blot analysis using 20 pg of total RNA
similar to that of hck (Ziegler et al., 1987; Quintrell et al., obtained from EBV-transformed B cell lines from two XLA obligate
1987; Holtzman et al., 1987) a src subfamily gene. carriers, the Burkitt’s lymphoma line, RAJI, and three patients with
The majority of SPK protein is located in the cytoplasm, XLA. The genotype and sex of each individual are indicated. The blot
as determined by subcellular fractionation and indirect im- was probed with the 2.0 kb human BPK partial cDNA containing the
munofluorescence staining (D. C. S., S. T. and 0. N. W., unique region of BPK, then stripped and reprobed with b-actin cDNA
to assess RNA loading. The location of BPK and actin transcripts are
unpublished data). This result is consistent with the pre- indicated. Exposure time was 24 hr.
Deficient Cytoplasmic Tyrosine Kinase in XLA
207

linked locus controlling region or promoter/enhancer se- Identification of molecules interacting with BPK should
quences outside the BPK gene might exert control over help define its possible roles in B lineage signaling path-
BPK expression. Examples of this include mutation within ways. These interactions may be important in understand-
the P-globin gene locus controlling region (Tuan et al., ing the basis of humoral immunodeficiencies and possibly
1985; Driscoll et al., 1989) and evidence for control of of abnormal B cell proliferation in malignancy and autoim-
severe hemophilia A expression by mutation outside the munity. BPK gene therapy of XLA patients may be useful
coding region of the factor VIII gene (Higuchi et al., 1991). in their management.
The block in pre-B cell development in XLA could involve
either abnormal pre8 expansion or exaggerated cell Experimental Procedures
death. Surrogate light chain (vLC) proteins encoded by
the )L5 and Vpre-B genes (Sakaguchi and Melchers, 1988; Screening and isolation of Murine BPK cDNA Clones
Kudo and Melchers, 1987) pair with IJIHC to form surface Poly(A) RNA was extracted from B iymphoid progenitor cells, whose
characterization has been described elsewhere (Scherle et al., 1990;
receptors only on pre-B cells (Lassoued et al., 1992). Li- Saffran et al., 1992) and was used to construct a cDNA library. The
gand interaction with these or other receptors may initiate cDNAs were synthesized using both an oligo(dT) primer and random
a signal for pre-B cell clonal expansion and differentiation. primers by the usual method (Sambrook et al., 1969). Double-stranded
Of note, disruption of the X5 gene in mice resulted in a cDNAs were ligated to EcoRl linkers, then inserted into the EcoRl
block in B cell development at the pre8 cell stage in a sites of 19110, followed by in vitro packaging. This cDNA library was
screened with the tyrosine kinase domain (302 bp fragment containing
manner analogous to XLA (Kitamura et al., 1992). This domains VI, VII, and VIII) of the human LTK gene (Maru et al., 1990).
block, as in XLA, was leaky and normal B cells were pro- Prehybridization and hybridization were carried out at 42OC in 30%
duced in low numbers. The signals initiating negative se- formamide, 5x SSPE, 5 x Denhardt’s solution, and 0.1% SDS. The
lection and apoptosis in pre-B cells remain poorly defined. filters were washed in 2x SSC and 0.1% SDS at room temperature.
After the screening and restriction analysis, cDNA inserts were sub-
Selection may involve interaction of self-ligand or other
cloned into the Ml3 vector and sequenced by Sequenase (US Bio-
molecules with immunoglobulin or other receptors on chemical). A cDNA library of the pre-B cell line 702/3 was kindly pro-
pre-B and newly formed immature B cells. If BPK expres- vided by Michael Gilly and Randolph Wail (UCLA).
