© 1999 American Heart Association, Inc.
Rapid Communication |
The Angiotensin II–Dependent Association of Jak2 and c-Src Requires the N-Terminus of Jak2 and the SH2 Domain of c-Src
From the Department of Pathology and Laboratory Medicine (P.P.S., M.S.A., K.H., K.E.B.), Emory University School of Medicine, Atlanta, Ga; Department of Medicine (S.J.F.), Division of Endocrinology and Metabolism, University of Alabama at Birmingham, Birmingham, Ala.
Correspondence to Kenneth E. Bernstein, Department of Pathology and Laboratory Medicine, 1639 Pierce Dr, 7107 WMB, Emory University School of Medicine, Atlanta, GA 30322. E-mail kbernst{at}emory.edu
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Abstract—The binding of angiotensin II (Ang II) to AT1 is known to increase the kinase activity of several nonreceptor tyrosine kinases including Jak2 and c-Src. In the present study, we demonstrate that treatment of vascular smooth muscle cells with Ang II results in a rapid and transient association of Jak2 and c-Src. This association is dependent on a catalytically active Jak2 kinase, because it is blocked both by pharmacological means and by the inability of a catalytically inactive Jak2 to associate with c-Src. c-Src bound tyrosine phosphorylated Jak2 but was unable to bind an equal amount of unphosphorylated Jak2 protein, indicating that the SH2 domain of c-Src mediates this association. In vivo studies indicated that c-Src binds the N-terminus of Jak2 as expression of a Jak2 molecule lacking the initial 240 amino acids, including 16 tyrosines, and was unable to bind c-Src. Lastly, using transiently transfected COS-7 cells, we found that Ang II treatment induced an association between c-Src and wild-type Jak2 but not between c-Src and the Jak2 molecule that lacks the initial 240 amino acids. Thus, our data suggest that in addition to increasing the kinase activities Jak2 and c-Src, treatment of cells with Ang II results in the physical association of Jak2 and c-Src; an association that is mediated by the SH2 domain of c-Src and the N-terminus of Jak2.
Key Words: angiotensin II • Jak2 • c-Src • tyrosine kinase • vascular smooth muscle cell
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Introduction |
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Angiotensin II (Ang II) is the effector molecule of the renin-angiotensin system.1 It is vital for maintaining a wide variety of physiological responses including salt and water balance, blood pressure, and vascular tone. These effects are transduced through a seven-transmembrane surface receptor called AT1. Activation of AT1 by Ang II results in the activation of several nonreceptor tyrosine kinases. For example, treatment of vascular smooth muscle cells (VSMCs) with Ang II activates two known Jak family tyrosine kinases: Jak2 and Tyk2.2 Similarly, treatment of cells with Ang II activates two known Src family tyrosine kinases: c-Src and Fyn.3 4
The Jak family of nonreceptor tyrosine kinases includes Jak1, Jak2,
Jak3, and Tyk2. Each protein is 
130 kDa in mass and contains seven
conserved Jak homology domains (JH1 through JH7). The Jak kinases were
initially shown to induce gene transcription through the signal
transducers and activators of transcription
(STATs).5 More recently, however, the Jaks have also been
shown to signal through classical signaling pathways such as the
Shc/Grb2/Sos pathway and through the serine/threonine kinase
Raf-1.6 7 Unlike almost all other protein tyrosine
kinases, members of the Jak family bear no SH2 or SH3 domains. In
contrast to the Jaks, members of the Src family of nonreceptor tyrosine
kinases are 55 to 62 kDa in mass and do possess SH2 and SH3 domains.
Although the expression of most Src family members is restricted to
hematopoietic cells, c-Src and Fyn are expressed in most tissues
including vascular smooth muscle. The Src family of tyrosine kinases
were initially shown to signal through the Shc/Grb2/Sos signaling
pathway but more recent studies indicate that they play a critical role
in transcriptional regulation by phosphorylating the STAT
proteins.8 9 10
Because the Jak and Src families of nonreceptor tyrosine kinases phosphorylate identical substrates, we hypothesized that treatment of cells with Ang II might result in a ligand-dependent association of Jak2 and c-Src. Such a physical association would allow these very different nonreceptor tyrosine kinases to phosphorylate the same substrate. In this study, we demonstrate that treatment of VSMCs with Ang II does in fact lead to a ligand-dependent association of Jak2 and c-Src, an association that is mediated by an amino terminus Jak2 phosphotyrosine and the SH2 domain of c-Src. These studies support the hypothesis of a ligand-dependent physical association of the Jak and Src nonreceptor tyrosine kinases and, for the first time, define the region of each molecule that mediates this specific physical interaction.
