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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 5 (March 1), 1998:
pp. 1496-1507
Tec and Jak2 Kinases Cooperate to Mediate Cytokine-Driven
Activation of c-fos Transcription
By
Yoshihiro Yamashita,
Sumiko Watanabe,
Akira Miyazato,
Ken-ichi Ohya,
Uichi Ikeda,
Kazuyuki Shimada,
Norio Komatsu,
Kiyohiko Hatake,
Yasusada Miura,
Keiya Ozawa, and
Hiroyuki Mano
From the Department of Molecular Biology, the Divisions of Hematology
and Cardiology, Jichi Medical School, Tochigi; and the Department of
Molecular and Developmental Biology, Institute of Medical Science,
University of Tokyo, Tokyo, Japan.
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ABSTRACT |
Although transcriptional activation of the c-fos
proto-oncogene plays an intrinsic role in the mechanism of blood cell
growth, it is still obscure how protein-tyrosine kinases (PTKs)
regulate the cytokine-driven c-fos activation
pathway. We present here that Tec PTK is
tyrosine-phosphorylated and activated by granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation in a human
GM-CSF-dependent cell line. Moreover, we could show that introduction
of Tec into mouse BA/F3-hGMR  cells can profoundly activate the
c-fos promoter in response to GM-CSF or to interleukin-3
(IL-3). In contrast, introduction of a kinase-deleted Tec could
suppress cytokine-driven c-fos activation, indicating that Tec
is directly involved in the regulation of c-fos transcription.
Interestingly, strong activation by Tec of the c-fos promoter
was blocked by the co-expression of dominant negative Jak2. The
molecular interaction between Tec and Jak2 was then investigated both
in mammalian and insect cell systems, revealing that they can not only
bind to each other, but either of the two can phosphorylate the other.
Thus, Tec and Jak2 can "cross-talk" in a complexed way to mediate
cytokine-driven c-fos activation.
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INTRODUCTION |
TEC PROTEIN-TYROSINE kinase (PTK) is the
prototype of a recently emerging subfamily among nonreceptor PTKs
including Tec, Btk, Emt/Itk/Tsk, Txk, and Bmx.1,2 In
contrast to the Src-family kinases, none of the Tec-family members
carry the myristylation signals or the C-terminal tyrosine residues
corresponding to Tyr-527 in c-Src. One of the characteristic feature of
the Tec-family members is that they (with the exception of Txk) contain
a relatively long N-terminal unique region comprising a pleckstrin
homology (PH) domain3 and a Tec homology (TH)
domain.4 Tec-family members are, to date, the only PTKs
containing the PH domain in their structures. Subsets of phospholipids
have been shown to bind to the PH domain of Btk5,6 and Tec
(T. Shirai and Y. Fukui, personal communication), and this
PH-phospholipid interaction is supposed to play an important role in
the recruitment of the PTKs to cell membrane and/or in the
regulation of the kinase activities, as already proven in the case of a
serine/threonine kinase, c-Akt/PKB/Rac.7
Although physiological roles of the Tec-family members are still to be
revealed, accumulating evidence has suggested that Tec-family PTKs may
be involved in the growth and/or differentiation mechanism of
hematopoietic cells. First, many Tec-family members are abundantly
expressed in blood cells; for instance, Btk in myeloid cells and B
lymphocytes,8 Emt/Itk/Tsk in T lymphocytes,9 and Tec in all the lineages.2 Second,
mutations in Btk cause agammaglobulinemia in humans.10,11
Third, many Tec-family kinases have been shown to be implicated in the
intracellular signaling pathways of cytokines. We and our colleagues
have shown that Tec can be tyrosine-phosphorylated and activated in
response to interleukin-3 (IL-3), IL-6, stem cell factor (SCF),
granulocyte colony-stimulating factor (G-CSF), erythropoietin (EPO), or
thrombopoietin (TPO).12-17 Tec was shown to be physically
associated (either directly or indirectly) with the receptors for SCF
and IL-6. In addition to Tec, Btk and Emt/Itk/Tsk have also been shown
to be activated by growth/differentiation signals of
lymphocytes.18-20 Thus, currently one of the major concerns
in the research of Tec-family kinases is in which part of intracellular
machinery of cytokines they directly participate.
The c-fos proto-oncogene is one of the immediate early genes
induced by a wide range of cytokine stimulations, and can encode a
transcription factor containing a "leucine zipper"
structure.21 Although transcriptional activation of
c-fos is not directly involved in DNA synthesis, it is believed
to be one of the important mechanisms to maintain the growth of blood
cells.22 So far, two PTK-subfamilies have been suggested to
play a regulatory role in the c-fos transcription. Minami et
al23 have shown that Lck, a member of the Src-family, becomes activated by the stimulation with IL-2, and this PTK activation correlates well with the accumulation of c-fos transcripts. On the other hand, expression of a dominant negative form of Jak2 or Jak3
was demonstrated to suppress the c-fos transcription induced by
granulocyte-macrophage CSF (GM-CSF)24 or
IL-2,25 respectively. Although these data place
Src-family and Jak-family members in the c-fos regulation
pathways, little is still understood for the molecular mechanism by
which they activate the c-fos promoter. We and others have
already shown that Lyn, a member of the Src-family, can phosphorylate
and activate Tec and Btk in cells.26,27 Thus, the
Tec-family members are likely to work downstream of the Src-family kinases in vivo. Therefore, it would be an intriguing question whether
Tec is directly involved in the regulation of the c-fos promoter activity in the hematopoietic system.
To address this issue, here we transiently introduced a reporter
plasmid (pfos/luc), containing the promoter region of the c-fos gene and the luciferase cDNA, into mouse
BA/F3-HGMR cells28 which express the
high-affinity receptors for human GM-CSF. pSR plasmids carrying the
cDNAs of various nonreceptor PTKs were co-introduced to compare the
ability of each PTK to modulate c-fos promoter activity.