sion modulates one of these two opposing processes, then
deficient expression may alter the normal balance of the isolation of a Human BPK cDNA Clone
pre-B cell compartment during differentiation. A human cDNA library derived from the human erythroleukemia cell
In addition to the abnormal pre-B to B cell transition, line K562 (Mes-Masson et al., 1966) was screened for human BPK
later defects in B cell maturation may also exist in XLA. cDNAclones. A murine BPK probe corresponding to 1.6 kb of upstream
region was hybridized to filter lifts from approximately 5 x 105 plaques
The expression of BPK throughout the B lineage would be
generated from size-selected RNA (>2 kb). Hybridization conditions
consistent with its role at one or more stages. In XLA, there were 50% formamide, 4 x SSC at 42OC overnight. Filters were washed
is a predominance of immature B cells. B cells in X-linked two times in 2 x SSC for 15 min at room temperature, followed by two
immunodeficient (xid) mice also display an immature phe- more washes in 0.2 x SSC for 15 min at 5OOC. Four positive clones
were identified, which were screened again using the most upstream
notype and appear to have impaired B cell maturation
unique region murine BPK cDNA probe (0.6 kb). One of four clones
(Scher, 1982; Schultz and Sidman, 1987). The xid muta- was positive, called Kl-1, containing 2 kb of sequence including trans-
tion is syntenic to the human X chromosome region Xq21- lated sequences and the most 5’ untranslated region. Nucleic acid
q22 (Hillyard et al., 1992). Preliminary studies have dem- and protein sequences for the BPK cDNA clones are deposited in
onstrated normal BPK mRNA, protein expression, and GenBank.

kinase activity in B cells from xid mice (D. C. S., S. T.,

D. J. Pt., and 0. N. W., unpublished data). It will be interest- Preparation of Anti-BPK Antiserum
ing to determine if the xid phenotype represents a more A Drai-Hindlii fragment containing amino acids 25-173 of BPK was
subtle mutation in the BPK molecule. cloned into the bacterial expression piasmid pGEX-2T (Smith and
Johnson, 1966). Bacterial culturesexpressing thispiasmid weregrown
BPK is expressed throughout the B lineage and in my-
in TyE containing 100 @ml ampicillin and induced with 0.1 mM isopro-
eloid cells. The myeloid compartment remains normal in pyl+-D-thiogalactopyranoside for 5 hr. induced bacteria were lysed
XLA. It seems paradoxical that the primary effect of defi- by sonication in phosphate-buffered saline, 1% Triton X-100, 50 mM
cient BPK expression would be manifest only in B lineage EDTA. 1 mM phenyimethylsuifonyi fluoride, 100 U/ml aprotinin, and
development. Targeted gene disruption of the ubiquitously 50 ug/ml leupeptin, then centrifuged at 6000 x g for 5 min. Since the
induced protein was insoluble, the pellet was iysed in 0.1 M giycine-
expressed cytoplasmic protein-tyrosine kinases, c-src and NaOH (pH 9.0) containing 6 M urea, and fractionated by spin-column
c-abl, however, also resulted in surprisingly limited cell chromatography of Sephadex G-25 (Pharmacia) equilibrated in 0.1 M
abnormalities (Soriano et al., 1991; Tybulewicz et al., glycine-NaOH (pH 9.0) (Frorath et al., 1992). The passed fraction was
1991; Schwartzberg et al., 1991). The expression of other dialyzed overnight at 4OC against phosphate-buffered saline con-
taining 1% Triton X-100 and purified by affinity chromatography using
kinases with redundant function may explain the limited a glutathione-Sepharose 48 column (Pharmacia). Eluted fusion pro-
effect of targeted c-abl and c-src gene disruption. Redun- tein was further purified using preparative SDS gel electrophoresis.
dancy of protein kinase-mediated control of B lineage immunizations of New Zealand White rabbits were initiated bysubcuta-
growth and development is also likely to exist. Perhaps, neous injections of 200 ug of purified fusion protein emulsified in com-
plete Freund’s adjuvant, followed by boosting with the same amount
the most surprising consequence of deficient BPKexpres-
of protein in incomplete Freund’s adjuvant every 2 weeks. Collection
sion may be that alternative mechanisms for control of of sera began 4 weeks after the initial injection. Specificity and titer of
pre-B cell expansion do not exist or have only a limited the sera were examined by immunoprecipitating in vitro translated
effect in overcoming the lack of BPK expression. BPK protein labeled with (Y9jmethionine (see Results).
Cell
288

Isolation of a Human BPK Cosmld Clone lines D. C. and C. C. were derived by transformation with supernatants
A human DNA cosmid library (Denny et al., 1989) in the cosmid vector from the EBV-producing line MCUV as described (Conley et al., 1986).