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Materials and Methods |
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Cell Culture
VSMCs were grown in DMEM+10% FBS at 37°C in a 5% CO2 humidified atmosphere and used between passages 8 to 22; 100-mm dishes at
75% confluence were
growth-arrested by incubation in serum-free DMEM for 48 hours before
use. COS-7 cells were cultured in the same medium. BSC-40 cells were
grown in DMEM+10% newborn calf serum. Cell culture reagents
were obtained from Gibco/BRL. Inhibitors were purchased
from Calbiochem. Losartan was a generous gift from Dupont
Merck. All other reagents were purchased from Sigma Chemical Co.
Plasmid and GST Fusion Protein Constructs
pBOSwtJk2 (wild-type Jak2 [Jak2 WT]) and pBODJK2
VIII
(dominant-negative Jak2 [Jak2 DN]) were a generous gift from
Dr Don M. Wojchowski (Pennsylvania State University) and were
previously described.11 Construction of the pRC-Jak2-WT,
pRC-Jak2-ATD, and pRC-Jak2-PKD plasmids has been described
elsewhere.12 The pRC-Jak2-AFL vector was made by digesting
pRC-Jak2-WT with AflII and closing with ligase. The vector
expressing wild-type AT1 cDNA
(pZeo/AT1) has been previously
described.13 The c-Src fusion proteins GST/SH2,
GST/SH3, and GST/SH2+SH3 were a generous gift from Dr Sarah J. Parsons
(University of Virginia).
Cell Transfection
COS-7 cells were transiently transfected exactly as previously
described.13 For BSC-40 cell transfection, cells were
seeded in 100-mm dishes and transfected at near confluency with either
4 µg pRC-Jak2-WT, 16 µg pRC-Jak2-ATD, 16 µg pRC-Jak2-AFL, or 8
µg pRC-Jak2-PKD in the presence of 20 µL lipofectin. Cells were
infected with vaccinia virus clone vTF7-3 as described below. The total
amount of transfected plasmid was normalized using empty expression
vector.
Vaccinia Virus–Mediated Jak2 Overexpression
Jak2 constructs were overexpressed using the vaccinia
virus–mediated transfection/infection protocol.14 15
Briefly, 100-mm dishes of nearly confluent BSC-40 cells were
transfected as described. After 4 hours, vTF7-3 was added at a
multiplicity of infection of 1.0 and incubated for 1 hour. The medium
was removed, and cells were incubated overnight in DMEM+10% newborn
calf serum. At 18 to 20 hours after infection, lysates were prepared
and samples were immunoprecipitated as described. Vaccinia clone vTF7-3
was generously provided by Dr Bernard Moss (National Institutes of
Health).
GST Fusion Protein Expression and Purification
The GST/Src fusion proteins and GST controls were expressed and
purified using Glutathione Sepharose 4B (Pharmacia Biotech) as
previously described.13
Protein-Protein Complex Formation and GST Pull-Down Assays
To prepare lysates, cells were washed with two volumes of
ice-cold PBS containing 1 mmol/L
Na3VO4 and lysed in 1.0 mL
ice-cold gentle lysis buffer (25 mmol/L Tris [pH 7.5], 10%
glycerol, 1% NP-40, 140 mmol/L NaCl, and protease
inhibitors). Lysates were gently sonicated and incubated on
ice for 1 hour. Samples were spun at 12 000g for 5 minutes
at 4°C, and supernatants were normalized using the
Dc protein assay (Bio-Rad). Normalized lysates
(400 µg/mL) were immunoprecipitated with 2 µg of antibody and 20
µL of a 50% slurry of Protein A/G Plus agarose beads (Santa Cruz
Biotechnology) for 6 to 16 hours at 4°C. The immunoprecipitating
anti-Src (N-16) and anti-Jak2 (HR758) polyclonal antibodies were
purchased from Santa Cruz Biotechnology. Immune complexes were washed 3
times with wash buffer (25 mmol/L Tris [pH 7.5], 150 mmol/L
NaCl, and 0.1% Triton X-100) and resuspended in SDS sample buffer. For
GST/Src pull-down assays, COS-7 cell lysates were precleared with 5
µg of Sepharose-bound GST for 1 hour at 4°C. To each sample, 0.2
µg of Sepharose-bound GST or GST/Src fusion protein was added and
incubated for 30 to 60 minutes at 4°C. Beads were washed 4 to 5 times
with wash buffer and resuspended in sample buffer. All sample
buffer–containing proteins were separated on SDS-PAGE (National
Diagnostics) and transferred onto nitrocellulose membranes
(Schleicher & Scheull).