Interestingly, we could observe that Tec was one of the most potent
PTKs in the ability of the reporter gene activation. On the contrary,
introduction of a kinase-deleted Tec could suppress the cytokine-driven
c-fos activation in a dose-dependent manner. Because Jak2
expression also activated the c-fos promoter in our assay, we
next investigated the functional and physical interaction between Tec
and Jak2 in the context of c-fos activation mechanism. Co-expression of dnJak2 could block the Tec-driven c-fos
activation, suggesting that Jak2 may work at a point downstream of Tec.
Surprisingly, in both 293 cells and insect cells we could show that Tec
and Jak2 can not only associate with, but also phosphorylate, each other. Our data indicate that the Src-, Tec-, and Jak-family members functionally interact to transduce the cytokine-driven c-fos
activation mechanism.
(C) BA/F3-hGMR cells
(1 × 107) were transfected with the pfos/luc
reporter plasmid (0.5 µg) with various amounts of pSR-Tec KD (TKD) as indicated at the bottom (in micrograms). After starvation in cytokine-free medium, cells were further cultured for 3 hours without (no factor) or with GM-CSF (+GM-CSF; 5 ng/mL) or mouse IL-3
(+IL-3; 25 U/mL). Total amount of plasmid DNA for each transfection was adjusted to be equal by adding the pSR vector DNA (pSR). The
mean value plus SD of the luciferase activities in triplicate samples
from each fraction is shown as arbitrary units. (D) BA/F3-hGMR cells (1 × 107) were transfected with the
pfos/luc reporter plasmid (2 µg) together with of pSR
(pSR), pSR-Tec (Tec), and pSR-dnRas (dnRas) plasmids at the
amounts indicated at the bottom (in micrograms). Luciferase activity
was assayed in the samples from the BA/F3-hGMR cells without (no
factor) or with the stimulation of GM-CSF (+GM-CSF) or IL-3
(+IL-3). The mean value plus SD of the luciferase activities in
triplicate samples from each fraction is shown as arbitrary units. (E)
BA/F3 cells (1 × 107) were transfected with the pFR-luc
reporter plasmid (1 µg) and the pFA-Elk fusion transactivator plasmid
(0.1 µg) by electroporation together with 30 µg each of pSR (V),
pSR-Tec (Tec), or pSR-TecKD (KD). After starvation in
cytokine-free medium for 5 hours, cells were further cultured for 3 hours without (no factor) or with IL-3 (+IL-3; 25 U/mL), and
subjected to the luciferase assay. (F) BA/F3 cells
(1 × 107) were transfected with the pFR-luc plasmid (1 µg) plus the pFA-Elk plasmid (0.1 µg) together with 30 µg each of
pSR (V) or pSR-Tec (Tec). After starvation in cytokine-free
medium for 4 hours, cells were cultured for 1 hour with DMSO
[PD( )] or 50 µmol/L of PD98059/DMSO [PD(+)]. Cells were
further cultured for 3 hours in the presence (+IL-3) or absence (no
factor) of IL-3.(G) The structure of the c-fos promoter
fragment. The c-fos promoter contains four known regulatory
elements, namely, the sis-inducible element (SIE), serum
response element (SRE), fos AP-1 binding element (FAP), and
calcium and cyclic AMP response element (Ca/CRE). (H) BA/F3 cells
(1 × 107) were transfected with 5 µg of pSR-Tec
(Tec) and 2 µg of pfos/luc (WT) or the pfos/luc
mutants carrying point mutations at SIE (mSIE), SRE (mSRE), FAP (mFAP),
or Ca/CRE site (mCRE). After starvation in cytokine-free medium for 5 hours, cells were further cultured for 3 hours without (no factor) or
with IL-3 (+IL-3; 25 U/mL), and subjected to the luciferase assay.
(I) BA/F3-hGMR cells (1 × 107) were transfected
with the pfos/luc reporter plasmid (2 µg) together with of
pSR (pSR), pSR-dnJak2 (dnJak) and pSR-Tec (Tec) plasmids at
the amounts indicated at the bottom (in micrograms). Luciferase activity was assayed in the samples from the BA/F3-hGMR cells without (no factor) or with the stimulation of GM-CSF (+GM-CSF) or
IL-3 (+IL-3). (J) BA/F3-hGMR cells (1 × 107
cells) were transfected with the pfos/luc reporter plasmid (2 µg) together with of pSR (pSR), pSR-Jak2 (Jak2) and
pSR-TecKM (TecKM) plasmids at the amounts
indicated at the bottom (in micrograms). Luciferase activity was
assayed in the samples from the BA/F3-hGMR cells without (no
factor) or with the stimulation of GM-CSF (+GM-CSF) or IL-3
(+IL-3).
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MATERIALS AND METHODS |
Cell lines.
BA/F3 cells29 were maintained in RPMI 1640 medium
(GIBCO-BRL, Gaithersburg, MD) supplemented with 10% fetal calf serum
(FCS) and 25 U/mL of mouse IL-3. The BA/F3-hGMR cells and a human GM-CSF-dependent cell line, UT-7,30 were maintained in the
same medium with 10% FCS and 1 ng/mL of human GM-CSF. 293 cells
(American Type Culture Collection [ATCC], Rockville, MD) were
maintained in Dulbecco's modified Eagle medium/F12 (DMEM/F12;
GIBCO-BRL) containing 10% FCS and 2 mmol/L L-glutamine. Sf21 cells
(Invitrogen, San Diego, CA) were grown in suspension at 28°C in the
SF-900 II serum-free medium (GIBCO-BRL) without CO2 supply.
For the stimulation experiments, UT-7 cells were cultured in the
starvation medium (RPMI 1640 medium with 0.5% FCS, 100 µg/mL
transferrin [Boehringer Mannheim, Mannheim, Germany] and 100 µg/mL
bovine serum albumin [Boehringer Mannheim]) at the concentration of 5 × 105 cells/mL for 12 hours, then at the concentration of
1 × 107 cells/mL in the same medium for 0.5 hour. The
cells were stimulated with 10 ng/mL of human GM-CSF for the period of 5 minutes unless otherwise indicated.
Immunoprecipitation and in vitro kinase assay.