pWE-15 (Stratagene, La Jolla, CA) was screened for clones that cross-
hybridized with murine BPK cDNA. Plaque lifts were screened by hy- Proteln Analysis
bridization using a BfK probe corresponding to 1.6 kb of upstream Metabolic labeling, immunoprecipitation, and in vitro kinase assays
region sequence. Hybridizations were performed overnight at 42OC in were performed as described previously (Konopka et al., 1964; Ko-
40% deionized formamide, 4 x SSC, and 10% dextran sulfate. Filters nopka and Witte, 1965). For the in vitro kinase assay, SDS was re-
were washed two times in 2 x SSC for 15 min at room temperature, moved from the kinaselysis buffer (1% Triton X-100, IO mM phosphate
followed by two washes at 50°C in 0.2 x SSC for 15 min. Four clones buffer [pH 7.01, 150 mM NaCI), since it greatly reduced BPK kinase
were identified and rescreened using a specific probe corresponding activity. Optimum kinase activity was achieved in the presence of 20
to 0.6 kb of the most upstream unique region of the gene. Two clones mM Mn” as a cofactor.
hybridizing with both probes were identified and one 28 kb clone,
designated C3-1, was selected for further analysis and used as a hu- RNA Analysls
man BPK-specific probe. A restriction map was constructed by partial RNA was extracted from cells by standard procedures described pre-
restriction digestion and hybridization with T3 and T7 oligonucleotide viously (McLaughlin et al., 1987). For Northern analysis, 20 pg of total
probes flanking the cloning site on either side of the insert. For the RNA was electrophoresed on formaldehyde-agarose gels, transferred
Southern analysis in Figure 1, 10 ug of genomic DNA was restriction to nitrocellulose, and probed as indicated in the figure legends.
digested overnight at 37OC. The digested DNA was electrophoresed
on an 0.8% agarose gel, transferred to nitrocellulose, and hybridized Acknowledgments
with the indicated probes.
Subclones of C3-1 were derived by digestion of cosmid DNA with We thank Michael Gilly and Randolph Wall for the 70213 cDNA library;
Taql and ligation of fragments to the Cial site of the pGEM7Zf (+) Yoshiro Maru for human LTK probe; Jami McLaughlin and Kathleen
vector. Colonies were screened using the upstream 0.6 kb unique Burke for preparation of B lymphoid progenitors and construction of
region probe of murine WK. One positive clone was identified con- the cDNA library; James lhle for personal communication; Charles
taining BPK exonic sequence from the unique region of the gene. Sawyers, Steve Smale, Danny Afar, Tim Hughes, and Mikhail Gishizky
Plasmid DNA prepared from this clone was digested with Xbal (to for critical reading of the manuscript; Chris Denny for providing the
remove additional intronic sequences), gel purified, and religated. This human cosmid library and helpful discussions; Robert Nussbaum for
subclone. designated U4X3, contained a 1.2 kb insert and was used DNA from patients with choroideremia; M. B. Passage for technical
as a human BPK unique region genomic probe. assistance; Numan Parada for photography; Bill Biggs for computer
design expertise and Kris Vensei and Julia Shimaoka for great assis-
Somatic Cell Hybrid Analysis tance in preparation of the manuscript. S. T. is an Associate with the
Somatic cell hybrids were isolated from the fusion of mouse A9 cells Howard Hughes Medical Institute. D. C. S. was an Associate with the
or Chinese hamster 1102 cells (each deficient in hypoxanthine phos- Howard Hughes Medical Institute and is a Fellow with the Leukemia
phoribosyltransferase) with normal human cells or human cells with X Society of America. D. J. R. is supported by a National Institutes of
chromosome/autosome translocations (obtained from the Mutant Cell Health Immunology Training Grant. J. W. B. is an Assistant Investigator
Repository, Camden, NJ) using methods previously described (Mo- and M. D. C. and 0. N. W. are Investigators with the Howard Hughes
handas et al., 1986). Clones 994X and 74.7 contained a structurally Medical Institute. This work was supported by the Howard Hughes
normal human X chromosome only in the A9 and 1102 background, Medical Institute and Grants Al25129, A130879, and CA13148.
respectively. The following clones retained only truncated human X
chromosomes: 31-1 (A9 x GM7792, t(X;2O)(qll;qli)); 1 I-4 (1102 x Received December 18, 1992; revised December 31, 1992.
GM1409, t(X;g)(q13;q34)); 50-28 (A9 x GM1696, t(X;7)(q21;p22));
32-23 (A9 x GM2621, t(X;12)(q22;q24)); 100-I (A9 x GMOO89, References
t(X;lg)(q22;ql3.3)); 33-16 (A9 x GM0097A, t(X;l)(q26;q12)). The GM
numbers refer to the Mutant Cell Repository directory. Allen, R. C., and Belmont, J. W. (1992). Dinucleotide repeat polymor-
DNA from each hybrid (10 ug) was digested with Hindlll overnight phism at the DXS178 locus. Hum. Mol. Genet. 1, 216.
at 37OC, separated by agarose gel electrophoresis (0.8% agarose gel), Anker, R., Conley, M. E., and Pollok, B. A. (1969). Clonal diversity in
transferred to nitrocellulose by vacuum blotting, ultraviolet cross- the B cell repertoire of patients with X-linked agammaglobulinemia. J.
linked, and prehybridized at 42’C, as described (Sambrook et al., Exp. Med. 169, 2109-2119.