Western Blotting
Membranes were blotted as previously described.13
Proteins were visualized with enhanced chemiluminescence according to
the manufacturer's instructions (Amersham). Protein bands were
quantitated using UN-SCAN-IT digitizing software (Silk Scientific).
Western blotting polyclonal anti-Jak2 and monoclonal
anti-phosphotyrosine (clone 4G10) were from Upstate Biotechnology.
Monoclonal anti-pp60c-src (clone GD-11) was a
generous gift from Dr Sarah J. Parsons (University of Virginia).
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Results |
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Ang II–Dependent Association of Jak2 and c-Src
Ang II, acting through the seven-transmembrane AT1, has been shown to activate Jak2 and c-Src kinase activities as measured by autophosphorylation or phosphorylation of synthetic substrates.2 3 To determine whether Ang II could induce a physical association between Jak2 and c-Src, quiescent VSMCs were treated with Ang II for 0 to 60 minutes. The cell lysates were then immunoprecipitated with anti-Src-pAb (Src N16), which does not cross-react with other Src tyrosine kinases, and the immunoprecipitates were Western-blotted with anti-Jak2-pAb (Figure 1A
). These data demonstrated an Ang
II–dependent association between Jak2 and c-Src. We confirmed equal
precipitations across all lanes by blotting the same membrane with
anti-Src-mAb (Figure 1A). The three experiments
represented in Figure 1A were quantitated by
densitometry and plotted as a function of fold increase with respect to
unstimulated controls (Figure 1B). We found that maximal
association between Jak2 and c-Src was 10-fold more than
unstimulated controls and occurred 5 to 40 minutes after Ang II
treatment. To demonstrate antibody specificity, we repeated the
experiments but immunoprecipitated the lysates with either the same
anti-Src-pAb or with control rabbit immunoglobulin (Ig) G. The use of
IgG resulted in a loss of specific Jak2 signal that correlated with the
lack of precipitated c-Src protein (Figure 1C). In experiments
in which we reversed the order of antibody addition (immunoprecipitated
VSMC lysates with anti-Jak2-pAb and Western-blotted with anti-Src-mAb),
we observed a similar Ang II–dependent association between Jak2 and
c-Src (data not shown). The Ang II–dependent association of Jak2 and
c-Src appears specific for these two molecules, because no association
was observed when anti-Src-pAb immunoprecipitates were Western-blotted
with anti-Jak1-mAb (data not shown).
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Finally, to determine whether AT1 was mediating
the Ang II–dependent association of Jak2 and c-Src, we pretreated
VSMCs with the AT1-specific inhibitor
losartan. Cells were then treated with Ang II, and the
resulting anti-Src-pAb immunoprecipitates were Western-blotted with
anti-Jak2-pAb (Figure 1D
). Losartan completely blocked
the Ang II–dependent association of Jak2 and c-Src. However, the loss
of Jak2 signal was not due to decreased immunoprecipitation of c-Src,
given that blotting the same membrane with anti-Src-mAb demonstrated
roughly equal precipitation (Figure 1D). Collectively, the data
in Figure 1 indicate that treatment of VSMCs with Ang II results
in an AT1-specific, ligand-dependent association
of Jak2 and c-Src.
