(Received for publication, September 1, 1994; and in revised form, October 24, 1994)
From the
Src-family tyrosine kinases share structural and amino acid
sequence homology, particularly in the catalytic domain as well as in
the SH2 and SH3 domains of the regulatory region. However, each
src-family member also contains a unique domain which is specific to
and characteristic of each individual tyrosine kinase. These unique or
specific domains may contribute to the functional specificity of each
src-family kinase. To address this possibility, we analyzed the kinase
activities and substrate specificities of the lymphoid src-kinase,
pp56, and a mutant of pp56
lacking its specific domain. Our data show that both the
wild type enzyme and the specific domain-deleted mutant displayed
similar affinities for ATP and the non-physiological substrate
denatured enolase. However, the specific domain-deleted mutant failed
to phosphorylate a number of physiological substrates of
pp56
. In addition, the ability of
pp56
to mediate induction of the interleukin-2
promoter was strongly impaired upon deletion of its specific domain.
Thus, the unique domain is not required for the intrinsic kinase
activity of pp56
, however, it influences
substrate preference and contributes to the unique physiological
function of this src-family tyrosine kinase.
The src-family of non-receptor tyrosine kinases act as signaling molecules in a wide array of cellular activation processes (for review, see (1) and (2) ). The existence of the various src-family kinases reflects the requirement for different cell-surface receptors to transmit cell type-specific intracellular signals(3, 4, 5, 6) .
The
structural basis for the functional specificity of src-kinases is being
actively investigated. Src-kinases share a general common organization:
an NH-terminal myristoyl group(7) , followed by a
non-homologous region (unique or specific domain), two regulatory
regions (SH2 and SH3 domains), the conserved catalytic domain (SH1) and
a COOH-terminal regulatory region involved in modulating the enzyme
activity (reviewed in (1) ). Regarding the involvement of any
of these regions in determining the enzyme specificity, SH2 and SH3 are
good candidates since these regions have the abilities to bind
different signaling molecules(8, 9, 10) .
However, the contribution of SH2 and SH3 domains may not be sufficient
to determine src-kinases specificity, since SH2 and SH3 domains of
src-kinases share more than differ in their
agonists(11, 12) . Another region that is likely to
confer specificity is the unique domain since it has a different amino
acid sequence for each member. In this regard, we and others have
already shown that the unique domains of pp56
,
pp55
and pp59
are
implicated in specific receptor
interaction(13, 14, 15, 16, 17) .
Thus, the interaction of the kinase with a specific transmembrane
receptor could be a mechanism to confer specificity. However, this
mechanism does not explain all the cases in which src-kinases are
essential. For instance, pp56
binds specifically
to CD4 and CD8(13, 14, 15) , and shares with
pp59
the ability to bind to the
TcR(
)(16, 18) . However, pp56
is essential for TcR-mediated IL-2 production in Jurkat
cells and for the induction of differentiation of early
thymocytes(3, 4, 19) , processes in which
neither CD4 nor CD8 seem to participate. In these and other processes,
the unique domain could be involved in the specific function of
src-kinases through a novel mechanism different from specific
transmembrane receptor binding. To test this hypothesis, we have
analyzed the function of the specific domain of
pp56
.
pp56 is a
src-kinase first identified by its overexpression in the LSTRA thymoma
cell line(20) . After its initial identification,
pp56
was found to be present in all T cells and
some B cells but rarely in cells of non-lymphoid origin(20) .
Regarding its function, pp56
seems to be crucial
for T cell maturation (3, 4, 5, 21) and is also involved
in the activation of mature T cells (22) . In particular,
pp56
seems to play a central role in the
induction of IL-2 production required for T cell proliferation (23, 24) and in mediating the intracellular signals
delivered by CD4 and CD8 co-receptors(15) .
In the present
report we have compared wild type pp56 and a
mutant lacking its unique domain. We have examined their substrate
specificities, kinetic constants, and functional activities in T cells.
We present data indicating that the specific domain of
pp56
is involved in regulating substrate
preference and is relevant for the specificity of pp56
biological function.
Anti-phosphotyrosine antibody (4G10) was
produced and generously donated by Dr. B. Druker (D.F.C.I., Harvard
Medical School, Boston, MA). UCHT1 anti-CD3 Ab was described previously (25) and generously donated by Dr. D. Cantrell. Anti-GAP Ab,
anti-COOH-terminal-pp56 Ab, and
anti-NH
-terminal-pp56
Ab, were obtained from
Upstate Biotechnology Incorporated.