1989). Filters were hybridized with the U4X3 subclone of the human Bolen, J. B., Thompson, P. A., Eisenman, E., and Horak, I. D. (1991).
BPK cosmid C3-1, after random primer labeling (Stratagene, Prime-It) Expression and interaction of the src family of tyrosine kinases in T
at 42OC overnight. Filters were washed at 55OC in 0.2X SSC for 45 lymphocytes. Adv. Cancer Res. 57, 103-149.
min, and autoradiography was performed.
Bruton, 0. C. (1952). Agammaglobulinemia. Pediatrics 9, 722-727.
DNA from four patients with the X-linked eye disorder, choroidere-
mia, was obtained as a gift from Dr. R. Nussbaum (University of Penn- Burkhardt, A. L., Brunswick, M., Boien, J. B., and Mond, J. J. (1991).
sylvania, Department of Genetics). DNA from each patient (10 ug) was Anti-immunoglobulin stimulation of B lymphocytes activates src-re-
digested with Hindlll and used for Southern blot analysis as described iated protein-tyrosine kinases. Proc. Natl. Acad. Sci. USA 88, 7410-
above. 7414.
Campana, D., Farrant, J., Inamdar, N., Webster, A. D. B., and Janossy,
Southern Blot, Pulsed-Field Ciel Electrophoresls, G. (1990). Phenotypic features and proliferative activity of B cell pro-
and YAC Analysis genitors in X-linked agammagiobulinemia. J. Immunoi. 145, 1675-
The preparation of high molecular weight DNA, digestion with restric- 1660.
tion enzymes, electrophoresis, and Southern blotting was performed Conley, M. E. (1985). B cells in patients with X-linked agammaglobu-
using standard techniques (Sambrook et al., 1969). Construction of linemia. J. Immunol. 734, 3070-3074.
YACs (Allen and Belmont, 1992) and pulsed-field gel electrophoresis
of genomic DNA and YACs were performed as previously described Conley, M. E. (1992). Molecular approaches to analysis of X-linked
immunodeficiencies. Annu. Rev. immunol. 70, 215-238.
(Parolini et al., 1993). Probes used for Southern analysis were pre-
pared as described above. Contey, M. E., Brown, P., Pickard, A. R., Buckley, R. H., Miller, D. 8,
Raskind, W. H., Singer, J. W., and Fialkow, P. J. (1986). Expression
Cell Lines and Patient Samples of the gene defect in X-linked agammaglobulinemia. N. Engl. J. Med.
The normal cell lines Ill-6 and 111-7, carrier cell lines ELB-5, EL&IO, 315, 564-567.
and EKG4.59, and XLA cell lines BT-8, BDBBM, and BDB-30 were Cooper, M. D., Nishimoto, N., Lassoued, K., Nunez, C., Nakamura,
derived by transformation with the EBV-producing 895.8 marmoset T., Kubagawa, H., and Volanakis, J. E. (1993). Antibody deficiencies
cell line as previously described (Kubagawa et al., 1988). The XLA cell reflect abnormal B cell differentiation, Prog. Immunol., in press.
Deficient Cytoplasmic Tyrosine Kinase in XLA
269

Cremers, F. P. M., van de Pal, D. J. FL, Diergaarde. P. J., Wieringa, jewsky, K. (1992). A critical role of I5 protein in B cell development,
B., Nussbaum, R. L., Schwartz, M.. andRopers, H.-H. (1989). Physical Cell 69, 823-831.
fine mapping of the choroideremia locus using Xq21 deletions associ- Kmiecik, T. E., and Shalloway, D. (1987). Activation and suppression of
ated with complex syndromes. Genomics 4, 41-46. ppso”” transforming ability by mutation of its primary sites of tyrosine
Denny, C. T., Shah, N., Ogden, S., Willman, C.. McConnell, T., Crist, phospholylation. Cell 49, 65-73.