A Catalytically Active Jak2 Kinase Is Required for Jak2/c-Src
Association
To determine whether the kinase activity of Jak2 and/or c-Src was
required for this association, quiescent VSMCs were pretreated with
either DMSO (control), the Jak2 kinase inhibitor AG-490, or
the c-Src specific kinase inhibitor PP1.16 17
The concentration and the duration of inhibitor
pretreatment used in the present study were based on our published
and unpublished work that defined the lowest doses that fully inhibited
Jak2 and c-Src kinase activities, respectively.18 19 Cells
were then left untreated or treated with Ang II for the indicated
times. The resulting lysates were immunoprecipitated with anti-Src-pAb
and Western-blotted with anti-Jak2-pAb to assess the Ang II–dependent
association of Jak2 and c-Src (Figure 2A). An Ang II–dependent association was
seen in the control cells and the PP1-treated cells. However,
ligand-dependent association between Jak2 and c-Src was blocked by
AG-490. To verify that AG-490 did not inhibit the immunoprecipitation
of c-Src, we blotted the same membrane with anti-Src-mAb and confirmed
that all lanes were precipitated equally well (Figure 2A). To
further test the inhibitory effect of AG-490 and the role
of Jak2 kinase in mediating the association between Jak2 and c-Src, we
pretreated VSMCs with varying doses of AG-490 and then stimulated them
with Ang II. The anti-Src-pAb immunoprecipitates were Western-blotted
with anti-Jak2-pAb to assess the Ang II–dependent association of Jak2
and c-Src (Figure 2B). AG-490 blocked the Ang II–dependent
association of Jak2 and c-Src in a dose-dependent manner, with maximal
inhibition occurring at 100 µmol/L. This is the same dose that
fully inhibits the Ang II–induced Jak2 tyrosine
phosphorylation in VSMCs (data not
shown).
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The data in Figure 2A and 2B suggested that the catalytic
activity of Jak2, but not c-Src, is required for the Ang II–dependent
association of Jak2 and c-Src. This hypothesis was tested with an
alternative approach that made use of a Jak2 DN protein. The
dominant-negative construct contains two point mutations in the kinase
domain rendering the molecule catalytically inactive and thus incapable
of autophosphorylation.11 In this
experiment, COS-7 cells were transiently transfected to express either
no Jak2 (empty vector control), Jak2 WT, or the dominant negative Jak2
protein (Jak2 DN). The large amount of transfected plasmid used in this
experiment results in Jak2 autophosphorylation through
oligomerization of the Jak2 molecules rather than by a ligand dependent
mechanism.18 The resulting lysates were then
immunoprecipitated with anti-Jak2-pAb, separated on SDS-PAGE, and
transferred onto nitrocellulose. The membrane was first blotted with
anti-Jak2-pAb to confirm equal precipitation of both the wild-type and
dominant-negative proteins (Figure 2C, top). We then examined
the level of tyrosine phosphorylation of the Jak2
proteins by blotting the same membrane with anti-Tyr(P)-mAb (Figure 2C, middle). As expected, Jak2 WT was heavily
phosphorylated on tyrosine whereas Jak2 DN was not.
Finally, the same membrane was blotted with anti-Src-mAb to evaluate
Jak2/Src association (Figure 2C, bottom). We repeatedly observed
that c-Src specifically bound kinase-active,
tyrosine-phosphorylated Jak2 WT but not the equally
abundant kinase-inactive, unphosphorylated Jak2 DN
protein. The level of Jak2/Src association was quantitated in several
experiments (Figure 2D). Cells transfected with Jak2 WT, but not
Jak2 DN, were found to have significantly increased Jak2/Src
association compared with empty vector controls. Collectively, the data
in Figure 2 indicate that a catalytically active Jak2 protein,
capable of tyrosine autophosphorylation, is necessary
for efficient association of Jak2 and c-Src in vivo.
The SH2 Domain of c-Src Binds Jak2 in a Tyrosine
Phosphorylation–Dependent Manner
To determine which region(s) of c-Src binds Jak2, we performed
Jak2 pull-down experiments using GST/Src fusion proteins composed of
either the SH2, SH3, or SH2+SH3 domains of c-Src. That is, we again
transfected COS-7 cells with either empty vector control, Jak2 WT, or
Jak2 DN. The resulting lysates were normalized, divided equally, and
incubated with either GST, GST/SH2, GST/SH3, or GST/SH2+SH3. The fusion
proteins were collected by centrifugation, separated on
SDS-PAGE, and transferred onto nitrocellulose. The membrane was then
blotted with anti-Jak2-pAb to measure Jak2 binding (Figure 3A). We observed that only the fusion
proteins containing the SH2 domain of c-Src bound Jak2, and this
binding was tyrosine phosphorylation–dependent. That
is, the SH2 domain of c-Src bound only the kinase-active,
tyrosine-phosphorylated Jak2 WT but did not bind the
equally abundant kinase-inactive, unphosphorylated Jak2
DN protein. To eliminate the possibility that the Jak2 DN protein was
not expressed, equivalent amounts of cell lysate from each of the three
transfected conditions were immunoblotted with
anti-Jak2-pAb (Figure 3B). Both Jak2 proteins were expressed at
similar levels. Thus, the data in Figure 3 strongly suggest that
the region of c-Src that binds Jak2 is the SH2 domain, and this binding
is tyrosine phosphorylation–dependent.
