identification of phosphorylation sites within the sh3 domains of tec family tyrosine kinases

Identification of phosphorylation sites within the SH3 domains

of Tec family tyrosine kinases

Beston F. Norea,*, Pekka T. Mattssona,b, Per Antonssona, Carl-Magnus Backesjoa,Anna Westlundc, Johan Lennartssond, Henrik Hanssone, Peter Lowf,

Lars Ronnstrandd, C.I. Edvard Smitha

aKarolinska Institutet, Clinical Research Center (CRC) at Novum, Huddinge University Hospital, SE-141 86 Huddinge, SwedenbDepartment of Biochemistry and Food Chemistry, University of Turku, FIN-20014 Turku, Finland

cKaroBio AB, SE-141 57 Huddinge, SwedendLudwig Institute for Cancer Research, SE-751 24 Uppsala, Sweden

eDepartment of Biotechnology, Royal Institute of Technology (KTH), SCFAB, SE-106 91 Stockholm, SwedenfDepartment of Neuroscience, Karolinska Institutet, SE-171 77 Stockholm, Sweden

Received 19 July 2002; received in revised form 2 October 2002; accepted 6 November 2002

Abstract

Tec family protein tyrosine kinases (TFKs) play a central role in hematopoietic cellular signaling. Initial activation takes place through

specific tyrosine phosphorylation situated in the activation loop. Further activation occurs within the SH3 domain via a transphosphorylation

mechanism, which for Bruton’s tyrosine kinase (Btk) affects tyrosine 223. We found that TFKs phosphorylate preferentially their own SH3

domains, but differentially phosphorylate other member family SH3 domains, whereas non-related SH3 domains are not phosphorylated. We

demonstrate that SH3 domains are good and reliable substrates. We observe that transphosphorylation is selective not only for SH3 domains,

but also for dual SH3SH2 domains. However, the dual domain is phosphorylated more effectively. The major phosphorylation sites were

identified as conserved tyrosines, for Itk Y180 and for Bmx Y215, both sites being homologous to the Y223 site in Btk. There is, however,

one exception because the Tec-SH3 domain is phosphorylated at a non-homologous site, nevertheless a conserved tyrosine, Y206. Consistent

with these findings, the 3D structures for SH3 domains point out that these phosphorylated tyrosines are located on the ligand-binding

surface. Because a number of Tec family kinases are coexpressed in cells, it is possible that they could regulate the activity of each other

through transphosphorylation.

D 2002 Elsevier Science B.V. All rights reserved.

Keywords: Immunodeficiency; Tyrosine kinase; Btk; Itk; Tec; Bmx; Signal transduction; X-linked agammaglobulinemia; XLA

1. Introduction

Tec family protein tyrosine kinases (TFKs) together with

Src family kinases (SFKs) are multifunctional non-receptor

protein tyrosine kinases (PTKs), making up about 40–45%

of all cytoplasmic PTKs identified in the human genome

[1,2]. There are five related members of Tec PTKs in

humans, Bruton’s tyrosine kinase (Btk), Itk, Tec, Bmx and

Txk/Rlk. The domain architecture of these kinases consists

of five domains, three of which, SH3, SH2 and SH1 (kinase

domain), are found in the SFKs [2–5]. Apart from Txk,

which lacks the pleckstrin homology (PH) domain [6], the

presence of a PH domain and a Tec homology domain (TH)

at the N-terminal end distinguishes this family from SFKs

[2–5]. TH domains are comprised of a Btk motif and a

poly-proline (PP) region, which are thought to regulate and/

or stabilize kinase activity [7].

The central role of TFKs in cellular signaling was

directly recognized after identification of the BTK gene,

mutations in which cause the human immunodeficiency

1570-9639/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.

doi:10.1016/S1570-9639(02)00524-1

Abbreviations: XLA, X-linked agammaglobulinemia; Xid, X-linked

immunodeficiency; Btk, Bruton’s tyrosine kinase; PTK(s), protein tyrosine

kinase(s); PPSH3, extended Btk-SH3 domain with N-terminal poly-proline

regions; PP, poly-proline; Amp, Amphiphysin; IVK, in vitro kinase assay;

IP, immunoprecipitation; pY, phosphotyrosine; rhBtk, full-length recombi-

nant human Btk; 2D-mapping, two-dimensional tryptic phosphopeptide

mapping

* Corresponding author. Tel.: +46-8-5858-3657;

fax: +46-8-5858-3650.

E-mail address: [email protected] (B.F. Nore).

www.bba-direct.com

Biochimica et Biophysica Acta 1645 (2003) 123–132

disease, X-linked agammaglobulinemia (XLA) [8,9] and a

milder form, X-linked immunodeficiency (Xid), in mouse

[10,11]. XLA exhibits an intrinsic defect in B-cell develop-

ment and maturation at level pre-B transition, leading to a

dramatic reduction of peripheral B-cells [12]. The role of

Tec kinases in signal transduction is best illustrated for Btk,

particularly in the context of B-cell antigen receptor (BCR)

engagement. Itk plays an analogous role in T-cell signaling,

as shown for Itk knockout mice where T-cell development

and T-cell receptor signaling responses are impaired [13,14].