W., Carroll, A., and Witte, 0. N. (1989). Localization of preferential sites Koch, C. A., Anderson, D., Moran, M. F., Ellis, C., and Pawson, T.
of rearrangement within the BCR gene inphiladelphia chromosome (1991). SH2 and SH3 domains: elements that control interactions of
positive acute lymphoblastic leukemia. Proc. Natl. Acad. Sci. USA 86, cytoplasmic signaling proteins. Science 252, 668-674.
42544268.
Konopka, J. B., and Witte, 0. N. (1985). Detection of cab/ tyrosine
Driscoll, M. C., Dobkin, C. S., and Alter, B. P. (1969). $&Thalassemia kinase activity in vitro permits direct comparison of normal and altered
due to a de nova mutation deleting the 5’ 8-globin gene activation- abl gene products. Mol. Cell. Biol. 5, 31163123.
region hypersensitive sites. Proc. Natl. Acad. Sci. USA 86,7470-7474.
Konopka, J. B., Davis, R. L., Watanabe, S. M., Ponticelli, A. S., Schiff-
Dymecki, S. M., Niederhuber, J. E., and Desiderio, S. V. (1999). Spe Maker, L., Rosenberg, N., and Witte, 0. N. (1984). Only site-directed
cific expression of tyrosine kinase gene, b/k, in B lymphoid cells. Sci- antibodies reactive with the highly conserved scr-homologous region
ence 247,332~336. of the v-abl protein neutralize kinase activity. J. Virol. 57, 223-232.
Eisenman, E., and Bolen, J. 8. (1990). Src-related tyrosine protein Kubagawa, H., Burrows, P. D., Grossi, C. E., Mestecky, J., andCooper,
kinases as signalling components in hematopoietic cells. Cancer Cells M. D. (1988). Precursor B cells transformed by Epstein-Barr virus
2, 303-310. undergo sterile plasma-cell differentiation: J-chain expression without
Faust, E. A., Saffran, D. C., Toksoz, D., Williams, D. A., and Witte, immunoglobulin. Proc. Natl. Acad. Sci. USA 85, 875-679.
0. N. (1993). Distinctive growth requirements and gene expression Kudo, A., and Melchers, F. (1987). A second gene, Vpre-B in the 56
patterns distinguish progenitor 6 cells from pre-B cells. J. Exp. Med., locus of the mouse, which appears to be setectively expressed in pre-B
in press. lymphocytes. EMBO J. 6,2267-2272.
Frorath, 8.. Abney, C. C., Berthold, H., Scanarini, M., and Northe- Kwan, S.-P., Kunkel, L., Bruns, G., Wedgewood, Ft. J., Latt, S., and
mann, W. (1992). Production of recombinant rat interleukin-6 in Esche Rosen, F. S. (1986). Mapping of the X-linked agammaglobulinemia
richia co/i using a novel highly efficient expression vector pGEX-3T. locus by use of restriction fragment-length polymorphism. J. Clin. In-
BioTech 72, 556-563. vest. 77, 649-652.
Fu, S. hf., Hurley, J. N., McCune, J. M., Kunkel, H. G., and Good, Kwan, S.-P., Terwilliger, J., Parmley, R., Raghu, G., Sandkuyl, L. A.,
R. A. (1980). Pm-8 cells and other possible precursor lymphoid cell Ott, J., Ochs, H., Wedgwood, R., and Rosen, F. (ISgO). Identification
linesderived from patients with X-linked agammaglobulinemia. J. Exp. of closely linked DNA marker, DXS178, to further refine the X-linked
Med. 752, 1519-1526. agammaglobulinemia locus. Genomics 6, 238-242.
Gregory, R. J., Kammermeyer, K. L., Vincent, W. S., Ill, and Wads- Lassoued, K., Nunez, C., Kubagawa, H.. and Cooper, M. D. (1992).
worth, S. G. (1987). Primary sequence and developmental expression Analysis of human surrogate light chain with monoclonal antibodies.
of a novel Drosophila melanogaster src gene. Mol. Cell. Biol. 7,21 lQ- FASEB J. 6.4448.
2127. Lau, Y. L., Shields, J. G., Levinsky, R. J., and Callard, R. E. (1989).
Guioli, S., Arveiler, B., Bardoni, B., Notarangelo, L. D., Panina, P., Epstein-Barr-virus-transformed lymphoblastoid cell lines derived from
Duse, M., Ugazio, A., de Saint Basile, G., Mandel, J. L., and Camerino, patients with X-linked agammaglobulinaemia and Wiskott-Aldrich syn-
G. (1989). Close linkage of probe ~212 (DXS178) to X-linked agamma- drome: responses to B cell growth and differentiation factors. Clin.
globulinemia. Hum. Genet. 84, 19-21. Exp. Immunol. 75, 190-195.
