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`
`Biol. Chem., Vol. 385, pp. 409–413, May 2004 • Copyright 䊚 by Walter de Gruyter • Berlin • New York
`
`Short Communication
`
`The Btk inhibitor LFM-A13 is a potent inhibitor of Jak2
`kinase activity
`
`kinase (Erk1/2) pathways but also tyrosine kinases like
`Lyn, a member of the Src-family, and Bruton’s tyrosine
`kinase (Btk), a member of the Tec-family (Chin et al.,
`1998; Schmidt et al., 2004).
`Tec-family members harbour an N-terminal pleckstrin
`homology (PH) domain, a proline-rich (PR) domain, a
`SRC homology 3 (SH3) domain, a Src homology 2 (SH2)
`domain and a kinase domain (SH1). Upon membrane
`recruitment via its PH domain, activation of Tec-family
`members occurs through phosphorylation of tyrosine
`residues in the kinase domain by Src kinases (amino acid
`551 in Btk; Rawlings et al., 1996). This phosphorylation
`event is believed to release the intramolecular interaction
`between the PR and the SH3 domain, thereby releasing
`the kinase domain from structural restraints (Andreotti et
`al., 1997; Brazin et al., 2000). Trans-phosphorylation
`results in activation of the kinase and subsequent auto-
`phosphorylation of a tyrosine residue within the SH3
`domain (amino acid 223 in Btk; Rawlings et al., 1996).
`Both membrane recruitment and kinase activation are of
`paramount importance for Btk function (Kurosaki and
`Kurosaki, 1997; Nisitani et al., 1999; Saito et al., 2001).
`Btk has been shown to play a role in phospholipaseC-
`g1/2 activation (Takata and Kurosaki, 1996; Fluckiger et
`al., 1998; Rawlings, 1999) and cytoskeleton organisation
`via WASP (Guinamard et al., 1998). Furthermore, it has
`been shown that Tec and Jak1 are able to cross-phos-
`phorylate each other, indicating that Tec family kinases
`may have a modulatory effect on Jak kinases or vice ver-
`sa (Takahashi-Tezuka et al., 1997; Yamashita et al., 1998).
`To analyse the role of Btk in EpoR signalling, we used
`the chemical compound a-cyano-b-hydroxy-b-methyl-
`N-(2,5-dibromophenyl)propenamide or in short LFM-A13.
`This inhibitor was reported to bind specifically to the cat-
`alytic pocket of the Btk kinase domain and not of other
`related tyrosine kinases like Jak1, Jak3 and Src kinases
`(Mahajan et al., 1999). Erythroid progenitors (cell line R10)
`were factor deprived in the presence and absence of
`100 mM LFM-A13, the recommended concentration for
`this inhibitor (Mahajan et al., 1999), and subsequently
`stimulated with Epo or left unstimulated. Cells treated
`with LFM-A13 during starvation did not show any abnor-
`malities with respect to cell shape and viability compared
`to untreated cells (data not shown). While Epo efficiently
`induced phosphorylation of the EpoR, Jak2, Btk, Stat5
`and Erk1/2, this was significantly reduced in the pres-
`ence of 100 mM LFM-A13 (Figure 1A, B). The co-immu-
`noprecipitating bands in the EpoR immuno-precipitate
`representing Stat5 and Shc (Figure 1A; identified by spe-
`cific antibodies) were absent when cells were pre-treated
`with LFM-A13. Thus LFM-A13 severely impairs EpoR
`phosphorylation and the recruitment of proteins. Since
`
`Emile van den Akker1,a, Thamar B. van Dijk1,b,
`Uwe Schmidt2,c, Lamberto Felida1, Hartmut
`Beug2, Bob Lo¨ wenberg1 and Marieke von
`Lindern1,*
`1 Department of Hematology, Erasmus MC, P.O. Box
`1738, NL-3000 DR Rotterdam, The Netherlands
`2 Institute of Molecular Pathology, Dr. Bohr-Gasse 7,
`A-1030 Vienna, Austria
`
`* Corresponding author
`e-mail: m.vonlindern@erasmusmc.nl
`
`Abstract
`
`LFM-A13, or a-cyano-b-hydroxy-b-methyl-N-(2,5-di-
`bromophenyl)propenamide, was shown to inhibit Bru-
`ton’s tyrosine kinase (Btk). Here we show that LFM-A13
`efficiently inhibits erythropoietin (Epo)-induced phospho-
`rylation of the erythropoietin receptor, Janus kinase 2
`(Jak2) and downstream signalling molecules. However,
`the tyrosine kinase activity of immunoprecipitated or in
`vitro translated Btk and Jak2 was equally inhibited by
`LFM-A13 in in vitro kinase assays. Finally, Epo-induced
`signal transduction was also inhibited in cells lacking Btk.