Jurkat cells and JCam1 cells
were grown in RPMI 1640 medium supplemented with 10% heat-inactivated
fetal calf serum, L-glutamine (2 mM), HEPES pH 8.0
(10 mM), streptomycin (50 µg/ml), and penicillin (50
units/ml) (RPMIc) at 37 °C, 5% CO.
For Escherichia coli expression, mutants were cloned into Gex-2T (G-pp56 constructs) as described previously(26) . For
electroporation of JCam1 cells, wild type and mutated pp56
were cloned into pEF BOS vector(27) . The constructs were
created by inserting the 1.5-kilobase NcoI to EcoRI fragment from G-pp56
constructs into the XbaI site of pEF BOS. Prior to ligation all the overhanging
ends were blunted using Klenow enzyme. Orientation was determined in
each case by restriction analysis. NFAT-CAT (28) has been
previously described; it contains three copies of the sequence
5`-GGAGGAAAAACTGTTTCATACAGAAGGCGT-3` (corresponding to the sequence
from position -284 to -258 relative to the ATG of the IL-2
gene) upstream of the IL-2 minimal promoter driving the reporter gene
CAT. This sequence contains the ARRE-2 site of the human IL-2 enhancer,
which is the binding site for NFAT(29) .
Kinase reactions
were performed as follows: 10 µl containing 50 ng ( Fig. 1and Table 1) or 25 ng ( Fig. 2and Fig. 3) of purified kinase were preincubated at 25 °C for 1
min and then mixed with: (a) 20 µl of 2 kinase
reaction mixture and (b) 10 µl containing 10 µg of
acid denatured enolase, or the corresponding substrates (indicated in
the figures). The mixture contained 50 mM Tris-HCl, pH 7.4, 10
mM MnCl
, and serial dilutions of the ATP stock
(100 µM ATP, 10 µCi/µl of
[
-
P]ATP, 3000 Ci/mmol). For the enzymatic
analysis ( Table 1and Fig. 1), reactions were incubated at
25 °C for 2 min (mixed every 30 s) and terminated by addition of 10
µl of 100 mM EDTA pH 8.0. For substrate specificity
analysis ( Fig. 2and Fig. 3), reactions were performed
similarly but incubated for 15 min. For kinase reactions using
immunopurified anti-pp56
(from 2
10
cells, Fig. 4), immunoprecipitates were prepared as in (7) , and in vitro reactions were performed as above.
Substrate and enzyme were resolved by SDS-PAGE. For the determination
of kinetic parameters, phosphate incorporated into enolase was
quantitated by liquid scintillation counting. V
and K
were estimated as described
previously(26) . Peptides (1.5 mM final concentration)
were used as substrates as described(33) .
Figure 1:
Purification and kinase activity of the
different bacterially expressed G-pp56 mutants. A, the diagram represents pp56
protein.
The different relevant domains are indicated. The position of the
different mutations prepared and their nomenclature are shown at the
bottom. B, wild type and mutated G-pp56
proteins were produced in E. coli X90. The
different fusion proteins present in the bacterial lysates were
purified using glutathione-Sepharose beads (200 ng obtained from 400
µg of total soluble protein). Purified proteins were analyzed by
SDS-PAGE followed by Coomassie Blue-staining. C, 50-ng
aliquots of the different samples were tested for their ability to
transfer phosphate into enolase in vitro and resolved by
SDS-PAGE. The resulting gel was analyzed by autoradiography. D, autophosphorylation of the different mutants in vivo was analyzed by comparison of similar volumes (20 µl) of each
of the crude extracts separated by SDS-PAGE and analyzed by Western
blot using anti-phosphotyrosine Abs.
Figure 2:
Comparison of the in vitro substrate specificity of
specific-pp56
and wild
type-pp56
. Wild type and mutated
pp56
proteins were produced and purified as
explained under ``Experimental Procedures.'' 25 ng of each
mutant or wild type enzyme were tested in vitro for their
ability to phosphorylate each of the indicated substrates (detailed
under ``Experimental Procedures''). Kinase reactions were
resolved by SDS-PAGE and analyzed by autoradiography. Substrates are
indicated on the left and G-pp56
mutants on the
top. A control in the absence of substrate (lanes 1) or enzyme (lanes 2) is run in each case.