Hanks, S. K., Quinn, A. M., and Hunter, T. (1988). The protein kinase Levitt, D., Ochs. H., and Wedgwood, R. J. (1984). Epstein-Barr virus-
family: conserved features and deduced phylogeny of the catalytic induced lymphoblastoid cell lines derived from the peripheral blood of
domains. Science 247, 42-52. patients with X-linked agammaglobulinemia can secrete IgM. J. Clin.
Harnden, D. G., and Klinger, H. P., eds. (1985). An international system Immunol. 4, 143-150.
for human cytogenetic nomenclature (Basel, Switzerland: Karger). Lipman, D. J., and Pearson, W. R. (1985). Rapid and sensitive protein
Hatakeyama, M., Kono, T., Kobayashi, N., Kawahara, A., Levi% S. D., similarity searches. Science 227, 1435-1441.
Perlmutter, Ft. M., and Taniguchi, T. (1991). Interaction of the IL-2 Malcolm, S., de Saint Basile, G., Arveiler, B., Lau, Y. L., Szabo, P.,
receptor with the srofamily kinase ~56”‘: identification of novel inter- Fischer, A., Griscelli, C., Debre, M., Mandel, J. L., Callard, R. E.,
molecular association. Science 252, 1523-1528. Robertson, M. E., Goodship, J. A., Pembrey, hf. E., and Levinsky,
Higuchi, M., Kazazian, H. H., Jr., Kasch, L., Warren, T. C., McGinniss, R. J. (1987). Close linkage of random DNA fragments from Xq 21.3-
M. J., Phillips, J. A., III, Kasper, C., Janco, R., and Antonarakis, S. E. 22 to X-linked agammaglobuknemia (XLA). Hum. Genet. 77,172-174.
(1991). Molecular characterization of severe hemophilia A suggests Mano, H.. Ishikawa, F., Nishida, J., Hirai, H., and Takaku, F. (1999).
that about half the mutations are not within the coding regions and A novel protein-tyrosine kinase, tee, is preferentially expressed in liver.
splice junctions of the factor VIII gene. Proc. Natl. Acad. Sci. USA 88, Oncogene 5, 1781-1786.
7405-7409.
Marth, J. D., Peet, R., Krebs, E. G., and Perlmutter, R. M. (1985). A
Hillyard, A. L., Doolittle, D. P., Davisson, M. T., and Roderick, T. H., lymphocyte-specific protein-tyrosine kinase gene is rearranged and
eds. (1992). Locus Map of the Mouse with Comparative Map Points of overexpressed in the murine T cell lymphoma LSTRA. Cell 43, 393
Human on Mouse Chromosomes (Bar Harbor, Maine: The Jackson 404.
Laboratory).
Maru, Y., Hirai, H., and Takaku, F. (1990). Human Itk: gene structure
Holtzman, D. A., Cook, W. D., and Dunn, A. R. (1967). Isolation and
and preferential expression in human leukemic cells. Oncogene Res.
sequence of a cDNA corresponding to a src-related gene expressed
5, 199-294.
in murine hemopoietic cells. Proc. Natl. Acad. Sci. USA 84, 8325-
8329. McLaughlin, J., Chianese, E., and Witte, 0. N. (1987). In vitro transfor-
Hutchcroft, J. E., Harrison, M. L., and Geahlen, R. L. (1992). Associa- mation of immature hematopoietic cells by the P210 BCRIABL onco-
tion of the 72-kDa protein-tyrosine kinase PTK72 with the B cell antigen gene product of the Philadelphia chromosome. Proc. Natl. Acad. Sci.
USA 84, 8558-6562.
receptor. J. Biol. Chem. 267, 8813-8619.
Kaplan, J. M., Marden, G., Bishop, J. M., and Varmus, H. E. (t988). Mensink, E. J. B. M., Thompson, A., Schot, J. D. L., van de Greef,
The first seven amino acids encoded by the v-src oncogene act as a W. M. M., Sandkuyl, L. A., and Schuurman, R. K. B. (1986). Mapping
myristoylation signal: lysine 7 is a critical determinant. Mol. Cell. Biol. of a gene for X-linked agammaglobulinemia and evidence for genetic
8, 2435-2441. heterogeneity. Hum. Genet. 73,327-332.