SFKs are ubiquitous PTKs regulating multiple cellular

signaling pathways [15,16]. It has been shown that SFKs

directly phosphorylate TFKs in the activation loop [17–19].

This phosphorylation site was mapped for human Btk on

Y551 [17] and for mouse Itk on Y511 [19]. Data from

multiple sequence alignments of TFKs deduced putative

homologous sites for other family members in human,

mouse and other species [20].

For Btk activation, membrane recruitment is a crucial

step associated with rapid, but transient, phosphorylation of

two major regulatory tyrosine residues, Y551 in the activa-

tion loop and subsequent autophosphorylation at Y223 in

the SH3 domain [17,21]. SH3 domain phosphorylation

provides a negative charge (as well as steric exclusion) to

this region, which can be severely unfavorable for the

interaction with ligand(s) [22]. The role of Btk-SH3 phos-

phorylation on Y223 in regulation of protein–protein inter-

actions and signal transduction has not yet been resolved,

despite the fact that numerous interacting proteins have been

identified [2,5,23]. It has been postulated that the two poly-

proline motifs in the TH domain stabilize and could interact

with the adjacent SH3 domain [24,25]. Recently, further

evidence provided an explanation to why the TH region of

Btk makes intermolecular (trans) interactions, whereas the

corresponding interaction in the related Itk kinase with only

one poly-proline motif is intramolecular (cis) [26–28].

However, it remains to be shown whether such an inter-

action takes place and/or influences signaling in vivo. In

Btk, the SH3 domain mutation Y223F results in enhanced

fibroblast transformation, implying that the SH3 domain

may play a negative regulatory role [21].

To determine a reliable substrate and to examine the

specificity for TFKs activity, we utilized a transphosphor-

ylation assay with recombinant SH3 proteins of Btk, Itk, Tec

and Bmx. For this purpose, we applied a steady-state in vitro

kinase assay (IVK) to follow the extent of g32P-ATP label-

ing of different SH3 domains at identical conditions. Our

initial analysis showed that transphosphorylation activity of

Btk is not restricted to its own SH3 domain, but also found

toward other family member SH3 domains. Like Btk, we

found that both Itk and Tec phosphorylate their own SH3

domains, but differentially transphosphorylate other TFK

members. The IVK activity of Btk, Itk and Tec is restricted

to SH3 domains of family members, whereas non-related

SH3 domains, such as Grb2 [29–31] and Amphiphysin

(Amp) [32], are not phosphorylated at any other tyrosine.

Our results show that the specificity in tyrosine phosphor-

ylation of TFK-SH3 domains in vitro is high. Therefore, we

believe that SH3 domain phosphorylation is required for a

distinct function in signaling.

2. Materials and methods

2.1. Cell culture and antibodies

The human B-cell line Ramos and T-cell Line Molt4

were cultured in RPMI 1640 supplemented with 10% FBS,

50 Ag/ml streptomycin and 50 units/ml penicillin (Gibco-

Invitrogen). Anti-Btk and anti-Itk were polyclonal rabbit

antibodies raised against their C- and N-terminal regions,

respectively. Anti-Tec was a polyclonal antibody kindly

provided by Dr. H. Mano (Jichi Medical School, Japan).

2.2. Cloning

The human Btk, Tec, Itk and Bmx SH3 domain con-

structs were cloned in pGEX-4T-3 vector as described

earlier [22]. A DNA fragment encoding residues 212–275

of hBtk containing tyrosine 223 mutated to phenylalanine

was amplified by PCR from pBluescript-hBtk Y223A. The

mutation was created by the overlap extension PCR techni-

que [33]. The fragment was cloned into pGEX-4T-3 vector

(Pharmacia). The sequence and presence of the mutation

was verified by sequencing. The human Btk gene was

cloned in baculovirus expression system pFASTBAC HT

with N-terminal 6xHis tag (Invitrogen).

2.3. Expression and purification of SH3 domains

Expression of protein was induced by the addition of

isopropyl D-thiogalactopyranoside (IPTG) to the XL1-Blue

strain of Escherichia coli [34]. After induction, the cells were

harvested and lysed by sonication at 4 jC in PBS buffer

containing 1% Triton X-100, 0.1 mM aprotinin, 1 mM

leupeptin and 1 mM EDTA. The GST-SH3 fusion products

were bound to glutathione-Sepharose, and digested with 100

units thrombin (Sigma-Aldrich). The GST-SH3 fusion pro-

teins were directly concentrated for gel filtration on a Super-

dex-200 column (Pharmacia). The purified Tec family SH3

proteins were finally concentrated and the protein solution

was stored at � 70 jC. The Btk (Y223A) SH3, Grb2-SH3

(AA159–217) and Amp-SH3 (AA545–694) proteins were

purified using the same procedure.