`Taken together, we conclude that LFM-A13 is a potent
`inhibitor of Jak2 and cannot be used as a specific tyro-
`sine kinase inhibitor to study the role of Btk in Jak2-
`dependent cytokine signalling.
`
`Keywords: cytokine signalling; erythropoiesis; tyrosine
`kinase.
`
`Cytokine receptors are devoid of intrinsic kinase activity
`and require the association with cytoplasmic tyrosine
`kinases to transmit signals upon ligand binding. Janus
`tyrosine kinase 2 (Jak2) activity is crucial for most cyto-
`kine receptors including the erythropoietin receptor
`(EpoR; Parganas et al., 1998). Epo binding to the EpoR
`induces a conformational change which juxtaposes the
`associated Jak2 kinases resulting in cross-phosphoryla-
`tion, activation and subsequent phosphorylation of the
`EpoR and induction of multiple signalling intermediates
`(for review see Wojchowski et al., 1999). These include
`signal transducer and activator of transciption 5 (Stat5),
`protein kinase B (PKB), the mitogen-activated protein
`
`Present addresses:
`a Institut Curie, Centre Universitaire, Baˆ t. 110, F-91405 Orsay,
`France
`Department of Cell Biology and Genetics, Erasmus MC, NL-
`000 DR Rotterdam, The Netherlands
`Institute of Immunology, University of Vienna, A-1235 Vienna,
`ustria
`
`c A
`
`b 3
`
`

`

`410 E. van den Akker et al.
`
`IP, Erythroid cell line R10
`+
`+
`Epo:
`-
`+
`-
`
`LFM-A13:
`
`150 kDa -
`
`100 kDa - ~ ♦ Stat5
`
`75 kDa - .. ♦ EpoR
`
`A
`
`EpoRIP
`
`anti-pTyr
`
`50 kDa -
`
`anti-EpoR
`
`♦ Sh e
`
`Jak21P
`
`Btk IP
`
`B
`
`anti-Stats
`
`anti-She
`
`I anti-pTyr . b I
`
`anti-Jak2 ....... -
`
`I anti-pTyr
`anti-Btk
`
`WCL, Erythroid cell line 1/11
`+
`+
`+
`
`-
`-
`
`Epo:
`LFM-A13:
`
`-
`C
`
`J
`
`~;;;~i~;;,;;;
`anti-p-Statsl
`anti-Stats I •
`j J j
`-
`
`anti-p-Erk
`
`anti-Erk
`
`Figure 1 Epo-induced phosphorylation of EpoR, Jak2, Btk,
`Stat5 and Erk1/2 is inhibited by pre-treatment with 100 mM LFM-
`A13.
`R10 cells were factor deprived in plain Iscoves medium (Invitro-
`gen, Breda, The Netherlands) for 4 h in the presence or absence
`of LFM-A13 (100 mM) and subsequently stimulated with Epo
`(5 U/ml for 10 min; 40–80=106 cells/ml) at 378C. Ten volumes
`of ice-cold PBS were added to stop the reaction. Preparation of
`cell extracts,
`immunoprecipitations, SDS-polyacrylamide gel
`electrophoresis and Western blots were performed as described
`by van Dijk et al. (2000).
`(A) The EpoR, Jak2 and Btk were immunoprecipitated to analyse
`Epo-induced phosphorylation on Western blots. The upper pan-
`els represent anti-phosphotyrosine stained blots; the lower pan-
`els represent the same blots re-probed with the antibody used
`in the immunoprecipitation (anti-EpoR, anti-Jak2 and anti-Btk)
`as indicated. In the EpoR immunoprecipitate the position of size
`markers is indicated and arrows indicate the EpoR and known
`proteins co-immunoprecipitating. Btk (lower band) is indicated
`with an arrow in the Btk immunoprecipitate. (B) In the upper
`panels, Stat5 and Erk1/2 phosphorylation was detected with
`phospho-specific antibodies in whole cell lysates, lower panels
`represent the total amount of Stat5 and Erk1/2 detected with
`anti-Stat5 and anti-Erk1/2, respectively. Antibodies against the
`murine EpoR (噛SC-697), Erk1/2 (噛SC-94), Stat5 (噛SC-836) and
`phosphotyrosine (PY99, 噛SC-7020) were purchased from Santa
`Cruz (tebu-bio, Heerhugowaard, The Netherlands), phospho-
`specific Erk1/2 (噛9106L) from Cell signalling (Westburg, Leus-
`den, The Netherlands), antibodies against mouse Jak2
`(噛06–255), phospho-specific Stat5 (噛05–495) from Upstate Bio-
`technology (Campro-Scientific, Veenendaal, The Netherlands).