Kitamura. D., Kudo, A., Schaal, S., Miiller, W.. Melchers, F., and Ra- Mes-Masson, A.-M., McLaughin, J., Daley, G., Paskind, M., and Witte,
0. N. (1966). Overlapping cDNA clones define the complete coding Smith, D. B., and Johnson, K. S. (1966). Single-step purification of
region for the P210& gene product associated with chronic myeloge- polypeptides expressed in Escherichia co/i as fusions with glutathione
nous leukemia cells containing the Philadelphia chromosome. Proc. S-transferase. Gene 67, 31-40.
Natl. Acad. Sci. USA 83, 9766-9772. Soriano, P., Montgomery, C., Geske, R., and Bradley, A. (1991). Tar-
Mohandas, T., Heinzmann, C., Sparkes, R. S., Wasmuth, J., Edwards, geted disruption of the C-SIC proto-oncogene leads to osteopetrosis in
P., and Lusis, A. J. (1966). Assignment of human 9hydroxy-3-methyl- mice. Cell 64, 693-702.
glutaryl coenzyme A reductase gene to ql3q23 region of chromosome Taniguchi, T., Kobayashi, T., Kondo, J., Takahashi, K., Nakamura, H.,
5. Somat. Cell Mol. Genet. 72, 69-94. Suzuki, J., Nagai, K., Yamada, T., Nakamura, S-l., and Yamamura,
Nada, S., Okada, M., MacAuley, A., Cooper, J. A., and Nakagawa, H. H. (1991). Molecular cloning of a porcine gene syk that encodes a
(1991). Cloning of a complementary DNA for a protein-tyrosine kinase 72-kDa protein-tyrosine kinase showing high susceptibility to proteoly-
that specifically phosphorylates a negative regulatory site of p6oc”“. sis. J. Biol. Chem. 266, 15790-15796.
Nature 357, 69-72. Tsuchiya, S., Konno, T., Tada, K., and Ono, Y. (1960). Epstein-Barr
Nishimoto, N.. Kubagawa, H., and Cooper, M. D. (1991). Comparison virus-induced lymphoblastoid cell lines from patients with primary im-
of pre-B cell differentiation in normal and X-linked agammaglobulin- munodeficiency diseases. Stand. J. Immunol. 77, 155-162.
emit (XLA) individuals. Fed. Proc. 5, 1346. Tuan, D., Solomon, D., Li, Q., and London, I. M. (1965). The “B-like
Okada, M., Nada, S., Yamanashi, Y., Yamamoto, T., and Nakagawa, globin” gene domain in human erythroid cells. Proc. Natl. Acad. Sci.
H. (1991). CSK: a protein-tyrosine kinase involved in regulation of src USA 82,6364-6366.
family kinases. J. Biol. Chem. 266, 24249-24252. Tybulewicz, V. L. J., Crawford, C. E., Jackson, P. K., Bronson, R. T.,
Parolini, O., Hejtmancik, J. F., Allen, R. C., Belmont, J. W., Lassiter, and Mulligan, R. C. (1991). Neonatal lethality and lymphopenia in mice
G. L., Henry, M. J., Barker, D. F., and Conley, M. E. (1993). Linkage with a homozygous disruption of the C-~/J/ protwncogene. Cell 65,
analysis and physical mapping near the gene for X-linked agamma- 1153-1163.
globulinemia at Xq22. Genomics, in press. Veillette, A., Bookman, M. A., Horak, E. M., and Bolen, J. B. (1966).
Pawson, T., and Gish, G. D. (1992). SH2 and SH3 domains: from The CD4 and CD6 T cell surface antigens are associated with the
structure to function. Cell 71, 359-362. internal membrane tyrosine-protein kinase ~56”. Cell 55, 301-306.
Pearl, E. R., Vogler, L. B., Okos, A. J., Crist, W. M., Lawton, A. R., Ill, Velazquez, L., Fellous, M., Stark, G. R., and Pellegrini, S. (1992). A
and Cooper, M. D. (1976). B lymphocyte precursors in human bone protein tyrosine kinase in the interferon al6 signaling pathway. Cell 70,
marrow: an analysis of normal individuals and patients with antibody- 313-322.
deficiency states. J. Immunol. 720, 1169-1175. Vincent, W. S., Ill, Gregory, R. J.,and Wadsworth, S. C.(1969). Embry-
Piwnica-Worms, H., Saunders, K. B., Roberts, T. M., Smith, A. E., and onic expression of a Drosophila src gene: alternate forms of the protein
Cheng, S. H. (1967). Tyrosine phosphorylation regulates the biochemi- are expressed in segmental stripes and in the nervous system. Genes
cal and biological properties of pp6o”n. Cell 49, 75-62. Dev. 3,334-347.