2.4. Expression and purification of human Btk (hBtk)

Spodoptera frugiperda (Sf9) cells (Invitrogen) were

maintained as suspension cultures in siliconized (Sig-

macotek, Sigma-Aldrich) 5 L shake flasks in 1 L of Sf900II

serum-free medium (Gibco-Invitrogen). When the culture

reached a cell density of 2� 106 cells ml� 1, the cells were

B.F. Nore et al. / Biochimica et Biophysica Acta 1645 (2003) 123–132124

infected at a multiplicity of infection of 5 with a recombinant

Autographa californica nuclear polyhedrosis baculovirus

containing the Btk gene. After 48 h, the cells were harvested

and then immediately frozen in liquid nitrogen and stored at

� 70 jC until processed. The production of full-length

recombinant human Btk (rhBtk) was performed by melting

the insect cell pellet lysate buffer containing 50 mM HEPES,

pH 8.0, 10 mM h-mercaptoethanol, 1 mM EDTA, 1 mM

PMSF, 1% NP-40, 0.5% Na-deoxycholate, 0.1 mM aproti-

nin, 1 mM leupeptin, 300 mM NaCl and two EDTA-free

Complete inhibitor cocktail tablets (Roche). The cells were

disrupted by mild sonic oscillation (Raytheon, USA) and the

cell debris was pelleted by centrifugation. The 6x polyhis-

tidine stretch in the N-terminal rhBtk facilitated the Btk

purification using Ni-affinity column (Qiagen, USA) (J.A.

Marquez, manuscript in preparation). The polyhistidine

stretch was cleaved by rTEV protease and Btk was further

purified on a Superdex-200 column (Pharmacia).

2.5. Immunoprecipitations (IP) and in vitro kinase assay

Molt4 were used for immunoprecipitation of Itk, and

Ramos cells for Btk and Tec. One to 5� 107 cells were

lysed in modified RIPA buffer (50 mM HEPES-buffer pH

7.5, 1% digitonine, 1 mM Na3VO4, 1 mM Na2MoO4, 1 mM

PMSF, 1 mM DTT) and one tablet of EDTA-free Complete

inhibitor cocktail (Roche) per 5 ml solution. Btk, Itk or Tec

was immunoprecipitated by incubation with 5 Ag of poly-

clonal antibodies for 1.5 h with rotation, at 4 jC. Followingaddition of 40 Al Protein A SepharoseR, the suspensions

were rocked for 2 h at 4 jC. Beads were washed five times

for 5 min each with modified RIPA buffer without digito-

nine. Subsequent to the IP procedure, immunoprecipitated

beads with Btk, Itk and Tec were washed twice with kinase

buffer (50 mM HEPES-buffer, pH 7.5; 10 mM MnCl2, 4

mM MgCl2, 0.05 mM Na3VO4, 0.1 mM Na2MoO4, 0.1 mM

PMSF, 1 mM DTT). The beads were resuspended in 20 Alkinase buffer containing 50 AM cold ATP and 1–10 Agrecombinant proteins. In a similar manner, in vitro kinase

with purified hBtk, 200 ng of purified Btk combined with

20 Al kinase buffer containing 50 AM cold ATP and 5–20

Ag recombinant proteins. Finally, 1–20 ACi g32P-ATP was

added to the reaction mixtures with gentle vortex for 20

min. Reactions were terminated by the addition of SDS-

polyacrylamide gel loading buffer. Proteins were resolved

on 16% SDS-PAGE and transferred to either HQ-PVDF or

nitrocellulose membranes (Millipore).

2.6. Analysis of tryptic phosphopeptide and radio-Edman

sequencing

Tryptic phosphopeptides were digested on nitrocellulose

membranes according to methods previously described by

Hansen et al. [35] and they were separated on a Hunter thin

layer electrophoresis system (HTLE-700) at 2000 V for 40

min under water cooling. The second dimension chromatog-

raphy was performed in isobutyric acid buffer for 16 h

[36,37]. Briefly, nitrocellulose membrane slices of 32P-

labeled SH3 protein were excised, washed extensively with

double distilled H2O, and then soaked in 0.5% polyvinylpyr-

olidone (Sigma) for 30 min at 37 jC. The membranes were

then washed four times with 50mMNH4HCO3. Immobilized

proteins were digested twice with 1 Ag trypsin (Promega) in

200 Al 50 mM NH4HCO3. Supernatants were separated and

the membranes were washed twice with NH4HCO3 and then

vacuum dried. Dried pellets were dissolved in 7 Al, pH 1.9

buffer (4.6 formic acid:15.6 acetic acid:18 H2O). The phos-

phopeptides were detected with a Fuji phosphoimager. Eluted

phosphopeptides [35] were coupled to Sequelonk-AAmem-

brane (Millipore) by use of carbodiimide coupling, according

to standard procedures as described by the manufacturer.