The cDNA of mouse Tec type IV,12 mouse Jak2,31
dominant negative Jak2,24 mouse Lyn A,32 Syk
with an N-terminal gp120 epitope tag,33 or dominant
negative Ras was ligated with the pSR expression vector to generate
pSR-Tec, pSR-Jak2, pSR-dnJak2, pSR-Lyn, pSR-Syk, or
pSR-dnRas, respectively. To construct the cDNA encoding a
kinase-deleted Tec (Tec KD), Tec cDNA was digested by
Bpu1102I, blunt-ended by T4 DNA polymerase, and the 3 -fragment
encoding the kinase domain was removed. Introduction of the expression
plasmids into 293 cells was performed by the calcium phosphate method.
UT-7 or 293 cells were rinsed once with ice-cold phosphate-buffered
saline (PBS) supplemented with 0.1 mmol/L
Na3VO4, and resuspended into the 1%-lysis
buffer (1% Nonidet P-40, 50 mmol/L Tris-HCl, 7.4, 150 mmol/L NaCl, 1 mmol/L NaF, 1 mmol/L Na3VO4, 200 U/mL
aprotinin, and 1 mmol/L phenylmethylsulfonyl fluoride). After
incubation on ice for 30 minutes, cell extracts were centrifuged to
remove insoluble materials. Tec or Jak2 was immunoprecipitated from 1.5 to 2 mg of the cell lysates by anti-Tec serum12 or
anti-Jak2 sera (Santa Cruz Biotechnology, Santa Cruz, CA and Upstate
Biotechnology, Lake Placid, NY), respectively, and was eluted into the
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
sample buffer. Where indicated, cells were solubilized by the
0.1%-lysis buffer containing 0.1% of NP-40 instead of 1%.
For the in vitro kinase assay, the immune complexes were washed three
times with the 1%-lysis buffer, three times with the kinase buffer (20 mmol/L Tris-HCl, 7.4, 50 mmol/L NaCl, 10 mmol/L MgCl2, 2 mmol/L MnCl2), and finally incubated with 0.37 MBq of [ -32P]ATP (Amersham, Arlington Heights, IL) for 15 minutes at 30°C. For the assay of Jak2 activity, a synthetic
substrate of Jak2 (Upstate Biotechnology) was added to the reaction (20 µg/experiment). Samples of the Jak2 kinase assay were subjected to
Tricine-SDS-PAGE.
Immunoblotting.
Total cell lysates (10 µg/lane) and the immune complexes were
separated through 7.5% SDS-PAGE and electroblotted onto polyvinylidene difluoride (PVDF) membranes (Immobilon; Millipore, Bedford, MA). The
membranes were incubated for 1 hour at room temperature in TBST (20 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 0.05% Tween 20) with 4%
bovine serum albumin (Fraction V; Sigma, St Louis, MO). The membranes
were then incubated with anti-Tec serum (1:10,000 dilution), anti-Jak2
serum, anti-Lyn serum,26 anti-gp120 epitope tag antibody
(H902), or anti-phosphotyrosine antibody (4G10; Upstate Biotechnology)
for 1 hour at room temperature in TBST. Specific bindings of the
antibodies were visualized by the ECL detection system (Amersham)
according to the manufacturer's instructions.
Metabolic labeling and phosphoamino acid analysis.
UT-7 cells were cultured at the concentration of 1 × 107
cells/mL in phosphate-free RPMI 1640 medium (GIBCO-BRL) supplemented with 5% dialyzed FCS (GIBCO-BRL) and 37 MBq/mL of
[32P]orthophosphate for 1 hour, and then stimulated with
human GM-CSF for 5 minutes. Tec was immunoprecipitated from the cells,
blotted onto a PVDF membrane, and incubated in 1 N KOH at 55°C
according to the method of Kamps and Sefton.34 Phosphoamino
acid contents of pp70Tec were determined as described
earlier.12
Luciferase reporter assay.
With the c-fos promoter-luciferase plasmid (pfos/luc)
as a reporter, the expression plasmid of each kinase was introduced into BA/F3-hGMR cells by electroporation according to the method of Watanabe et al35 with minor modifications. Briefly, 1 × 107 of BA/F3-hGMR cells were resuspended into 200 µL of OPTI-MEM I medium (GIBCO-BRL) and mixed with the expression
vector DNAs (5 µg per construct unless otherwise indicated) plus the
pfos/luc reporter plasmid (2 µg). Total amounts of plasmid
DNAs in each set of electroporation were adjusted to be equal by adding
the appropriate amounts of the blank vector DNA. After electroporation with the GenePulser apparatus (BioRad, Hercules, CA) at the condition of 200 V and 960 µF, cells were resuspended into 30 mL of RPMI 1640 medium with 10% FCS and cultured for 5 hours. The samples were further
cultured for 5 hours either unstimulated or stimulated with 25 U/mL of
mouse IL-3 or 5 ng/mL of human GM-CSF. The luciferase activities were
measured by using the Luciferase Assay System (Promega, Madison, WI),
and are shown as relative light units/min/µg of protein. The Elk
activity was assayed in BA/F3 cells by using the PathDetect in vivo
reporting system (Stratagene, La Jolla, CA). The MEK1 inhibitor
(PD98059; New England Biolabs, Beverly, MA) was dissolved in dimethyl
sulfoxide (DMSO) and add to the culture at the concentration of 50 µmol/L. The pfos/luc mutants were constructed by inserting
the mutant promoter fragments36 into pGL3-Basic plasmid
(Promega).
Recombinant baculoviruses.
The cDNAs of Tec and Jak2 were inserted into the pFastBacHT and
pFastBac1 plasmids (both from GIBCO-BRL), respectively. The recombinant
baculoviruses based on these plasmids were generated by the Bac-to-Bac
baculovirus expression systems (GIBCO-BRL), and were used to infect
Sf21 cells at the multiplicity of infection (MOI) of 1.0. After 48 to
72 hours of culture, cells were harvested and lysed as described above.
| |
RESULTS |
Tec is involved in the signaling pathway of GM-CSF receptor.
