(Received for publication, September 22, 1995; and in revised form, January 10, 1996)
From the
Activation of CD28 on T lymphocytes initiates a cascade of intracellular events, which in concert with activation of the T cell receptor, culminates in production of cytokines and a functional immune response. One of the earliest biochemical changes observed following stimulation of CD28 is tyrosine phosphorylation. We have demonstrated that both the LCK and the EMT/ITK/TSK (EMT) intracellular tyrosine kinases are activated following cross-linking of CD28. Utilizing somatic cell mutants lacking LCK, we demonstrate that functional LCK is required for CD28-induced activation of EMT as evidenced by increased tyrosine phosphorylation and kinase activity. In support of a role for LCK in EMT activation, reconstitution of a LCK-negative Jurkat T cell line by transfection with normal LCK recreates CD28-mediated EMT activation. Furthermore, co-transfection of LCK and EMT into COS-7 cells showed that EMT becomes phosphorylated in the presence of LCK. In addition, increases in EMT association with CD28 were eliminated in a LCK-negative Jurkat cell line, but were restored following transfection of wild type LCK. The data are most compatible with a model in which LCK, either directly or indirectly, initiates EMT activation and association with CD28 following ligation of CD28.
Co-stimulation of T lymphocytes requires the cooperation of two
signals delivered by antigen presenting cells: one stimulatory signal
derived from interaction of the T cell receptor (TCR) ()complex with antigen in the context of the major
histocompatibility complex and a second co-stimulatory signal from the
ligation of accessory molecules(1) . In the absence of the
co-stimulatory signal, T cells fail to undergo clonal expansion and
instead ultimately enter an abortive pathway characterized by antigen
desensitization, anergy, or programmed cell
death(1, 2, 3, 4) . The interaction
of CD28 on T lymphocytes with B7.1 (CD80) or B7.2 (CD86) on
antigen-presenting cells is the most potent identified co-stimulatory
signal. Indeed, cross-linking of CD28 can prevent activation-induced
desensitization, anergy, and programmed cell
death(4, 5, 6, 7, 8) .
Upon activation of CD28, there is a rapid and immediate increase in tyrosine phosphorylation of a number of specific substrates(8, 9, 10, 11, 12) . However, because CD28 does not contain an intrinsic kinase domain, it must activate intracellular tyrosine kinases. In addition, cross-linking of CD28 leads to the induction of a number of early signaling events, including increases in cytosolic free calcium(7, 8) , activation of RAS(13) , activation of mitogen-activated protein kinase (13) , activation of phosphatidylinositol 3`-kinase(14, 15, 16, 17, 18) , activation of JNK kinase (also known as stress-activated kinase) (19) and activation of RAF kinase(20) . Although it is clear that cross-linking of CD28 can induce a number of early signals, the role that activation of these biochemical changes plays in the ability of CD28 to synergize with the TCR to induce a functional T cell response remains unclear. Furthermore, the mechanisms leading to activation of these enzymes by CD28 and in particular the order of activation of each of these enymes is unknown.
We have demonstrated recently that EMT/ITK/TSK (EMT), a TEC family protein tyrosine kinase, becomes activated after CD28 cross-linking, as evidenced by a transient increase in tyrosine phosphorylation and kinase activity. In addition, stimulation of CD28 results in a rapid increase in the association of EMT with CD28(21) . Thus EMT has the potential to play a role in CD28 signal transduction. LCK is also activated after stimulation of CD28, suggesting that this kinase may have a signaling role through CD28 in addition to its dual role downstream of both the TCR and CD4/8(21, 22) .
The TEC family of intracellular
kinases currently consists of members that contain SH2 and SH3 SRC
homology (SH) domains but lack the negative regulatory tyrosine present
at the carboxyl terminus and the myristoylation site found at the amino
terminus of SRC family members. Thus the TEC family of tyrosine kinases
must be regulated in a different manner from the SRC family of tyrosine
kinases. BTK and EMT contain, in addition to the SH2 and SH3 domains, a
pleckstrin homology (PH) domain. The exact function of this domain is
currently unknown, but it may play a role in the ability of BTK and EMT
to associate with other molecules such as protein kinase
C(23, 24) . Both BTK and EMT have restricted patterns
of expression; BTK is expressed mainly in mast cells and B cells and
EMT is expressed primarily in mast cells and T
cells(25, 26) . BTK is involved in B cell signal
transduction; mutations in BTK have been causally linked to X-linked
agammaglobulinemia, a severe human B cell
immunodeficiency(27) . In addition, since cross-linking mouse
FcRI leads to activation of BTK (28) , this kinase may
also play a role in mast cell activation.