Radio-Edman degradation was performed using an Applied

Biosystems gas-phase sequencer (Model 477A) as described

by Blume-Jensen et al. [38].

3. Results

3.1. Purification of SH3 domains

Introducing purified SH3 domains of Btk, Itk, Tec and

Bmx enable us to study transphosphorylation and to identify

a reliable substrate in vitro for quantifying kinase activity.

The SH3 domains were expressed and purified to homoge-

neity on glutathione-Sepharose columns followed by gel

filtration (Fig. 1A). Three Btk-SH3 domain variants were

Fig. 1. Purification of recombinant proteins. (A) Proteins were fractionated

on the Phastgels (8–20% gradient gels) system and stained with Coomassie

blue G-250. SH3 domains of GST-Grb2 (C-terminal) and GST-Amphiphy-

sin (Amp-SH3) domains were thrombin cleaved without further purifica-

tion. The quantity of protein loaded was between 9 and 12 Ag per lane. (B)

Autophosphorylation of rhBtk in vitro. The g32P-ATP-labeled rhBtk was

separated on 7.5% SDS-PAGE and blotted on PVDF membrane. The

amount of Btk loaded was 100 ng per lane. Lanes 1–3 represent three

batches of purified human Btk. Radiolabeled rhBtK were detected and

visualized by autoradiography.

B.F. Nore et al. / Biochimica et Biophysica Acta 1645 (2003) 123–132 125

produced, a wild-type SH3 domain, a Y223A mutation form

lacking the transphosphorylation site [21] and an extended

version of Btk-SH3 domain (PPSH3), containing both PP

region (GSSHRKTKKPLPPTPEEDQILKKPLPPEPA-

AAPV-) at the N-terminal. For recombinant Btk production,

human full-length Btk protein (rhBtk) was expressed in Sf9

insect cells using a baculovirus vector. The rhBtk was

purified with a Ni-affinity resin and was found to have

intact kinase activity (Fig. 1B).

3.2. Transphosphorylation specificity of Btk

Initial examination of SH3 domain phosphorylation was

done by IVK assay using immunoprecipitation of endoge-

nous Btk in Ramos cells (Fig. 2A). We followed the

phosphorylation level of purified SH3 domains of Btk,

Itk, Tec and Bmx under identical assay conditions. We

found that Btk preferentially phosphorylates its own SH3

domain, but also phosphorylates the Bmx-SH3 domain

strongly (Fig. 2A). Despite the fact that Bmx has an SH3-

like domain with an extended duplicated sequence, it con-

tains tyrosines and other amino acid residues conserved

among TFK-SH3 domains. Although the Itk-SH3 domain is

more related to the Btk-SH3 sequence, it is phosphorylated

to a lower degree in comparison with Btk-SH3 (Fig. 2A).

Moreover, the Tec-SH3 domain is a poor substrate for Btk

(Fig. 2A). Collectively, these data demonstrate that Btk

differentially transphosphorylates TFK-SH3 domains in

trans (Fig. 2A).

It has been shown earlier that the major Btk trans-

phosphorylation site is at position Y223 within the Btk-

SH3 domain [21]. Therefore, we included the Btk-SH3

Y223A domain mutant as a negative control. As expected

(Fig. 2A), Btk did not significantly phosphorylate the SH3

domain mutant. We observed that the transphosphorylation

of the Btk-SH3 domain is blocked significantly when the

PP peptide of Dynamin [39,40] is included in the IVK (Fig.

2B). It has been shown that the TFK-SH3 domains, like

SFK SH3 domains, preferentially bind to PP-rich motifs

with the consensus sequence R/KXXPXXP [41–44].

Because dynamin peptide (STPMPPPVDDSWLQVQ-

SVPAGRRSPTSSPTPQRRAPAVPPARPGSRGPAPGP-

PPAGSALGGAPPVPSRPGASPDPFGPPPQVPSRPN-

RAPPGVPSRSGQASPSRPESPRPP) contains two

potential recognition motifs R/KXXPXXP for the SH3

domains of TFKs [41,42,44], this strongly suggests that

the catalytic domain of Btk is unable to access and mediate

catalysis to the SH3 domain in trans due to the Dynamin PP

peptide interactions. However, we do not know whether this

interaction involves the recombinant SH3 domain, or the

full-length Btk molecule or both. To address the question of

whether the transphosphorylation activity of Btk is selec-

tive, we examined Grb2-SH3 (C-terminal) [29–31] and

Amphiphysin-SH3 [32] domain transphosphorylations. As

shown in Fig. 2B, these domains were not detectably

phosphorylated. This observation implicates that the phos-

phorylation event occurs at specific tyrosine site.