Quintrell, N., Lebo, R., Varmus, H., Bishop, J. M., Pettenati, M. J., Whitlock, C. A., and Witte, 0. N. (1962). Long term culture of B lympho-
LeBean, M. M., Diaz, M. O., and Rowley, J. D. (1967). Identification of cytes and their precursors from murine bone marrow. Proc. Natl. Acad.
a human gene (HCK) that encodes a protein-tyrosine kinase and is Sci. USA 79, 3606-3612.
expressed in hemopoietic cells. Mol. Cell. Biol. 7, 2267-2275. Wilks, A. F., Harpur, A. G., Kurban, R. R., Ralph, S. J., Zurcher, G.,
Rolink, A., and Melchers, F. (1991). Molecular and cellular origins of and Ziemeicki, A. (1991). Two novel protein-tyrosine kinases, each
B lymphocyte diversity. Cell 66, 1061-1094. with a second phosphotransferase-related catalytic domain, define a
Saffran, D. C., Faust, E. A., and Witte, 0. N. (1992). Establishment of new class of protein kinase. Mol. Cell. Biol. 77, 2057-2065.
a reproducible culture technique for the selective growth of B cell Yamanashi, Y., Kakiuchi, T., Mizuguchi, J., Yamamoto, T., and Toyo-
progenitors. Curr. Top. Microbial. Immunol. 182, 34-44. shima, K. (1991). Association of B cell antigen receptor with protein
Sakaguchi, N., and Melchers, F. (1966). 25, a new light chain-related tyrosine kinase lyn. Science 257, 192-194.
locus selectively expressed in pre-B lymphocytes. Nature 324, 579- Young, J. C., and Martin, G. S. (1964). Cellular localization of c-fps
562. gene product NCP96. J. Virol. 52, 913-916.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1969). Molecular Clon- Ziegler, S. F., Marth, J. D., Lewis, D. B., and Perlmutter, R. M. (1967).
ing: A Laboratory Manual (Cold Spring Harbor, New York: Cold Spring Novel protein-tyrosine kinase gene (hck) preferentially expressed in
Harbor Laboratory Press). cells of hematopoietic origin. Mol. Cell. Biol. 7, 2276-2265.
Samelson, L. E., Philips, A. F., Loung, E. T., and Klausner, R. D.
(1990). Association of the fyn protein tyrosine kinase with the T cell GenBenk Acceeeion Number
antigen receptor. Proc. Natl. Acad. Sci. USA 87, 4366-4362.
Schaller, M. D., Borgman, C. A., Cobb, B. S., Vines, R. R., Reynolds, The accession number for the sequence reported in this paper is
A. B., and Parsons, J. T. (1992). p~125”~, a structurally distinctive L06967.
protein-tyrosine kinase associated with focal adhesions. Proc. Natl.
Acad. Sci. USA 89.5192-5196.
Scher, I. (1962). The CBA/N mouse strain: an experimental model
illustrating the influence of the X-chromosome on immunity. Adv. Im-
munol. 33, 1-71.
Scherle, P. A., Dorshkind, K., and Witte, 0. N. (1990). Clonal lymphoid
progenitor cell lines expressing the BCR/ABL oncogene retain full
differentiative function. Proc. Natl. Acad. Sci. USA 87, 1906-1912.
Schultz, L. D., and Sidman, C. 1. (1967). Genetically determined mu-
rine models of immunodeficiency. Annu. Rev. Immunol. 5, 367-403.
Schwartzberg, P. L., Stall, A. M., Hardin, J. D., Bowdish, K. S., Hu-
maran, T., Boast, S., Harbison, M. L., Robertson, E. J., and Goff,
S. P. (1991). Mice homozygous for the ablm’ mutation show poor viabil-
ity and depletion of selected B and T cell populations, Cell 65, 1165-
1175.
Siliciano, J. D., Morrow, T. A., and Desiderio, S. V. (1992). itk, a T-cell-
Specific tyrOSine kinase gene inducible by interleukin 2. Proc. Natl.
Acad. Sci. USA89, 11194-11196.