To investigate whether Tec is involved in the signaling mechanism
mediated by GM-CSF receptor (GMR), Tec was immunoprecipitated from a
human GM-CSF-dependent cell line, UT-7, with or without the GM-CSF
stimulation, and was immunoblotted with anti-phosphotyrosine antibody
(P-Tyr Ab). As shown in the upper panel of Fig
1A, GM-CSF stimulation of UT-7 cells for 5 minutes could clearly induce tyrosine-phosphorylation of Tec (indicated
by an arrow) and a Tec-associated p56. The identity of this p56 is yet
to be determined although we confirmed that p52shc and
p56lyn, both of which are known to be associated with Tec,
have the same electrophoretic mobility with that of the "p56."
The same membrane was reblotted with anti-Tec serum to prove that
equivalent amounts of Tec were precipitated (lower panel). We could not
detect a significant level of Btk expression in UT-7 cells by using an anti-Btk antibody (M-138; Santa Cruz Biotechnology). We next examined the time course of Tec phosphorylation. Tec was immunoprecipitated from
UT7 cells with various periods of GM-CSF stimulation, and probed with
P-Tyr Ab. As shown in Fig 1B, tyrosine-phosphorylation of Tec was
induced as rapidly as 1 minute after the stimulation, reached to the
maximum level in 5 to 10 minutes, and decreased thereafter. Thus, the
phosphorylation of Tec in response to GM-CSF is rapid and transient. To
examine whether the kinase activity of Tec is also affected in response
to GM-CSF, Tec was immunoprecipitated from UT-7 cells with or without
GM-CSF stimulation and subjected to an in vitro kinase assay without
exogenous substrates. As shown in Fig 1C and D, stimulation with GM-CSF
for 5 minutes could enhance the auto-phosphorylation activity of Tec.
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| Fig 1.
Tec is involved in the signaling pathway of GM-CSF
receptor. (A) UT-7 cells (1 × 107) were cultured in the
starvation medium for 12 hours and then stimulated with 10 ng/mL of
human GM-CSF (+) for 5 minutes or left unstimulated (). Tec was
immunoprecipitated from each fraction (Tec), subjected to 7.5%
SDS-PAGE, and immunoblotted with anti-phosphotyrosine antibody
(P-Tyr). Total cell lysates (TCL; 10 µg/lane) and the immunoprecipitates by normal rabbit serum (NRS) prepared from the same
set of cells were also analyzed. The position of Tec is indicated by an
arrow. The molecular weight standards (×10 3) are shown
at the left. The same membrane was reblotted with anti-Tec serum to
show the amounts of Tec precipitated (lower panel). (B) UT-7 cells were
stimulated with GM-CSF (10 ng/mL) for 0, 1, 5, 10, or 20 minutes as
indicated at the top. Tec was immunoprecipitated from each fraction
(1 × 107 cells), and was immunoblotted with
anti-phosphotyrosine antibody (P-Tyr) or anti-Tec serum (Tec).
(C) Tec was immunoprecipitated from 1 × 107 of UT-7 cells
with (+) or without () 5 minutes of GM-CSF stimulation, and was
subjected to an in vitro kinase assay. Autophosphorylation of
pp70Tec is shown. (D) Specific kinase activity of the Tec
protein (32P-incorporation/protein amount) with (+) or
without () the GM-CSF stimulation was calculated by densitometric
analysis and shown as arbitrary units. (E) Tec was immunoprecipitated
from UT-7 cells (1 × 107), with (GM) or without ()
the GM-CSF stimulation (10 ng/mL), metabolically labeled with
[32P]orthophosphate (37 MBq/mL), and was analyzed by
7.5% SDS-PAGE. The proteins were blotted onto a PVDF membrane, and
heated in 1 N KOH to decrease the backgrounds of serine- and
threonine-phosphorylation. The position of Tec is indicated. (F)
pp70Tec in (E) was subjected to the phosphoamino acid
analysis. The positions of free phosphate (Pi), phosphoserine (p-Ser),
phosphothreonine (p-Thr), and phosphotyrosine (p-Tyr) are indicated at
the right.
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To directly estimate the phosphotyrosine contents, Tec was
immunoprecipitated from UT-7 cells metabolically labeled with
[32P]orthophosphate, separated through 7.5% SDS-PAGE,
blotted onto a PVDF membrane, and incubated in 1 N KOH to enrich the
signals of phosphotyrosine. Autoradiography of the membrane could show that GM-CSF can induce phosphorylation of pp70Tec (Fig 1E).
The phosphoamino acid contents of this pp70Tec were then
examined by thin-layer chromatography, showing that phosphorylation of
tyrosine residues was actually induced by the stimulation with GM-CSF
(Fig 1F). These data imply that Tec is involved in the intracellular
signaling mechanism mediated by GMR.
Tec is involved in cytokine-driven activation of c-fos
transcription.
To examine whether Tec mediates cytokine-driven activation of the
c-fos gene, the pfos/luc plasmid in which the
luciferase expression is controlled by the c-fos promoter was
transfected into BA/F3-hGMR cells by electroporation together
with the pSR-based expression plasmid of Syk, Lyn, Jak2, or Tec. As
shown in Fig 2A,stimulation of the vector-transfected
BA/F3-hGMR cells with either GM-CSF or IL-3 could enhance the
luciferase reporter activity. Co-introduction of the Syk kinase with an
N-terminal tag33 did not affect the luciferase activity,
suggesting Syk is not involved in the c-fos activation
mechanism in BA/F3 cells. In contrast, introduction of Lyn kinase
significantly elevated the luciferase activity of the unstimulated
basal level. However, cytokine stimulation of the cells could not
further enhance the reporter activity. This lack of
cytokine-responsiveness in Lyn-transfected cells was confirmed in
repeated experiments. As previously reported, introduction of Jak2
could elevate the reporter activity of the unstimulated state as well
as of cytokine-stimulated states. Interestingly, Tec introduction
elevated the reporter activity of the unstimulated state similar to the
level obtained by the Lyn-transfection. In contrast to the case of Lyn,
Tec expression could also strongly enhance the reporter activity in
response to GM-CSF or IL-3. Appropriate expression of each kinase was
confirmed by the immunoblot analysis of the total cell lysates (Fig
2B).
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| Fig 2.