Herein, we demonstrate that CD28-mediated EMT activation and EMT association with CD28 is greatly decreased in cells lacking functional LCK. Reconstitution of LCK kinase activity by enforced expression of the normal human LCK reconstituted ligand-induced EMT activation and increased EMT association with CD28, confirming a role for LCK in EMT activation. In addition co-expression of EMT and LCK in COS-7 cells lead to tyrosine phosphorylation of EMT. Thus, EMT appears to be located downstream of LCK in the signaling pathways activated by CD28.
Figure 1:
CD28-induced tyrosine phosphorylation
of EMT requires functional LCK. Jurkat T cells were stimulated by
cross-linking CD28 with 10 µg/ml anti-CD28 (9.3) antibody and 10
µg/ml RAM for the indicated times. Similar results were observed in
the absence of RAM (see (18) and data not presented). The
cells were then lysed as described under ``Materials and
Methods.'' A, EMT was immunoprecipitated with anti-EMT
serum as described under ``Materials and Methods.'' The
precipitates were loaded on a 10% SDS-PAGE gel, transferred to
Immobilon, and Western-blotted with anti-phosphotyrosine antibody
(4G10). B, the blot was stripped with 1% SDS and reprobed with
anti-EMT antibody. C, total phosphotyrosine-containing
proteins were immunoprecipitated with polyclonal anti-phosphotyrosine
antibodies prepared in this laboratory, loaded on SDS-PAGE, and blotted
with anti-phosphotyrosine antibody (4G10). Immunoreactivity was
detected by ECL in all cases. Parental Jurkat (panel a), JCaM
1.6 (panel b), and LCK transfected JCaM 1.6 (panel c)
were treated similarly; the result shown represents one of three
similar experiments. In panel d, data from panels
a-d were converted to relative levels by densitometry and
normalized to EMT protein levels determined by densitometry. The
relative level of tyrosine phosphorylation is presented as the -fold
increase over basal levels of EMT tyrosine phosphorylation. ,
cell line: Jurkat, stimulation: CD28, readout: EMT Tyr(P);
, cell
line: JCaM1, stimulation: CD28, readout, EMT Tyr(P);
, cell line:
JCaM1 LCK, stimulation: CD28, readout: EMT
Tyr(P).
Figure 2:
Activation-induced increase in EMT
tyrosine kinase activity. The relative increase in kinase activity of
EMT was determined after stimulation of Jurkat cells as described in
the legend to Fig. 1. EMT was immunoprecipitated and kinase
activity was determined utilizing the SRC peptide as substrate as
described under ``Materials and Methods.'' Kinase activity is
presented as -fold increase over that observed in unstimulated cells to
allow comparison between experiments. The results represent mean and
standard error of the mean of three repeats of three independent
experiments. , cell line: Jurkat, stimulation: CD28, readout:
EMT immunoprecipitation kinase;
, cell line: Jcam1, stimulation:
CD28, readout: EMT immunoprecipitation kinase;
, cell line: Jcam1
LCK, stimulation: CD28, readout, EMT immunoprecipitation
kinase.
Figure 3: EMT association with CD28 following CD28 activation. EMT association with CD28 was determined by lysing JCaM1.6 cells (a) or JCaM1.6 cells transfected with LCK in Nonidet P-40 lysing buffer after cross-linking CD28 for the indicated time periods (b). CD28 was immunoprecipitated from cell lysates with anti-CD28 (9.3, 10 µg/ml) and RAM (10 µg/ml) and Western-blotted with anti-EMT as described under ``Materials and Methods.'' Activation of the parental Jurkat cell line with anti-CD28 for 5 min provides a positive control for CD28-induced EMT association with CD28. The data represent one of three similar experiments. ipt, immunoprecipitation.
To confirm that the decreased activation of EMT and reduced association of EMT with CD28 in the JCaM 1.6 line was due to a lack of functional LCK, we determined whether the response to CD28 cross-linking was restored in a JCaM 1.6 cell line transfected to express normal human LCK. Previous studies had demonstrated that expression of LCK in JCaM 1.6 restores anti-TCR-mediated signaling and interleukin-2 production (29) . In JCaM 1.6 LCK transfectants, CD28-induced tyrosine phosphorylation, and specifically, tyrosine phosphorylation of EMT induced by cross-linking CD28 was completely restored (Fig. 1, panels c and d). Similarly, anti-CD28 mediated increases in EMT kinase activity were restored in the JCaM 1.6 LCK transfectants (Fig. 2). In addition, the increase in association of EMT with CD28 was restored (Fig. 3b). The ability of transfected LCK to reconstitute CD28-mediated EMT activation confirms that the defect in EMT activation in JCaM 1.6 was indeed a consequence of lack of functional LCK.
Figure 4: LCK tyrosine phosphorylation of EMT in COS-7 cells. COS-7 cells were transfected with expression vectors encoding EMT/ITK in the presence or absence of LCK as described under ``Materials and Methods.'' Cells were harvested and EMT was immunoprecipitated and separated on a 10% SDS-PAGE gel and transferred to Immobilon membrane, which was then probed with anti-phosphotyrosine antibodies (top). The blot was then stripped and reprobed with anti-EMT antibodies (bottom).