To address the question if the PP region in Btk interacts

with the adjacent SH3 domain in cis, we have also

generated an extended Btk-SH3 domain, containing intact

motifs in the N-terminal PP region (PPSH3). When PPSH3

protein is used in the IVK assay, we found that the SH3

domain phosphorylation level is unaffected (Fig. 2B). The

data imply that the PP motif of Btk does not have a

significant binding affinity in cis.

3.3. Transphosphorylation specificity of Itk

The Itk transphosphorylation activity was analyzed on

endogenous Itk obtained from the human T-cell line Molt4

Fig. 2. In vitro kinase assay of immunoprecipitated Btk. Btk in Ramos cells

was immunoprecipitated (IP) with anti-Btk and IP-beads used in a 20-Alkinase assay with 2 Ag of SH3 polypeptides in each reaction (A and B). The

phosphorylation level was monitored and visualized by autoradiography.

Lane 4, 0.6 Ag PP peptide of Dynamin (PPR) was included. The data

representing three independent experiments are shown for this and

subsequent figures.

Fig. 3. In vitro kinase assay of immunoprecipitated Itk. Itk in Molt4 cells

was immunoprecipitated (IP) with anti-Itk and IP-beads used in a 20-Alkinase assay with 2 Ag of SH3 polypeptides in each reaction. Lane 7, 0.6 AgPP peptide of Dynamin (PPR) was included. The phosphorylation level was

monitored and visualized by autoradiography.


(Fig. 3). We found that Itk efficiently phosphorylates both

Btk and Itk SH3 domains (Fig. 3). In other words, both Itk

and Btk SH3 domains are good substrates for Itk in vitro.

This observation raises the possibility that Itk and Btk might

regulate each other in cells coexpressing these kinases, such

as mast cells. Itk phosphorylated Bmx-SH3 to a low extent

(Fig. 3), in contrast to Btk, which efficiently phosphorylates

Bmx-SH3 (Fig. 2A). The Tec-SH3 domain is the least

suitable Itk substrate of the four TFK-SH3 domains used

(Fig. 3, lane 4). Furthermore, we observe that the Btk-SH3

mutant Y223A was not phosphorylated by Itk (Fig. 3, lane

5). Itk was unable to phosphorylate two non-conserved

tyrosines at the C-terminal Grb2-SH3 domain (Fig. 3, lane

6). This is in accordance with the idea that TFK-SH3

domains are the preferred substrates. The phosphorylation

level is reduced considerably by addition of Dynamin PP

peptide (Fig. 3, lane 7).

3.4. Transphosphorylation specificity of Tec

The endogenous kinase activity of Tec was investigated

from immunoprecipitation of Tec in Ramos cell extracts. In

a similar manner, we performed the kinase assay using

purified TFK-SH3 domains to determine a potential sub-

strate. Here, we noticed that Btk-SH3 is phosphorylated to a

similar extent as the Tec-SH3 domain (Fig. 4), but Btk does

not phosphorylate the Tec-SH3 domain appreciably (Fig.

2A). This observation may suggest that Tec has a broader

substrate specificity reflecting the broader tissue expression

of Tec. In addition, we found that Itk-SH3 and Bmx-SH3

domains are also phosphorylated by Tec (Fig. 4), but to a

smaller extent. Moreover, we could show that Tec does not

phosphorylate the Amphiphysin-SH3 domain, implying that

the location of tyrosine phosphorylation is conserved (Fig.

4, lane 3).

3.5. Transphosphorylation activity of rhBtk

For the subsequent experiments, we performed the IVK

assay using purified full-length rhBtk (Fig. 1B) to deter-

mine the transphosphorylation specificity and the consis-

tency with IP experiments of endogenous Btk. When we

performed the IVK assay for rhBtk with SH3 domain

substrates at identical concentrations as in the IP experi-

ments (Fig. 2A), namely 1–2 Ag, we found that the

transphosphorylation pattern is equivalent (not shown).

Moreover, the transphosphorylation pattern was found to

be consistent, even if the substrates are scaled-up 20 times

(Fig. 5). These observations demonstrate that the catalytic

activity of rhBtk has an identical substrate preference and

specificity as endogenous protein obtained from Ramos

cells (Fig. 2A).

3.6. Identification of transphosphorylation site(s)

To further characterize the recognition motif of phos-

phorylation, we decided to identify the phosphotyrosine

(pY) residue(s) in the SH3 domains. For this purpose, we

applied two-dimensional tryptic phosphopeptide mapping

(2D-mapping) using Hunter thin layer electrophoresis to

separate and resolve short phosphopeptides on TLC cellu-

lose plates.

In Fig. 6, we have summarized the results from 2D-

mapping experiments for Btk-SH3, Itk-SH3, Bmx-SH3 and

Tec-SH3 domains. The Btk-SH3 tryptic phosphopeptides

resolved on the 2D-TLC plate to three peptides labeled with32P (Fig. 6A). Among these phosphopeptides, only two show

a clear peptide sequence of VVALYDYMPMNANDLQL

and the pY was only identified in cycle 5, corresponding to

Y223 (Fig. 6A, inset) and this is in agreement with earlier

findings of the major transphosphorylation site within the

Btk-SH3 domain [21]. Here, we could confirm that Y223 is

the only site in the Btk-SH3 domain being detectably phos-

phorylated. In particular, we could exclude the possibility that

Y225 becomes phosphorylated, because the corresponding

tyrosine in Itk is conserved [20].