Tec is involved in the cytokine-driven activation of
c-fos proto-oncogene. (A) BA/F3-hGMR cells
(1 × 107) were transfected with the pfos/luc
reporter plasmid (2 µg) together with 5 µg each of the pSR
(Vector), pSR-Syk (Syk), pSR-Lyn (Lyn), pSR-Jak2 (Jak), or
pSR-Tec (Tec). After 5 hours of incubation in cytokine-free medium,
the cells were further cultured for 5 hours without (no factor) or with
5 ng/mL of human GM-CSF (+GM-CSF) or 25 U/mL of mouse IL-3 (+IL-3).
Luciferase activity was assayed in each fraction and calculated as
relative light units (RLU)/min/µg of protein. The mean value plus SD
of the luciferase activities in triplicate samples from each fraction
is shown as arbitrary units. (B) BA/F3-hGMR cells were
transfected with pSR-Syk, pSR-Lyn, pSR-Jak2, or pSR-Tec,
and cultured for 24 hours in the presence of IL-3. Total cell lysates
(10 µg/lane) were prepared from each set (+) and untransfected
BA/F3-hGMR cells (), and were immunoblotted with the
antibodies against the corresponding kinases.
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We then directly tested whether Tec is an intermediate in the
cytokine-driven c-fos activation pathway by using a
kinase-deleted Tec (TecKD). As shown in Fig 2C, introduction of
pSR-TecKD into BA/F3-hGMR cells suppressed the
c-fos promoter activity stimulated by GM-CSF or IL-3 in a
dose-dependent manner. These data strongly support the idea that Tec
directly mediates the cytokine-driven c-fos activation. It is
widely known that c-fos transcription is regulated via the
Ras-MAPK pathway. Therefore, we checked whether the Tec-driven
c-fos activation is transduced through Ras by coexpressing a
dominant negative form of Ras (dnRas). As shown in Fig 2D, coexpression
of dnRas could totally block the Tec-driven activation of the
c-fos gene. Thus, Tec is likely to drive the c-fos
activation through a Ras-regulated mechanism. By using the PathDetect
in vivo reporting system (Stratagene), we then asked whether Elk, a
transcriptional factor acting downstream of Ras, is involved in the
Tec-mediated c-fos activation. The pFA-Elk plasmid, encoding
the fusion protein consisting of the DNA binding domain of yeast GAL4
and the activation domain of Elk, was transfected into BA/F3 cells
together with Tec-expression plasmids and the reporter pFR-luc plasmid
in which expression of luciferase is controlled by a promoter
containing the GAL4-binding sites (Fig 2E). In the pSR-transfected
cells ("V" part), IL-3 stimulation resulted in the elevation of
luciferase activity, which suggests that Elk is activated in response
to IL-3. Introduction of Tec markedly increased the reporter activity
both in the unstimulated and stimulated states ("Tec" part). In
contrast, transfection of TecKD suppressed the luciferase activity,
indicating that Elk-pathway is involved in the Tec-driven c-fos
activation process. We also tested whether MEK1, an intermediate
between Ras and Elk, plays a role in this c-fos activation
mechanism. After electroporation with pFA-Elk and pFR-luc, BA/F3 cells
were cultured for 4 hours without IL-3, and then for 1 hour with an
MEK1 inhibitor, PD98059, before the IL-3 stimulation. As shown in Fig
2F, treatment with PD98059 significantly suppressed the Tec-driven
Elk-activation. In a separate line of experiment, we investigated what
kind of transcriptional factor(s) is responsible for the Tec-mediated c-fos transcription. The c-fos promoter fragment is
known to contain four cis-regulatory elements, namely, the
sis-inducible element (SIE), the serum response element (SRE),
the c-fos AP-1 binding element (FAP), and the calcium and
cyclic AMP response element (Ca/CRE) (Fig 2G). These regulatory
sequences are presumed to work in concert to control the c-fos
transcription in a tissue- and stimulus-specific fashion.36
By using the pfos/luc mutants in which point mutations were
introduced into individual regulatory elements (kind gifts of
T. Curran), we here analyzed how each element contributes
to the Tec-driven c-fos activation (Fig 2H). In accordance with
the results in Fig 2E, mutations at SRE, the binding site of the
Elk/TCF complex, significantly decreased the Tec-driven activation of
c-fos transcription. These lines of evidence support the idea
that Tec activates c-fos promoter through, at least in part,
the Ras-MEK1-Elk pathway.
Because both of the Tec and Jak2 kinases could enhance cytokine-driven
c-fos activation, we then tried to clarify whether Tec and Jak2
work in the same pathway or in a parallel manner to drive the
c-fos promoter. First, Tec was introduced into BA/F3-hGMR cells with or without dominant negative Jak2 (dnJak2) to examine whether Jak2 is involved in the Tec-driven pathway (Fig 2I).
Interestingly, expression of dnJak2 could suppress the cytokine-driven
as well as Tec-driven luciferase activity, indicating that Jak2 acts
downstream of Tec in the c-fos activation mechanism. We have
also tested the possibility of the Tec-Jak2 interaction in the reverse
direction. As shown in Fig 2J, introduction of a kinase-dead
TecKM (Lys-397 at the ATP-binding site is replaced with
Met) could slightly suppress the Jak2-driven activation of the
c-fos gene. Although we could reproducibly observe this weak
suppression (about 20% reduction), we do not yet have a strong proof
that Tec is involved in a part of the Jak2-driven mechanism in the
c-fos regulation.
Tec can phosphorylate Jak2 in cells.