`Recombinant human Epo was a kind gift from Ortho Biotech
`(Tilburg, The Netherlands). LFM-A13 was manufactured by
`Boehringer Ingelheim (Germany) as described by Mahajan et al.
`(1999).
`
`we found that Btk is required for efficient signal trans-
`duction by the ligand-activated EpoR (Schmidt et al.,
`2004), we expected LFM-A13 to inhibit Epo-induced sig-
`nal transduction.
`However,
`the strong effect of LFM-A13 on Epo-
`induced signalling could also be due to direct inhibition
`of Jak2 by LFM-A13. To investigate whether LFM-A13
`could act directly on Jak2, Btk and Jak2 were immuno-
`precipitated from lysates of factor-deprived cells and
`subjected to in vitro kinase assays in the presence of
`increasing amounts of LFM-A13. Surprisingly, the kinase
`activity of Btk and Jak2 (auto-phosphorylation) appeared
`to be inhibited with similar dose-response curves. Both
`kinases were already inhibited at 10 mM LFM-A13, while
`complete inhibition occurred at 200 mM LFM-A13 (Figure
`2A). The same results were obtained when Btk and Jak2
`were immunoprecipitated from Epo-stimulated cells (Fig-
`ure 2B). Epo stimulation leads to Btk and Jak2 phospho-
`rylation, resulting in a lower pool of non-phosphorylated
`Btk or Jak2, hence explaining the lower auto-phospho-
`rylation in the Epo-stimulated lanes. These data suggest
`that LFM-A13 directly inhibits Jak2 kinase activity.
`However, it could still be possible that the effect of
`LFM-A13 on Jak2 is mediated by Btk. If both proteins
`interact with each other, the presence of Btk in the Jak2
`immunoprecipitate could affect Jak2 auto-phosphoryla-
`tion. To rule out Btk interference in the in vitro kinase
`assay, we used in vitro transcription/translation (ITT) to
`synthesise recombinant Jak2 that was subsequently
`subjected to an in vitro kinase assay in presence or
`absence of LFM-A13. Expression of Jak2 was detected
`by immunoblotting using anti-Jak2 antibodies. Jak2 was
`
`A
`
`Btk IP, Jak2 IP,
`in vitro autophosphorylation
`
`LFM-A13 (µM ):
`
`0
`
`10
`
`20
`
`100
`
`200 1000
`
`[32P]-Btk
`
`[32P]Jak2
`
`B
`
`Btk IP, Jak2 IP,
`in vitro autophosphorylation
`
`LFM-A13:
`Epo (5U/ml):
`
`-
`
`+
`+
`
`+
`
`[ 32P]-Btk
`
`[ 32P]Jak2
`
`Figure 2 LFM-A13 inhibits in vitro auto-phosphorylation of Btk
`and Jak2.
`(A) Jak2 and Btk were immunoprecipitated from factor-depleted
`erythroid progenitors (R10) and tested for auto-phosphorylation
`in an in vitro kinase assay. Sepharose G beads (Sigma, Zwy¨ n-
`drecht, The Netherlands) with immunoprecipitated Jak2 and Btk
`complexes were split into 6 equal portions and subsequently
`subjected to an in vitro kinase assays as described by von Lin-
`dern et al. (2000) in the presence of increasing concentrations
`LFM-A13 as indicated on the top. (B) Jak2 and Btk were immu-
`noprecipitated from cells non-stimulated (y) or Epo-stimulated
`(q; 5 U/ml) and similarly subjected to an in vitro kinase assay.
`100 mM LFM-A13 was added to precipitates from stimulated
`cells.
`
`

`

`A
`
`Jak2 IP, Western blot
`
`A
`
`IP, Erythroid BM-derived cell lines
`
`LFM-A13 inhibits Jak2 kinase activity 411
`
`Jak2 template :
`
`anti-Jak2
`
`+
`
`R10
`control
`
`B
`
`Jak2 IP,
`in vitro autophosphorylation
`
`LFM-A13:
`Jak2 template:
`[32P]Jak2
`
`+
`+
`+
`- - - - - - -
`
`Figure 3 LFM-A13 inhibits auto-phosphorylation of recombi-
`nant Jak2 produced by in vitro transcription/translation (ITT).
`The in vitro transcription/translation was carried out using the
`TnT䊛 T7 coupled reticulocyte lysate system (噛L4611; Promega,
`Leiden, The Netherlands) according to the manufacturer’s
`protocol.