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The N-Terminus of Jak2 Is Required for Binding c-Src In
Vivo
The data in Figure 2 demonstrate that Jak2 must possess a
functional kinase domain to efficiently bind c-Src. Interestingly,
previous work in other cell systems has shown that the N-terminus of
Jak2 (JH7 and JH6 domains) mediates specific protein-protein
interactions between Jak2 and other signaling
molecules.20 21 In addition, work using Jak1/Jak2 chimeras
suggested that the N-terminus of Jak2 mediates specific protein-protein
interactions whereas the kinase domain simply
phosphorylates substrate that is bound by the N-terminus of
Jak2.22 We therefore hypothesized that the N-terminus of
Jak2 is required for binding c-Src. To test this, we made several Jak2
N-terminal deletant constructs (Figure 4A). We then used the vaccinia virus/T7
bacteriophage expression system to generate high-level expression of
all constructs. This allowed for the rapid mapping of the region of
Jak2 that binds c-Src. Specifically, we transiently transfected BSC-40
cells with the indicated Jak2 cDNA and then subsequently infected cells
with vaccinia virus clone vTF7-3 to significantly increase protein
expression. At 18 to 20 hours after infection, cells were lysed and
immunoprecipitated with anti-Src-pAb. After separation on SDS-PAGE and
transfer onto nitrocellulose, the samples were blotted with
anti-Jak2-pAb to assess Jak2/c-Src association. Cells transfected with
empty vector alone (pRC) contained a single, nonspecific 94-kDa band,
presumably from the vaccinia virus (Figure 4B). We observed that
Jak2 WT (pRC-WT), the AflII deletant (pRC-AFL), and the
pseudokinase deletant (pRC-PKD) all coimmunoprecipitated with c-Src. In
contrast, the amino terminal deletant (ATD) did not. We confirmed equal
precipitation of c-Src by blotting the same membrane with anti-Src-mAb
(Figure 4B). To rule out the possibility that the ATD protein
was being masked by the 94-kDa nonspecific band, we repeated the
experiment but reversed the order of antibody addition. Specifically,
BSC-40 cells were transfected/infected with the same constructs, but
this time we immunoprecipitated with anti-Jak2-pAb and then
Western-blotted with anti-Src-mAb. This protocol clearly showed that
c-Src did not coimmunoprecipitate with the ATD deletant whereas it
could associate with either Jak2 WT or the other two deletant
constructs (Figure 4C, top). To demonstrate that all Jak2
constructs were precipitated equally, we blotted the same samples with
anti-Jak2-pAb (Figure 4C, middle). In the experiment shown, both
the AFL and ATD deletants were precipitated at levels below that of WT
and PKD. However, both the AFL and ATD proteins were precipitated at
similar levels; the AFL deletant associated with c-Src whereas the ATD
protein did not. Finally, because Figure 3 indicated that the
SH2 domain of c-Src bound Jak2 in a tyrosine
phosphorylation–dependent manner, we Western-blotted
these Jak2 immunoprecipitates with anti-Tyr(P)-mAb to measure the
tyrosine phosphorylation of each Jak2 protein (Figure 4C, bottom). Other than the ATD construct, all Jak2 proteins
contained some readily visible degree of tyrosine
phosphorylation. With prolonged exposure, we could see
some tyrosine phosphorylation of Jak2 ATD, but it was
significantly less than the other deletant proteins (data not shown).
The lack of tyrosine phosphorylation of the ATD
construct is not due to a lack of catalytic activity of the molecule
because expression of similar ATD Jak2 proteins has been shown to be
catalytically active and capable of
autophosphorylation.21 23 In
addition, we have demonstrated that the ATD mutant mediates the Ang
II–dependent tyrosine phosphorylation of STAT1 in a
manner that is no different than Jak2 WT (data not shown).
Collectively, the data in Figure 4 indicate that the amino
terminal 240 amino acids of Jak2 are required for binding c-Src in
vivo. Furthermore, expression of the Jak2 molecule lacking the initial
240 amino acids (the c-Src binding region) is not heavily
phosphorylated on tyrosine, supporting the idea that
the interaction between Jak2 and c-Src is dependent on a Jak2
N-terminal phosphotyrosine and the SH2 domain of c-Src.