In a similar manner, we have analyzed Itk-SH3 tryptic

phosphopeptides (Fig. 6B) and the 2D-mapping was

resolved to one strong spot and a diffuse smeared band.

Fig. 4. In vitro kinase assay of immunoprecipitated Tec. Tec in Ramos cell

was immunoprecipitated (IP) with anti-Tec and IP-beads used in a 20-Alkinase assay with 2 Ag of SH3 polypeptides in each reaction. The


Fig. 5. In vitro kinase assay of rhBtk. Identical quantities of full-length

rhBtk protein (200 ng) were applied in a 20-Al kinase assay together with

20 Ag of SH3 polypeptides as substrates in each reaction. The


Phosphopeptide bands were digested with trypsin and subjected to 2D-

mapping analysis (described in Materials and methods).


Only, the strong band could produce a distinct peptide

sequence. The labeled tyrosine residue is found in cycle

10 and the pY residue was located in the following sequence

GSETVVIALYDYQTNDPQELAL (Fig. 6B, inset). This

pY corresponds to Y180 in the human Itk sequence. Multi-

ple sequence alignments of TFK-SH3 domains show that

Y180 is located at a conserved position, which is identical to

the Y223 location in Btk (Fig. 7).

As observed, the level of Bmx-SH3 phosphorylation is

high and even occasionally appears as a dual band (Fig. 5).

The tryptic phosphopeptide of Bmx-SH3 from the upper

band was resolved to a number of distinct spots (Fig. 6C),

but the lower band could not be separated as phosphopep-

tide spots (not shown). However, the analysis of all phos-

phopeptide spots from the upper band shows two major

phosphopeptide sequences, (highly labeled peptide)

IYGSQPNFNMQYIP and (weakly labeled peptide)

GSSSTSLAQYDSNS. As indicated, labeled tyrosines were

identified in cycles 2 and 10, respectively (Fig. 6C). The pY

positions correspond to the residues Y215 and Y223 in

Bmx. The phosphorylation site on Y215 corresponds to the

conserved tyrosine, which is found for both Btk (Y223) and

Itk (Y180). These findings show that these pY residues are

conserved in the TFKs (Fig. 7). However, the second Bmx-

SH3 phosphorylation site at Y223 differs from other TFKs

(Fig. 7). As described for the Bmx [45], the protein

sequence of both TH and SH3 domains are extended and

contains sequence repeats. One can expect that Bmx be

regulated differently due to these sequence differences and

as a consequence of double phosphorylations on the SH3

domain. Further experimental work is required to study the

importance of these phosphorylations in Bmx signaling and

SH3 domain interactions.

Although the transphosphorylation level of Tec-SH3

domain was always very low, we performed phosphopep-

tide analysis (Fig. 5). The phosphopeptide maps resulted in

a weak phosphopeptide spot (Fig. 6D). The phosphopeptide

sequence is found in this peptide (GQEYLILE) and the pY

eluted in cycle 4. This tyrosine is localized at Y206 in the

Tec sequence (Figs. 6D and 7). Despite the fact that this

tyrosine site is well conserved in all SH3 domains, tyrosine

phosphorylation at the identical position has not been

reported, so far, for any TFK (Fig. 7). However, our data

exclude the possibility of Y188 in the Tec-SH3 being

Fig. 6. Identification of pY sites using 2D-mapping. Phosphopeptide maps of the SH3 domains from Btk (A), Itk (B), Bmx (C) and Tec (D) are shown. Arrows

point to the major phosphopeptide spots, which were subjected to radio-Edman sequencing (inset). Peptide spots were eluted from the TLC plate and were

processed for radio-Edman sequencing (described in Materials and methods). The radio-Edman analysis (inset) is presented as a diagram of relative

radioactivity units (PSL) vs. the cycle number of eluted amino acid residues.


phosphorylated as residue, which is equivalent to the

conserved Btk-Y223, Itk-Y180 and Bmx-Y215 (Fig. 7).

Taken together, these data show that Tec-SH3 is not

phosphorylated at the common site found in the other

family members.

In addition to previously identified structures of Btk-SH3

[22] and Itk-SH3 domain [25], the Tec-SH3 domain struc-

ture has recently been published [46]. These structural

coordinates were used to visualize the conserved tyrosines

from these SH3 domains and the phosphorylated tyrosine

sites found in this work are decorated (Fig. 8). From the

structural model (Fig. 8), we found the Y206 on Tec-SH3

domain is positioned at the same side as Y223 in Btk and

Y180 in Itk. Our SH3 domain models show that the pY

identified in this work is located on the same surface. This

implies that these conserved tyrosines define a common

binding surface required for a specific interaction, which is

conserved between the TFK members.