Figure 3:
Analysis of the in vitro substrate specificity of
specific-pp56
and wild type
pp56
. The analysis of substrate specificity was
performed as in Fig. 2. Controls in the absence of enzyme (lanes 1) or substrate (lanes 2) are run in each
case.
Figure 4:
Expression of wild type and
specific-pp56
in JCam1 cells. A, the cDNAs encoding wild type and mutated
pp56
(50 µg) were electroporated into JCam1
cells. 24 h later cells were lysed, and similar volumes of the
different lysates were resolved by SDS-PAGE and developed by Western
blot using anti-COOH-pp56
antibodies. B, JCam1 lysates, prepared as in A, were
immunoprecipitated using anti-COOH-pp56
Abs.
Kinase reactions were carried out in pp56
immunoprecipitates, the reaction products were resolved by
SDS-PAGE, and the gel was visualized by
autoradiography.
CAT assays (34, 35) were performed using 5-µg aliquots of the different cellular lysates (previously optimized to be within the linear range).
As a first approximation to test the kinase activity of
the different mutants, 50 ng of each of the pure preparations were
tested in an in vitro kinase assay (Fig. 1C).
The kinase activity was also evaluated by comparison of similar volumes
of the lysates by anti-phosphotyrosine Western blotting (Fig. 1D). Both analysis indicated that
specific-pp56
displayed a kinase activity
that was roughly similar to that displayed by wild type
pp56
. Interestingly, active pp56
and wild
type pp56
also displayed similar phosphotransfer
activities (Fig. 1, C and D). This is probably
due to the fact that bacteria does not express p50
, an
enzyme that down-regulates pp56
activity(37) . To
compare quantitatively the kinase activity of
specific
-pp56
and wild type
pp56
, enzymatic parameters were estimated. To this end,
we first compared the kinase activity of the fusion protein
G-pp56
with the activity of bacterial pp56
upon cleavage of the glutathione S-transferase fragment,
and the activity of both preparations was similar(26) . Thus,
purified wild type and mutated G-pp56
proteins were used
for the enzymatic analysis. To determine the enzymatic parameters of
G-pp56
, we followed our previously described
method(26) . Briefly, the concentration of purified enzyme was
estimated by SDS-PAGE followed by Coomassie Blue staining. 50 ng of
G-pp56
were mixed with varying amounts of ATP and enolase
and subjected to kinase reaction. A time course of the reaction
revealed that the incorporation of phosphate was linear at least for
the first 5 min using between 10-100 ng of enzyme (not shown).
Therefore, for all the assays, 50 ng of enzyme and 2-min incubations
were used to remain in the linear range. To measure the K
of G-pp56
for ATP in
autophosphorylation reactions, enzyme amount was fixed (50 ng) and ATP
concentration was varied from 0.25 to 10 µM. To calculate
the K
for enolase, ATP concentration was fixed at
5 µM and enolase varied from 0.34 to 22 µM (corresponding to <0.1 to >3
K
for enolase of G-pp56
). To evaluate the
phosphotransfer activity, we determined the apparent V
for enolase phosphorylation in excess of ATP (5 µM ATP, corresponding to >5
K
).
Enzyme and substrate were resolved by SDS-PAGE. Initial velocity was
estimated by measuring the amount of phosphate incorporated into
G-pp56
or enolase by liquid scintillation counting. The
data obtained were evaluated by Eisenthal Cornish-Bowden(38) ,
and Lineweaver-Burk (39) approximations, which yielded similar
values in every case. The values obtained for wild type G-pp56
( Table 1and (26) ) were found to be similar to
those obtained using purified baculoviral pp56
(26, 31) . Using these conditions, we have
compared specific
-pp56
and wild
type-pp56
and found that they displayed similar
phosphotransfer activities (V
for enolase
phosphorylation, Table 1). Furthermore, the enzymatic parameters
indicated that the affinities (estimated by the K
)
of specific
-pp56
and wild type pp56
for ATP and enolase were similar. Thus, deletion of the specific
domain does not alter the affinities of pp56
for ATP and
enolase and the enzymatic activity of pp56
toward
enolase.