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The N-Terminus of Jak2 Is Required to Bind c-Src in Response to
Ang II
The data in Figure 4 indicate that the N-terminus of Jak2
is required for efficient binding of c-Src in vivo. However, those
experiments were done in the absence of Ang II. To determine whether
the N-terminus of Jak2 is required for an Ang II–mediated association
between Jak2 and c-Src, we transiently transfected COS-7 cells with
cDNAs encoding either AT1 and Jak2 WT or
AT1 and Jak2 ATD. In contrast to the COS-7 cell
transfections done in Figures 2C, 3A, and 3B, the
amount of Jak2 plasmid used here (Figure 5) was
significantly less to avoid Jak2 autophosphorylation in
the absence of ligand. At 48 hours after transfection, the quiescent
COS-7 cells were stimulated with Ang II, and the resulting lysates were
immunoprecipitated and blotted with anti-Jak2-pAb to assess Jak2
protein expression (Figure 5, top). Both
Jak2 WT and Jak2 ATD protein expression were observed as was the
endogenous Jak2 protein found in COS-7
cells.12 20 The samples were then blotted with
anti-Tyr(P)-mAb to measure the Ang II–induced tyrosine
phosphorylation of both Jak2 WT and Jak2 ATD (Figure 5, middle). In these experiments, the Jak2 WT protein was
tyrosine-phosphorylated in response to Ang II whereas
the ligand-dependent tyrosine phosphorylation of the
Jak2 ATD protein was significantly less. Finally, the membrane was
blotted with anti-Src-mAb to assess Jak2/c-Src association (Figure 5, bottom). Cells that were transfected with Jak2 WT and treated
with Ang II bound more c-Src than similarly treated cells transfected
with Jak2 ATD. These results indicate that the N-terminus of Jak2 is
required for efficient binding of c-Src in response to Ang II.
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Discussion |
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One hypothesis that is consistent with these findings is that in response to Ang II, a catalytically active Jak2 autophosphorylates tyrosines at the N-terminus of the molecule. This region then binds the SH2 domain of c-Src, allowing for ligand-dependent association of Jak2 and c-Src. The time course of Jak2/c-Src association appears to coincide with the duration of Ang II–mediated Jak2 tyrosine phosphorylation as previously described.2 13 As indicated earlier, one functional consequence of this ligand-dependent binding would be that both Jak2 and c-Src would be physically colocalized and could therefore act on the same substrate. Several molecules are known to be phosphorylated by both the Jak and Src families of tyrosine kinases. These include STAT1, STAT3, Raf-1, and Shc.6 7 8 9 Interestingly, it appears that the Jak and Src kinases may act synergistically, because they phosphorylate different tyrosine residues on a given substrate.7 With respect to Ang II, a recent report indicated that STAT1, Jak2, and Fyn form a ligand-dependent protein complex, in vitro.24 These data support the hypothesis of an Ang II–dependent physical colocalization of the Jak and Src family tyrosine kinases with a putative substrate. However, the specific protein domains that mediate this complex, the requirement for catalytically active Jak2 and the demonstration of an in vivo STAT1:Jak2:Fyn ternary complex, were not presented.
A natural question that might be posed from our data is whether Ang II mediates the formation of a ternary complex among Jak2:Src:AT1. We have previously demonstrated that Jak2 binds AT1 in response to Ang II.2 13 18 The present observation that Jak2 binds c-Src in response to Ang II lends itself to the possibility that some c-Src protein may also bind AT1 as well. This is currently under investigation.
Finally, these data are the first to demonstrate that in response to Ang II, Jak2 and c-Src form a specific protein-protein complex. We have defined the requirement of a catalytically active Jak2 for this association and the regions that mediate this binding. The data support the hypothesis that the N-terminus of Jak2 mediates specific protein-protein interaction and suggest that the tyrosine phosphorylation of the N-terminus of Jak2 is critical for this physical interaction consisting of a Jak2 phosphotyrosine and the SH2 domain of c-Src.
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Acknowledgments |
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This work was supported by National Institutes of Health grants DK39777, DK44280, DK45215, DK51445, and HL47035. Dr Sayeski has been supported by grants T32-DK07298 and F32-HL09678. We wish to thank Shaun Benford for administrative assistance.
Received February 1, 1999; accepted April 16, 1999.
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