To understand how Tec and Jak2 can functionally interact with each
other, we first examined the possibility that the former directly
phosphorylates the latter. A kinase-dead Jak2 (Jak2KE:
Lys-882 in the ATP-binding site is replaced with Glu) was expressed in
293 cells with or without Tec, immunoprecipitated by anti-Jak2 serum
and blotted with P-Tyr Ab. To our surprise, as shown in the upper
panel of Fig 3A, Jak2KE could
be phosphorylated by Tec in intact cells. Interestingly, the
tyrosine-phosphorylated p70Tec was also identified in the
anti-Jak2 immunoprecipitate, suggesting the physical interaction
between the two PTKs. The same membrane was reprobed with anti-Jak2
serum to prove that equivalent amounts of Jak2 were precipitated (lower
panel). This Tec phosphorylation of Jak2KE is not likely to
arise from a nonspecific reaction by over-expressed Tec proteins,
because Syk could not phosphorylate Jak2KE in a similar
experiment in 293 cells (data not shown). We also examined the ability
of Tec to phosphorylate Jak2 in the insect cell system. Sf21 cells
derived from Spodoptera frugiperda were infected with the
recombinant baculovirus expressing Jak2KE alone or in
combination with the Tec-expressing or TecKM-expressing
virus. After 2 days of incubation, Jak2KE was
immunoprecipitated from the cells and probed with P-Tyr Ab (upper
panel of Fig 3B). As expected, phosphorylation of Jak2KE
could be identified only when Jak2KE was coexpressed with
kinase-active Tec. In contrast, coexpression of TecKM did
not confer detectable tyrosine-phosphorylation on Jak2KE.
The same membrane was reblotted with anti-Jak2 serum to estimate the
amounts of Jak2 immunoprecipitated. These data favor the idea that Jak2
is a direct substrate of Tec in vivo. In these experiments, Jak2-phosphorylation by Tec was clearly and reproducibly observed when
we used, for immunoprecipitation, anti-Jak2 serum against the
C-terminal tail of Jak2 (C-20; Santa Cruz Biotechnology), not the one
against amino acid positions 758-776 (Upstate Biotechnology), which may
imply that the target site(s) of Tec is localized within or very close
to the 758-776 region.
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| Fig 3.
Tec can phosphorylate Jak2 in both mammalian and insect
cells. (A) Jak2KE was immunoprecipitated from 2 × 106 of 293 cells expressing Jak2KE with (T) or
without () Tec. Total cell lysates (TCL, 10 µg/lane) and anti-Jak2
immunoprecipitates (Jak IP) were electrophoresed and probed with
anti-phosphotyrosine antibody (P-Tyr) or anti-Jak2 serum (Jak2).
The positions of Jak2 (Jak2), Tec (Tec), and the Ig heavy chain (IgH)
are indicated at the right. (B) Jak2KE was
immunoprecipitated from Sf21 cells infected with Jak2KE
expressing baculovirus (JE) alone or in combination with
Tec-expressing (T) or TecKM-expressing (TM)
virus. The immunoprecipitates were separated through 7.5% SDS-PAGE and
probed with anti-phosphotyrosine antibody (P-Tyr) or anti-Jak2 serum
(Jak2). The positions of Jak2 (Jak2) and Tec (Tec) are indicated at
the right. (C) Jak2 was immunoprecipitated from Sf21 cells expressing
Jak2 (J) or Jak2KE (JE) either alone or in
combination with Tec (T). The immunoprecipitates were incubated with
[-32P]ATP and the synthetic Jak2-substrate, and
subjected to Tricine-SDS-PAGE. Phosphorylation of the Jak2-substrate is
shown. (D) Total cell lysates (TCL: 10 µg/lane) and the anti-Jak2
immunoprecipitates (Jak IP) were prepared from parental BA/F3 cells (P)
and two BA/F3 clones (1 and 2) stably expressing TecSH3, and
immunoblotted with P-Tyr Ab (upper panel) or anti-Jak2 serum (lower
panel). The position of Jak2 is indicated at the right. The positions of molecular weight standards (×103) are also shown at
the left.
|
|
We then tested whether this trans-phosphorylation of Jak2 by Tec
affects the kinase activity of the Jak2 protein. Jak2 was expressed in
Sf21 cells with or without Tec, immunoprecipitated by anti-Jak2 serum,
and was subjected to an in vitro kinase assay with a synthetic
substrate peptide. As shown in Fig 3C, coexpression of Tec did not
affect the phosphorylation of the Jak2-substrate. Because the
immunoprecipitated Jak2KE could not phosphorylate the
peptide at all (lane "JE + T"), phosphorylation of
the peptide in the other lanes was supposed to be carried out by Jak2,
not by the coprecipitated kinases from Sf21 cells. We observed that
Jak2 was expressed in equal amounts in each Sf21 fraction, as judged
from the immunoblotting of the total cell lysates with anti-Jak2 serum
(data not shown). Similar results of the in vitro kinase assay were
obtained with Jak2 expressed in 293 cells (data not shown).
Phosphorylation of Jak2 without the modulation of its activity may be
used in vivo for collecting signaling molecules to Jak2 protein. We
checked this possibility by using the BA/F3 cells expressing an
SH3-deleted active Tec (TecSH3).37 Jak2 was
immunoprecipitated from parental BA/F3 cells and two BA/F3
transfectants stably expressing TecSH3, and blotted with P-Tyr
Ab. As shown in Fig 3D, many tyrosine-phosphorylated proteins become
associated with Jak2 only when TecSH3 is coexpressed, which may
indirectly support the possibility above.
Jak2 can phosphorylate Tec at Tyr-518.
To investigate the phosphorylation reaction in the reverse direction
between the two kinases, TecKM was introduced into 293 cells with or without Jak2, and was analyzed for
tyrosine-phosphorylation (upper panel, Fig
4A). Because it was already known that Tec
can be directly phosphorylated and activated by Lyn PTK, a coexpression
experiment of Lyn kinase was used as a positive control. To our
surprise again, Tec could be in vivo phosphorylated by Jak2 as well as
by Lyn. The same membrane was then probed with anti-Tec serum to
estimate the amounts of Tec precipitated (lower panel). Therefore, Tec
and Jak2 can trans-phosphorylate each other.
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| Fig 4.
Jak2 can phosphorylate Tec at Tyr-518. (A) The
kinase-dead TecKM (TM) was expressed in 293 cells either alone or in combination with Jak2 (Jak2) or Lyn (Lyn)
kinase. Tec was immunoprecipitated from each fraction, and probed with
anti-phosphotyrosine antibody (P-Tyr) or anti-Tec serum (Tec).