The data presented are most consistent with a model wherein CD28-induced activation of the LCK tyrosine kinase is proximal in a cascade leading to CD28-induced activation of EMT. In support of this possibility, we have demonstrated that LCK kinase activity is increased following stimulation of CD28 (21, 22) and that LCK can lead to tyrosine phosphorylation of EMT in COS-7 cells (Fig. 4). Whether LCK directly phosphorylates EMT or EMT activation is a consequence of LCK-mediated phosphorylation of an intermediary molecule following CD28 activation is currently unknown. As Jurkat cells express both SRC and FYN(32) , these Src family tyrosine kinases, in contrast to LCK, are either not sufficient for CD28-mediated EMT activation or are not stimulated following CD28 ligation.
Regulation of EMT kinase activity may well be at the level
of tyrosine phosphorylation, since tyrosine phosphorylation of EMT and
EMT kinase activity demonstrated concurrent changes following
cross-linking of CD28 (Fig. 1d and Fig. 2). EMT
contains a tyrosine in a conserved internal site which becomes
autophosphorylated in Src family tyrosine kinases and likely positively
regulates kinase activity(26) . However, kinase assays revealed
that EMT is very inefficient at autophosphorylation as compared to Src
family tyrosine kinases(21) . Since EMT is inefficient in
autophosphorylation (21) and LCK has the ability to induce
tyrosine phosphorylation of EMT (Fig. 4), this phosphorylation
site in EMT could be a direct target for other kinases such as LCK.
Indeed a number of different tyrosine kinases are both positively and
negatively regulated by other tyrosine kinases in activation cascades.
For example, phosphorylation of the negative regulatory site of SRC
family kinases seems to be dependent on the action of CSK family
kinases(33, 34) . In turn, a number of different SRC
family kinases have been demonstrated to regulate non-SRC family
kinases. This is well documented for LYN and SYK in B
cells(35) , SRC and FAK in fibroblasts(36) , and FYN or
LCK and ZAP70 in T cells(2) . ZAP70 activation is, however, not
detectable after CD28 ligation. ()Further support for a
model in which tyrosine kinases are coordinately regulated in an
activation cascade is provided by the demonstration that functional
signaling by the platelet-derived growth factor receptor is dependent
on the presence of functional SRC family kinases(37) . It is
important to note, in terms of potential kinase activation cascades,
that the experiments presented herein, although demonstrating that LCK
is required for CD28-mediated EMT activation, do not address the
question of whether LCK is reciprocally regulated either negatively or
positively by EMT.
After CD28 activation, the receptor becomes tyrosine-phosphorylated (15, 18) . Signaling proteins such as phosphatidylinositol 3`-kinase and Grb2 bind to these phosphotyrosine residues through their SH2 domains (14, 15, 16, 17, 18, 38, 39) . As demonstrated previously(21) , EMT binds to CD28 constitutively and after activation the association is up-regulated presumably by SH2 domain interactions. This increase in association was greatly reduced in the JCaM 1.6 cell line and restored in the JCaM 1.6 cell line transfected with LCK. This suggests that functional LCK is required for CD28 cross-linking induced increases in EMT association with CD28. This may be a consequence of LCK either directly or indirectly tyrosine phosphorylating CD28. Recent results, using transfection into Spodoptera cells, supports LCK as the primary mediator of CD28 phosphorylation(38) . Furthermore, EMT, phosphatidylinositol 3`-kinase, and GRB2 association with CD28 in transfected Spodoptera cells required co-expression of LCK (38) .
Although the PH domain of both BTK and EMT associate with protein kinase C isoymes(24) , limited data are present to date on the interactions between TEC family kinases and other tyrosine kinases. Although EMT and BTK appear to associate with SRC family kinases (including LCK and FYN) in the yeast two-hybrid system and when expressed as bacterial fusion proteins(40, 41, 42) , it has been difficult to demonstrate interactions in intact cells(40, 41, 42) . Indeed, under conditions (using Nonidet P-40 buffers) in which we can readily demonstrate association of EMT with CD28 (Fig. 3, (21) ), we have been unable to demonstrate association of EMT with LCK, FYN, SRC, or TTK (all of which are expressed by Jurkat T cells; 10, 32, 43, 44) as indicated by co-immunoprecipitation (data not shown).
In summary, we have demonstrated that the ability of CD28 to optimally activate EMT is dependent on the presence of functional LCK. The data are most compatible with a model of CD28 signaling in which activated LCK mediates phosphorylation and activation of EMT and mediates EMT association with CD28. Thus EMT activation seems to be located downstream of LCK in a kinase cascade stimulated by CD28.