The results obtained (Table 2, Fig. 2and 3) show that both the wild type and
active pp56 enzymes were able to phosphorylate all of the
tested substrates. In contrast, the specific
-pp56
phosphorylated only a subset of these substrates. In fact, even
after long exposure, Fig. 2and Fig. 3demonstrate that
the physiological substrates GAP and MAPK as well as the polyaminoacids
poly-(EY) and poly-(KAEY) were very poorly utilized by
specific
-pp56
(see Table 2for
quantitatitative results). This altered substrate specificity is
unlikely to be due to a structural change that inactivates the kinase,
since the resulting mutant still phosphorylates cdc2 peptide, MBP,
enolase, and poly-(EAY) ( Fig. 2and Fig. 3, Table 2) and binds ATP with a similar affinity than the wild type
protein (Table 1). Thus, specific
-pp56
displayed a significantly different substrate specificity profile
from the wild type or activated pp56
enzymes.
Interestingly, no alteration in substrate specificity was obtained with
deletion mutants at SH2 or SH3 domains (our data not shown). Overall,
these results indicate that the specific domain contributes in
directing the ability of the pp56
enzyme to phosphorylate
substrate.
To compare the expression of the different
mutants, aliquots of the lysates from the different electroporated cell
samples, corresponding to similar number of cells, were analyzed by
Western blotting. As shown in Fig. 4A, the different
mutants were expressed to a similar extent in JCam1 cells. The
stability of wild type and mutated pp56 seems to be
similar since the expression of wild type and mutated proteins at 24 h,
analyzed by Western blotting (Fig. 4), was similar. Furthermore,
fractionation experiments performed as in (7) , indicated that
wild type pp56
and specific
-pp56
display a similar intracellular distribution (not shown). The
kinetic parameters from bacterial enzymes demonstrated that wild type
pp56
and specific
-pp56
display
similar catalytic activities (Table 1). We have also compared the
kinase activity of these mutants upon expression in T cells. As judged
by the kinase assay performed in anti-pp56
immunoprecipitates (Fig. 4B),
specific
-pp56
from transfected JCam1 cells
displayed a kinase activity similar to wild type pp56
.
Interestingly, active and wild type pp56
also displayed
similar kinase activities (discussed below).
Figure 5:
IL-2 promoter induction in resting and
activated JCam1 cells upon expression of pp56.
JCam cells were transfected with 5 µg of NFAT-CAT (dark
bars) or 5 µg of NFAT-CAT and 50 µg of pEF
BOS-pp56
expression vectors (dotted
bars), as described under ``Experimental Procedures.'' 6
h after electroporation the cells were activated using anti-CD3 Abs
(UCHT1) at 1 µg/ml. 18 h later, the cells were lysed and reporter
gene activity assessed. Percent conversion represents the ratio of
radioactivity extracted in the organic phase versus total
radioactivity in organic plus aqueous phases. Standard deviation from
three experiments is indicated.
From the analysis of CAT
activity present in cells electroporated with the different
pp56 mutants (Fig. 6), we have obtained several
conclusions: (i) the kinase activity of pp56
is required
for IL-2P activation since the mutant lacking kinase activity did not
induce the IL-2P. (ii) Overexpression of active pp56
induced the IL-2P similarly to wild type pp56
,
confirming that, in certain circumstances, overexpression of
pp56
substitutes for pp56
activation(42) . (iii) More importantly, the IL-2P
induction was significantly attenuated in cells overexpressing
specific
-pp56
compared to cells
overexpressing wild type or activated pp56
(Fig. 6). The fact that the biological activity of
specific
-pp56
was only 15% compared with wild
type pp56
, indicated that the specific domain is required
for pp56
to mediate IL-2 production.
Figure 6:
IL-2 promoter induction in resting and
activated JCam1 cells upon expression of
specific-pp56
. JCam1 cells were
transfected with 5 µg of NFAT-CAT alone or 5 µg of NFAT-CAT
plus 50 µg of mutated pEF BOS-pp56
expression vectors as indicated. After 24 h at 37 °C and
5% CO
, the cells were lysed and reporter gene activity
assessed. The percentage of conversion of radioactivity extracted in
the organic phase versus total radioactivity in organic plus
aqueous phases is represented. Standard deviation from four different
experiments is indicated.
The src-family tyrosine kinase pp56 mediates
early events in signal transduction induced via the T cell antigen
receptor (19) and via CD4 and CD8 co-receptors(15) .