3.7. Transphosphorylation of the SH3SH2 tandem domain

compared to the single SH3 domain

As described in earlier sections, the TFK-SH3 domains

are specifically phosphorylated, although the degree of

Fig. 7. Multiple sequence alignments of pY site in the Tec family SH3 domains. Multiple alignments of SH3 domains indicating phosphorylation sites (black

box) identified in this study. Sequence alignment was performed as described in Nore et al. [20].

Fig. 6 (continued ).


phosphorylation is variable. To further explore the trans-

phosphorylation selectivity, we purified tandem domains of

Btk-SH3SH2 and Itk-SH3SH2. As shown in Fig. 9, rhBtk

phosphorylates efficiently both SH3SH2 domains. In this

comparative analysis, both Btk-SH3SH2 and Itk-SH3SH2

were preferred, as compared to the Btk-SH3 domain alone.

This implies that SH3SH2 domains have additional inter-

actions with the cleft in the full-length kinases, in accord-

ance to earlier observations [47]. Furthermore, in agreement

with the experiments on single SH3 domains (Fig. 2), Btk-

SH3SH2 is more strongly phosphorylated than Itk-SH3SH2

domain (Fig. 9). The phosphorylation level of both Btk- and

Itk-SH3SH2 tandem domains is approximately 5- to 8-fold

higher as compared to the corresponding SH3 domain alone

(Fig. 9). The data demonstrate clearly the consistency of

transphosphorylation between SH3SH2 and SH3 domains;

despite that, SH3SH2 tandem domains are preferred sub-

strates.

4. Discussion

In this study, we show the significance of using purified

SH3 domains from TFKs as substrates in an IVK assay and

we observe that transphosphorylation catalysis for endoge-

nous kinases and for full-length rhBtk are identical. For Btk,

we found the transphosphorylation activity to be selective

for TFK-SH3 domains and the level of phosphorylation was

of the order BtkcBmx>Itk>Tec (Fig. 2A). For Itk, trans-

phosphorylation catalysis is also selective and the substrate

preference toward SH3 domains is in the order BtkcItk>Bmx>Tec (Fig. 3). We show that both Btk-SH3 and

Itk-SH3 domains are good substrates for Btk and Itk

kinases, suggesting the possibility that Btk and Itk can

modulate each other’s activity when they are coexpressed,

such as in mast cells. For Tec, the kinase activity was found

to have a broader specificity and the overall SH3 domain

phosphorylation was higher with the following order

TeccBtk>Itk>Bmx (Fig. 4). Taken together, we report

that the IVK activity of TFKs is rather specific. We have

found that TFK activity is restricted to SH3 domains of

family members and therefore the transphosphorylation

catalysis is a unique feature for these kinases (Figs. 2–4).

Because TFKs can be coexpressed in a single cell type,

for instance, Btk and Tec in B-cells and Itk and Txk in T-

cells, these kinases might compensate and/or modulate the

activity of each other via transphosphorylation catalysis.

Consistent with this idea, it has been shown that the double

knockout of Txk and Itk has a more severe phenotype such

as TCR responses, proliferation and cytokine production

[48] as compared to the Itk-deficient mice only [13,14]. In

analogy, Btk and Tec double knockout animals [49] show a

more severe B-cell deficiency compared to the mild Btk

deficient phenotype, Xid [10,11]. These observations could

mean that TFKs regulate each other, but could also reflect

that they may have redundant functions. Thus, it has been

shown that Itk, Tec and Bmx rescue Btk� / � chicken

lymphoma cells in terms of PLCg2-dependent Ca2 +

responses, MAPK activation and apoptosis signaling [50].

In addition, it has been suggested that Btk and Itk play

analogous roles in antigen receptor signaling pathways in

Fig. 9. Btk transphosphorylation of SH3SH2 tandem domains and single

SH3 domains from Btk and Itk. The IVK assay was done with 0.2 Ag rhBtk

in the presence of 4 or 10 Ag SH3SH2 domains of Btk or Itk together with 4

or 10 Ag of Btk-SH3 domain. The assay conditions were as described in

Materials and methods.

Fig. 8. Location of pYand conserved aromatic residues within the structure of the SH3 domains from Btk, Itk and Tec. The pY residues are highlighted with red

color. Conserved, but not phosphorylated tyrosines are colored yellow. The side chains of tryptophane (blue) and phenylalanine (green) residues that are located

in or close to the peptide binding site are indicated. The model was created using the software MolMol [57]. The pdb files used were: 1gl5 for Tec SH3 [46],

1awx for Btk SH3 [22] and 1awj for Itk SH3 [25].


accordance with a reciprocal pattern of expression in B-cells

and T-cells, respectively [2,51]. However, human B cells,

such as the Ramos cell line used in this study, coexpress Btk

and Tec, and yet the phenotype in XLA is very severe. This

argues against redundant functions although the expression

level may be a significant factor.