(B) TecKM (TM), TecKMSH3
(TM3), or TecKM,YF (TM,518F) was
expressed in 293 cells either alone () or in combination with Jak2
(J) or Lyn (L). Tec was immunoprecipitated from each fraction, and
blotted with anti-phosphotyrosine antibody (P-Tyr) or anti-Tec serum
(Tec). The positions of full-length Tec (T) and SH3-deleted (SH3)
forms are indicated at the right. (C) The amino acid sequences of the
Tec-family kinases, surrounding the tyrosine residues corresponding to
Tyr-518 in mouse Tec, are compared. The asterisk indicates the position
of the phosphorylated tyrosine. At the left shown are the numbers of
amino acid positions of mouse Tec,2 human
Btk,10,11 mouse Emt/Itk/Tsk, 9,41,42 human
Bmx,43 and human Txk.44 (D) pSR (V),
pSR-Tec (T), or pSR-TecKM (TM) was
transfected into 293 cells with or without pSR-Jak2 (J). Tec was
immunoprecipitated from each fraction, and incubated with [32P]ATP without exogenous substrates.
Autophosphorylation of pp70Tec in each sample is shown.
|
|
We then tried to map the phosphorylation site of Tec by Jak2. Yamashita
et al37 previously demonstrated that the deletion of the
internal SH3 domain results in hyperphosphorylation and activation of
Tec in vivo. In addition, Tec kinase has a tentative autophosphorylation site (Tyr-518) in the activation loop of its catalytic domain, corresponding to Tyr-416 in c-Src. Therefore, we
investigated the possibility that either of the SH3 domain or Tyr-518
is the target site of Jak2 and Lyn kinases. TecKM,
TecKMSH3 (the SH3 domain of TecKM is
deleted), or TecKM,YF (Tyr-518 of TecKM is
replaced with Phe) was expressed in 293 cells either alone or in
combination with Jak2 or Lyn. Tec was then immunoprecipitated and
probed with P-Tyr Ab. As shown in the upper panel of Fig 4B,
internal deletion of the SH3 domain did not decrease the
phosphorylation of TecKM by Jak2. On the other hand, the
Phe-substitution for Tyr-518 nearly completely abolished the
phosphorylation of TecKM protein
("TM,518F" part). Hence, Tyr-518 of Tec is the target
site of both Jak2 and Lyn. The same membrane was reblotted with
anti-Tec serum to prove that equivalent amounts of Tec proteins were
immunoprecipitated (lower panel). It is not likely that Jak2
phosphorylated Tec indirectly through Lyn (or other Src-family
kinases), because the kinase activity of Lyn was not affected by the
coexpression of Jak2 when transiently expressed in 293 cells (data not
shown). The amino acid sequences surrounding this Tyr-518 position in
Tec-family kinases are well conserved (Fig 4C). Therefore, it would not
be surprising if other members of the Tec-family are also controlled by
Src- and Jak-family kinases through a similar phosphorylation mechanism.
Because Jak2 and Lyn can phosphorylate the same residue (Tyr-518) of
Tec, we speculated that Jak2 may activate Tec as in the case of Lyn.
Tec or TecKM was expressed in 293 cells either alone or in
combination with Jak2. Tec was immunoprecipitated from each set and
subjected to an in vitro kinase assay to test its auto-phosphorylation
activity. Unexpectedly, as shown in Fig 4D, autophosphorylation level
of p70Tec was not altered irrespective of the presence of
Jak2 PTK. In contrast, coexpression of Lyn kinase could activate Tec as
reported previously (data not shown). Thus, although Jak2 and Lyn can
phosphorylate the same site of Tec, we observed only Lyn can activate
the Tec kinase under the sensitivity of our assay.
Tec can bind to Jak2 in insect cells.
We then tested whether Tec and Jak2 can physically associate with each
other in cells. Recombinant baculovirus expressing Tec or
TecKM was used to infect Sf21 cells either alone or in
combination with the virus expressing Jak2 or Jak2KE (Fig
5). Jak2 was immunoprecipitated from the
cells lysed by the 0.1% lysis buffer, and immunoblotted with anti-Tec
serum. Tec could be identified very weakly in the Jak2-immune complex, and more clearly found was TecKM in the Jak2-immune complex
(top panel). Also, Jak2KE was shown to associate with Tec
irrespective of the Tec-activity. Thus, Tec can weakly bind to Jak2 in
insect cells, and this interaction does not require the kinase activity
of Tec and Jak2. It should be noted that co-introduction of
kinase-active Tec always reduced the expression level of Jak2 in Sf21
cells (middle panel, and also confirmed in other repeated experiments).
Therefore, difference of the amounts of coprecipitated Tec may have
arisen from the different expression level of Jak2 (compare the
intensities of the bands between the top and middle panels).
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| Fig 5.
Tec can constitutively associate with Jak2 in Sf21 cells.
Sf21 cells were infected with baculovirus expressing Tec (T), Jak2 (J),
TecKM (TM), and Jak2KE
(JE) in the combinations indicated at the top. Jak2 was
immunoprecipitated from each cells lysed by the 0.1%-lysis buffer
(Jak IP), and probed with either anti-Tec serum (Tec) or
anti-Jak2 serum (Jak2). Total cell lysates (TCL: 10 µg/lane) of
each fraction were also probed with anti-Tec serum to estimate the
expression level of Tec.
|
|
We could not test the Tec/Jak2 interaction in the reverse
direction, since Jak2 nonspecifically bound to protein
A-Sepharose beads and glutathione Sepharose 4B (and even to
nickel agarose beads) when Jak2 was expressed abundantly in either 293 or Sf21 cells (data not shown).
| |
DISCUSSION |
In this report we have shown that Tec is involved in the signaling
pathway of GMR, especially in the c-fos activation machinery. Because Jak2 was previously shown to be an intermediate in the cytokine-driven c-fos activation pathway, both of Jak2 and Tec should play a role in the regulation of c-fos transcription.
Furthermore, our data with dnJak2 support an intriguing idea to place
Jak2 downstream of Tec in the mechanism of c-fos activation.