`(A) Jak2 was immunoprecipitated from ITT reactions in the
`absence (lane1) or presence (lane2) of Jak2 template or from the
`erythroid progenitor cell line R10 and analysed for their presence
`on Western blots using an anti-Jak2 antibody. (B) To test the
`effect of LFM-A13 on recombinant Jak2, immunoprecipitated
`ITT Jak2 was subjected to an in vitro kinase assay in the
`absence and presence of 100 mM LFM-A13 (lanes 2 and 3). Lane
`1 represents Jak2 immunoprecipitate on ITT-lysate with no Jak2
`template present.
`
`only present when the template was added to the ITT
`and the mobility of recombinant Jak2 was the same as
`immunoprecipitated Jak2 when compared to the ery-
`throid progenitor cell
`line R10 (Figure 3A). Auto-phos-
`phorylation of in vitro synthesised Jak2 in an in vitro
`kinase assay was fully inhibited by 100 mM LFM-A13
`(Figure 3B). This proves that LFM-A13 directly inhibits
`Jak2 kinase activity.
`Finally, we tested the effect of LFM-A13 in cells devoid
`of Btk.
`Immortalised cultures of erythroid progenitors
`established from Btky or Btkwt, p53-deficient bone mar-
`row cells, were factor deprived in the presence or
`absence of 100 mM LFM-A13 and stimulated with Epo or
`left unstimulated. Epo-induced phosphorylation of the
`EpoR is impaired in Btk-deficient cells (Schmidt et al.,
`2004; Figure 4A). However, the remaining Epo-induced
`phosphorylation of the EpoR is still blocked by LFM-A13.
`Importantly, Epo-induced phosphorylation of Jak2, Stat5
`and Erk1/2, was clearly inhibited by 100 mM LFM-A13 in
`Btk-deficient cells (Figure 4A, B), providing further evi-
`dence that LFM-A13 directly affects Jak2, independently
`of Btk. Finally, we tested whether LFM-A13 still showed
`some of its alleged specificity. COS cells were transfect-
`ed in duplo with the kinases Jak2, Tec, Btk and Lyn. Half
`of the cells were treated with LFM-A13, the other half left
`untreated. Cell lysates were harvested and examined for
`auto-phosphorylated kinase by Western blotting. LFM-
`A13 inhibited auto-phosphorylation of Jak2, Tec and Btk,
`but it did not affect Lyn kinase auto-phosphorylation (Fig-
`ure 5). Thus, LFM-A13 does not inhibit the activity of all
`tyrosine kinases.
`Both Jak2 and Tec-family kinases, including Btk, are
`prominent cytoplasmic tyrosine kinases in hematopoietic
`cells which co-operate in signalling pathways activated
`by e.g. cytokine receptors, the B cell receptor and the
`
`Btk-
`
`+
`
`Epo:
`LFM-A13:
`
`-
`-
`
`+
`+
`
`anti-Jak2
`
`Btkwt
`+
`+
`+
`anti-pTyr '---=----------'I I Jak2 IP
`I
`
`anti-pTyr
`anti-EpoR !=I =::::!!!!!!!!!!!!::::========!I EpoR IP
`
`B
`
`WCL, Erythroid BM-derived cell lines
`
`Epo:
`LFM-A13:
`
`anti-Erk
`
`anti-p-Stat5 I
`l:t
`anti-Stat5
`anti-p-Erk I
`Ira:
`
`+
`
`-
`l a I I
`
`+
`+
`
`I
`I I
`I
`I
`
`Figure 4 LFM-A13 inhibits Jak2, Stat5 and Erk1/2 phospho-
`rylation in Btk-deficient erythroid progenitor cells.
`The murine erythroid cell lines 2B6 (Btkwt) and 3G4 (Btky) were
`established from p53-deficient mouse bone marrow of wt and
`Btk-deficient mice, respectively, as previously described (von
`Lindern et al., 2001; Schmidt et al., 2004). These Btk-deficient
`cells were factor-depleted in the presence and absence of
`100 mM LFM-A13 as indicated and stimulated with Epo (5 U/ml;
`10 min) where indicated.
`(A) The EpoR and Jak2 proteins were immunoprecipitated and
`assayed for tyrosine phosphorylation wanti-p-Tyr (PY99); upper
`panelsx and for the presence of the EpoR and Jak2 by specific
`antibodies. (B) Lysates of the Btky cells (3G4) were assayed on
`Western blots using phospho-specific antibodies for Stat5 or
`Erk1/2. The lower panels indicate the same blots re-probed with
`anti-Stat5 or anti-Erk1/2 antibodies to check for equal loading.