To determine the location of SH3 phosphorylation sites,

we carried out phosphopeptide mapping of Btk, Itk, and

Bmx and Tec SH3 domains subsequent to transphosphor-

ylation. From tryptic peptide mapping analysis of Btk, we

confirmed that the Btk-SH3 phosphorylation site is located at

Y223 (Fig. 6A), a result identical to that achieved by the

previous phospho-Btk mapping [21]. We demonstrate here

that the transphosphorylation site of Itk-SH3 is located at

Y180 (Fig. 6B). In addition, sequence homology alignments

show that Itk-Y180 corresponds to Btk-Y223 [20]. For Bmx,

we obtained two phosphorylated sites, Y215 and Y223 (Fig.

6C). The Bmx-Y215 is a conserved tyrosine, which is

homologous to Btk-Y223 and Itk-Y180. In contrast, Bmx-

Y223 site is a unique site with no homology to SH3 domains

of other TFKs (Fig. 7). The Bmx-SH3 domain, unlike other

family members, has an extended repeat at the N-terminal

region [2,45] and the overall phosphorylation level is differ-

ent. On the other hand, Tec is widely expressed in different

tissues and was found to have a broad preference for the

TFK-SH3 domains, whereas Tec-SH3 protein was a poor

substrate for Btk and Itk kinases (Fig. 5). The weak trans-

phosphorylation site of Tec-SH3 domain was found at Y206

(Fig. 6D). The Y206 site is still a conserved tyrosine, which

has not been shown phosphorylated in any family member

SH3 domain (Fig. 7). On other hand, the common phos-

phorylated tyrosine found in Btk-Y223, Itk-Y180 and Bmx-

Y215 is not phosphorylated in the Tec-SH3 domain. The

majority of phosphorylation sites are conserved tyrosines

between all TFK members. They are located at a common

binding surface site, as illustrated from the three-dimensional

model for Btk, Itk, and Tec SH3 domains (Fig. 8). When the

Btk-SH3 domain is autophosphorylated, its ability to take

part in protein interactions, such as with WASP, is altered as

compared to the non-phosphorylated fusion protein [52]. In

addition, the BCR-induced calcium mobilization and PLCg2

phosphorylation are abrogated partially by Btk mutation on

tyrosine 223 to phenylalanine (Y223F) [53]. Therefore, we

believe that SH3 domain phosphorylations regulate the

protein–ligand interactions for all Tec family members. In

accordance to the single SH3 domain phosphorylations, Btk-

SH3SH2 tandem domains are more strongly phosphorylated

than Itk-SH3SH2. This could mean that SH3SH2 tandem

domains have an additional interaction(s) with the substrate-

binding cleft in the kinase domain, or other parts of the

kinase, or, alternatively, that the SH3SH2 doublet alters

conformation so that the target tyrosine residue is more

exposed. Furthermore, we have not ruled out the possibility

of new phosphorylation sites in the doublet. However, the

isolated SH2 domain of Btk is not a substrate (unpublished

observations).

Taken together, our data show that transphosphorylation

of SH3 domains of the TFKs is a conserved mechanism,

which is not established in other SH3 domain-containing

proteins [54]. However, it has been demonstrated that two c-

Src SH3 domain mutants, Y133 and Y138, inhibit mito-

genic signaling, phosphorylation presumably being a pre-

requisite for the function [55,56]. On the other hand, these

two tyrosines in the Src SH3 domain are located at the SH3

domain C terminus, which is not homologous to the

phosphotyrosines reported in this work. Here, we show

the TFK transphosphorylation site to be specific and con-

served. Despite the fact that the role of SH3 domain

phosphorylations is still unclear, our data imply that TFK-

SH3 domains serve a conserved function. It has been shown

previously that the PXXP motif in the TH region of Itk may

associate in cis with its SH3 domain [25]. In the case of Btk,

such interactions can be observed in trans [26]. Further

examination of PP peptide interactions in two independent

studies [27,28] showed that Btk, in contrast to the Itk, forms

a dimer. In fact, this reflects the existence of double PP

stretches in Btk favoring asymmetric homodimer forma-

tions, whereas Itk containing a single PP motif allows

interactions in cis [27,28]. The identification of the pY

targets in the SH3 domain of the TFKs demonstrates another

level of regulation with both conserved and unique features.

Acknowledgements

We are indebted to Christer Wernstedt for radio-Edman

sequencing. The Swedish Cancer Foundation, Swedish

Medical Research Council, the European Union Grant

QLRT-2000-01395, the Paivikki and Sakari Sohlberg

Foundation, and Sigurd and Elsa Goljes Stiftelse supported

this work. Lars Ronnstrand holds a position as Senior

Researcher funded by the Swedish Research Council.

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identification of phosphorylation sites within the sh3 domains of tec family tyrosine kinases

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