How does Jak2 participate in the Tec-driven pathway to the
c-fos gene? A simple hypothesis is that Jak2 becomes activated via the phosphorylation by Tec, and drives the c-fos
transcription as an effector of the Tec kinase. However, this is
unlikely because coexpression of Tec could not affect the activity of
Jak2 in either mammalian or insect cells. The second explanation is
that Jak2 may be required to fully activate Tec through the
phosphorylation of Tec protein by Jak2. This assumption is again
unlikely because (1) coexpression of Jak2 could not activate Tec in
either 293 cells or Sf21 cells, and (2) coexpression of dnJak2 with Tec
in 293 cells did not suppress the kinase activity or
tyrosine-phosphorylation of Tec (data not shown). Therefore, Jak2 may
not be a direct second messenger of Tec, but should be required for the
appropriate function of Tec-substrates ("Substrate X" in Fig
6). There are several possible scenarios
for such interaction. Jak2 may be, for instance, prerequisite to
recruit the substrates of Tec into the cytokine receptor complex. It is
well known that cytokine receptors are the good substrates of
Jak-family kinases both in vitro and in vivo, and that a variety of
signaling molecules become associated with the receptors through the
phosphotyrosine-SH2 domain (or phosphotyrosine-binding [PTB] domain)
interaction.38 Thus, it is possible that the second
messengers for c-fos activation can become accessible to Tec
through the phosphorylation of receptors by Jak2. Another explanation
may be that Tec collects its substrates by phosphorylating Jak2 and
thereby making it bound to the Tec-substrates. In this scenario, Jak2
plays as a "bridge" to connect Tec and its effector molecules.
There would be, again, the other possibility that Jak2 is required to
phosphorylate the Tec-substrates and to let them associated with Tec.
To determine which interaction really takes place in vivo, we have to
identify the "Substrate X" responsible for the c-fos
activation, and we should also clarify the phosphorylation site(s) of
Jak2 by the Tec kinase.
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| Fig 6.
Cytokine-driven pathways to the c-fos
proto-oncogene. When activated by Lyn, Tec phosphorylates "Substrate
X" and triggers the signaling pathway linked to the c-fos
activation. Tec and Jak2 can trans-phosphorylate each other. The
biological significance of this phosphorylation in the context of
c-fos activation mechanism is not settled yet. Jak2 is required
for the appropriate function of "Substrate X" as well as for the
phosphorylation of STATs. The STATs activation may also have some roles
in the regulation of the c-fos transcription.
|
|
Analysis of various deletion mutants of human GMR common chain
(c) showed that a central area in the cytoplasmic region of c is necessary for cytokine-dependent Shc
phosphorylation, activation of Ras, and induction of the c-fos
gene.35,39 In fibroblasts, Shc is already known to bind to
Grb2 in a phosphorylation-dependent manner, and thereby to trigger the
recruitment of SOS guanine nucleotide exchanging factor and the Ras
activation.40 Therefore, Shc/Ras may be a key component to
drive the c-fos transcription also in blood cells. If this is
the case, Shc would be an intriguing candidate for the "Substrate
X" in Fig 6. Currently we have only a few data to support this
hypothesis. First, because expression of dnRas could suppress the
cytokine-driven as well as Tec-driven c-fos activation, Ras
itself or the Ras-regulated machinery should be a relay point of the
pathway to the c-fos gene. Second, we already proved that Shc
can be associated (either directly or indirectly) with Tec in cells.
However, it is yet to be shown whether Shc is a direct substrate of Tec
in vivo, and whether Shc plays a central role in the activation of
c-fos gene in the hematopoietic system.
Our mapping experiments evidenced that Tyr-518 is the major
phosphorylation site of Tec by both Jak2 and Lyn. The fact that Jak2 is
capable of phosphorylating Tec was also confirmed in another laboratory
(T. Matsuda and J.N. Ihle, personal communication). However, only Lyn could enhance the kinase activity of Tec in our
experiments. Although we do not have any evidence to explain this
discrepancy, several possibilities can be raised. First, as shown in
Fig 4A and B, stoichiometry of Tec-phosphorylation was always higher
when coexpressed with Lyn than with Jak2. Thus, low level of Tyr-518
phosphorylation by Jak2 may not be sufficient to demonstrate the
enhancement of autophosphorylation activity in the anti-Tec
immunoprecipitates. On the other hand, a weak but still significant
tyrosine-phosphorylation of TecKM,518F could be
reproducibly observed in a longer exposure of the film, when
coexpressed with either Lyn or Jak2 (data not shown). Thus, there may
be additional phosphorylation sites by Jak2 and Lyn, and these minor
sites may have a pivotal role in the regulation of Tec activity. It is
also possible that Lyn and Jak2 bind to Tec at different sites, and
that these bindings may render distinct allosteric effects on Tec
molecules.
Our report has shown the presence of a "cross-talk" between Tec
and Jak2 PTKs. Although Tec is the first PTK among non-Jak kinases
shown capable of phosphorylating Jak2, it would not be surprising if
the members of other PTK-families are also able to phosphorylate Jak
kinases. Growth of blood cells would be controlled through these
complexed networks among various PTKs, and our observation would be an
important information to decipher the control mechanisms.
| |
FOOTNOTES |
Submitted June 9, 1997;
accepted December 2, 1997.
Supported in part by the Grant-in-Aid for Scientific Research on
Priority Areas from the Ministry of Education, Science and Culture,
Japan, and by the grant from Haraguchi Memorial Cancer Research Fund.
Address reprint requests to Hiroyuki Mano, MD, PhD, Department of
Molecular Biology, Jichi Medical School, Yakushiji 3311-1, Minami-kawachi-machi, Kawachi-gun, Tochigi 329-04, Japan.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be here-by marked
"advertisement" in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We are grateful to J.N. Ihle for the kind gifts of Jak2 cDNAs and for
critical reading of this manuscript. We thank T. Curran for the
fos-promoter constructs, A. Kikuchi for dominant negative Ras
cDNA, T. Mustelin for tagged Syk cDNA, T. Yi for Lyn A cDNA, Kirin
Brewery (Tokyo, Japan) for mouse IL-3, and Sumitomo Pharmaceutical Company (Osaka, Japan) for human GM-CSF. The H902-production hybridoma cell line is the reagent no. 521 from the NIH AIDS Research and Reference Program (Bethesda, MD).
| |
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Abstract
Introduction
Abstract