`
`WCL, COS cells
`Btk
`--
`+
`
`Jak2 Tee
`+
`- +
`-
`
`-
`
`LFM-A 13:
`
`Jak2 ♦
`Btk ♦
`Tee ♦
`Lyn ♦
`
`Lyn
`- +
`
`150
`100
`75
`
`50
`
`Figure 5 LFM-A13 inihibits Jak2, Tec and Btk, but not Lyn
`auto-phosphorylation in COS cells.
`COS cells were transfected with pSG5-based expression plas-
`mids encoding Jak2, Tec, Btk or Lyn, using calcium-phosphate
`precipitates as previously described (Van Dijk et al., 2000). Two
`dishes were used for each construct and LFM-A13 (100 mM) was
`added 24 h after transfection to one of the dishes. Forty-eight
`h after transfection the cells were lysed and total cell lysates
`were examined for phosphorylated proteins on a Western blot
`using the anti-phosphotyrosine antibody PY99 (anti-pTyr). The
`kinases expressed in COS cells are indicated on top of the lanes,
`the position of the kinases is indicated by arrows. The position
`and of size markers is indicated at the right hand side (molecular
`mass in kDa).
`
`

`

`412 E. van den Akker et al.
`
`Fc´ receptor (Qiu et al., 2000). Moreover, Jak1 and Tec
`can associate and cross-phosphorylate each other (Tak-
`ahashi-Tezuka et al., 1997) and we recently demonstrat-
`ed that Btk similarly associates with Jak2 (Schmidt et al.,
`2004). Since lack of Btk impairs EpoR signalling, the
`effect of LFM-A13 on Epo-induced signal transduction
`was initially expected. However, since LFM-A13 inhibited
`intrinsic auto-phosphorylation of cellular or recombinant
`Jak2 in in vitro kinase assays and significantly decreased
`Epo-induced Jak2 phosphorylation in cells lacking Btk,
`we conclude that LFM-A13 directly inhibits Jak2 phos-
`phorylation independent of its effect on Btk.
`It was previously reported that the kinase activity of
`Jak1 and Jak3, close homologues of Jak2, was not inhib-
`ited by high concentrations (139 to 278 mM) of LFM-A13
`in in vitro kinase assays (Mahajan et al., 1999). Impor-
`tantly,
`the sequences of the kinase domains of Jak
`kinases are highly conserved. LFM-A13 incorporation
`into the catalytic site of Btk requires hydrogen bonds
`between the side chain of LFM-A13 and Arg525 and Asp539
`of Btk and a hydrophobic pocket for the aromatic ring.
`The kinase domains of Btk and Jak kinases both contain
`arginine and aspartic acid residues at comparable posi-
`tions, but there are several differences regarding the
`hydrophobic pocket trapping the aromatic ring of LFM-
`A13. Mahajan et al. (1999) discussed the consequences
`of these differences with respect to LFM-A13 binding
`affinity, but complete understanding of these affinities is
`still elusive. Jak1 and Jak3 kinase activity was shown to
`be unaffected by LFM-A13 (Mahajan et al., 1999) but no
`data was shown for Jak2, leaving open the possibility
`that Jak2 is the exception in the Jak kinase family with
`respect to LFM-A13 sensitivity.
`In conclusion, we have shown that LFM-A13 is not
`specific for Btk but also inhibits Jak2 kinase activity in
`vitro and in vivo. LFM-A13 is proposed to be a useful
`anti-leukaemic and anti-thrombotic agent (Uckun, 2002,
`2003). Although the successful use of STI571 to treat
`chronic myelogenous leukaemia proves that an inhibitor
`does not necessarily need to be specific in order to be
`effective (Mauro, 2002), it is important that its specificity
`is known. Using LFM-A13 to elucidate the role of Btk in
`signal transduction of Jak2-dependent cytokine recep-
`tors could result in wrong conclusions due to simulta-
`neous inhibition of Jak2 and Btk by LFM-A13.
`
`Acknowledgements
`
`The authors thank Rudi Hendriks for providing the Btk- and p53-
`deficient mice bone marrow, and Martine Parren-van Amelsvoort
`for technical assistance. This work was supported by grants
`from the Dutch Cancer Society (EUR 1999–2064), the Nether-
`lands Organisation for Scientific Research (NWO 901-08-338),
`the European Union (HPRN-CT-2000-00083), a fellowship of the
`Erasmus University Rotterdam (EUR) to T.B.vD. and a fellowship
`of the Dutch Academy of Arts and Sciences to M.vL.
`
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`Received October 6, 2003; accepted February 5, 2004
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