We have studied the involvement of the unique domain (also named
specific domain) of pp56
in determining its biological
function. A specific domain deletion mutant was constructed and
expressed in Jurkat cells lacking pp56
expression (JCam1
cells). These cells require pp56
to be functional. In
fact, the lack of pp56
expression correlates with their
inability to produce IL-2(19) . Expression of pp56
in this system induced the recovery of IL-2 production (Fig. 5). Transfection of the pp56
mutant lacking
the specific subdomain indicated that this domain is required for
pp56
to mediate its specific biological function, as
measured by its ability to induce the IL-2 promoter (Fig. 6).
Several explanations may account for the loss of function of the
mutant of pp56 lacking the specific domain. First, the
lack of the specific domain could affect the transmission of the TcR
signal to pp56
. However, this is unlikely because under
the overexpression conditions used in this analysis IL-2 promoter
induction is independent of TcR triggering (Fig. 5). Second, an
impaired association of pp56
with CD4 or CD8 (13, 14) as the cause for the defective biological
function is also unlikely since neither CD4 nor CD8 seem to participate
in the induction of IL-2 production of Jurkat cells. Third, the lack of
the specific domain could affect the activation of the enzyme. However,
under the overexpression conditions used in this analysis pp56
activation is not required to observe IL-2 promoter induction (Fig. 5). Finally the lack of pp56
function could
be due to a structural change. However, the enzyme seems to maintain
unaffected its ability to bind ATP (Table 1) and is able to
phosphorylate enolase, cdcd2, MBP, and poly-(EAY) ( Fig. 2and Fig. 3, Table 2). Thus, we favor the hypothesis that the
unique domain is essential for pp56
to trigger IL-2
production due to the involvement of this domain in regulating
substrate preference.
The mechanism through which the specific
domain contributes to the phosphorylation of certain substrates is not
evident. One possibility is that the specific domain contributes to
bind to and orientate the substrate in the enzyme so that it becomes
accessible to the catalytic domain. This is supported by the
observation that the specific domain of p56 binds MAPK
and GAP and that this association is not equally observed with
p59
(17) . In addition, MAPK has been found to
directly phosphorylate pp56
at residue 59 located within
the specific/unique domain(43) . The crystal structure of SH2
and SH3 domains of pp56
suggests that this enzyme may
form dimers(44) . If this is the case, it would be interesting
to know how the specific domain interacts the catalytic core in these
structures.
The highly efficient expression system used in this
study allowed us to analyze the biological function of the mutants in
the absence of any activation requirement. In fact, overexpression of
wild type-pp56 did not require TcR signaling and was
similar to active pp56
in their ability to induce IL-2P ( Fig. 5and 6). These results are in agreement with previous
reports showing that overexpression of src-kinases may result in a
phenotype similar to the one obtained with the activated
enzyme(42) . This could occur because overexpressed wild type
pp56
exceeds the amount of enzyme that endogenous
p50
is able to down-regulate (39) and thus,
behaves as the activated enzyme. Alternatively, the presence in the
cells of very high amounts of kinase, under these expression
conditions, may result in increased phosphotransfer activity similar to
the one observed upon enzyme activation.
Several authors have
suggested that pp56 biological function seems to depend
upon the ability of the regulatory domain to associate with cellular
proteins(45) . Our results along those lines illustrate that
the ability of the specific domain to regulate specific substrate
phosphorylation contributes to the enzyme function. However, our
results also indicate that the kinase activity is required for
pp56
to be functional, since no IL-2P induction was
observed in cells expressing inactive pp56
. The kinase
activity of pp56
is also critical for T cell
differentiation(3, 4) . Thus, only in particular
situations(45) , the ability of pp56
to bind
cellular proteins, conferred mainly by the regulatory domain, seems to
be sufficient for pp56
signaling. Under physiological
conditions, probably both the kinase activity and the ability to bind
cellular proteins cooperate to yield pp56
biological
function.
In conclusion, the specific domain of this src-family
tyrosine kinase is involved in functions specific for
pp56, such as CD4/CD8 binding (as we and others have
previously described(13, 14) ) or selection of
specific substrates ( Fig. 2and 3). The selection of specific
substrates by pp56
seems essential for the enzyme to be
functional in T cells. In fact, even in the cellular system used in
this report (independent of CD4/CD8 co-receptors, and bypassing TcR
signaling requirements) the lack of the specific domain results in
impaired pp56
biological function. Thus, substrate
selection, regulated by the specific domain, seems to be a novel
mechanism that contributes to determine the specific biological
function of src-kinases.