(Received for publication, October 19, 1995; and in revised form, December 11, 1995)
From the
Engagement of the T cell antigen receptor results in both its
phosphorylation and its ubiquitination. T cell antigen receptor
ubiquitination was evaluated in Jurkat, a well characterized human T
leukemia cell line. Treatment of cells with the tyrosine kinase
inhibitor herbimycin A resulted in an inhibition of receptor
ubiquitination. Consistent with this, pervanadate, which increases
cellular tyrosine phosphorylation, enhanced receptor ubiquitination. A
requirement for receptor-mediated tyrosine kinase activity for
ubiquitination was confirmed in cells lacking the tyrosine kinase
p56 and also in cells that are defective in
expression of CD45, a tyrosine phosphatase that regulates the activity
of p56
. The need for tyrosine kinase activation
for ubiquitination was not bypassed by directly activating protein
kinase C and stimulating endocytosis of receptors. These observations
establish ubiquitination of the T cell antigen receptor as a tyrosine
kinase-dependent manifestation of transmembrane signaling and suggest a
role for tyrosine phosphorylation in the ligand-dependent
ubiquitination of mammalian transmembrane receptors.
For many transmembrane receptors, including the multisubunit
TCR, ()signaling is initiated by ligand-induced
aggregation(1, 2) . The earliest obligate
intracellular event following TCR aggregation is the activation of the src-family protein tyrosine kinases, Lck
(p56
) and Fyn (p59
). Lck
and/or Fyn phosphorylate TCR subunits resulting in the association of a
third tyrosine kinase, ZAP-70 (70-kDa
-associated protein), with
the TCR and to subsequent activation
events(3, 4, 5) . CD45, a tyrosine
phosphatase that dephosphorylates key regulatory residues on Lck and
Fyn, is also implicated in the initiation of TCR-mediated
signaling(6) .
TCRs consist of six different polypeptides,
these include the antigen-recognition element, in most cells an
-
heterodimer, and a set of invariant signal transducing
subunits. The invariant subunits include CD3-
, -
, and -
and the structurally distinct TCR-
subunit, which exists within
the TCR as a disulfide-linked homodimer (4) . The minimal
signal transducing element of the TCR is the immunoreceptor
tyrosine-based activation motif (ITAM)(7) .
monomers have
three ITAMs, and each CD3 subunit has one. ITAMs include two tyrosine
residues 10 or 11 amino acids apart that are potential phosphorylation
sites. The
subunit is a particularly prominent substrate for
tyrosine phosphorylation; up to 5% of
subunits are phosphorylated
on multiple tyrosines upon TCR
engagement(8, 9, 10, 11) .
In addition to being a substrate for tyrosine phosphorylation when cross-linked by antibody or mitogen(11) , TCRs also are ubiquitinated. The covalent modification of proteins with chains of ubiquitin, a highly conserved 76-amino acid polypeptide, plays a central role in the targeting of abnormal proteins and a number of regulatory cytosolic and nuclear proteins for degradation in the 26 S proteasome(12, 13, 14, 15, 16) . Ubiquitination occurs via a multienzyme process involving families of enzymes termed E1-E3. E1 (ubiquitin-activating enzyme) is involved in the ATP-dependent charging of ubiquitin. The high energy thiol-ester bond between E1 and ubiquitin is transferred to an E2 (ubiquitin-conjugating enzyme). E2s either by themselves or in conjunction with E3s (ubiquitin protein ligases), transfer ubiquitin monomers or multiubiquitin chains to target proteins, where isopeptide linkages are formed with lysine residues.
The signals that lead to
ubiquitination of most naturally occurring substrates are unknown. The
N-end rule established a relationship between the N-terminal amino acid
of certain proteins and susceptibility to ubiquitination(14) .
For the cyclins and c-Jun, specific internal polypeptide sequences have
been implicated in targeting for
ubiquitination(17, 18) , and for cyclins as well as
IB
, serine phosphorylation also plays a role in this process (19, 20, 21, 22) .
The TCR is
distinguished from most ubiquitination substrates by its long half-life
and by being ubiquitinated in response to a specific external stimulus.
TCR ubiquitination occurs on multiple subunits and on multiple
intracellular lysines, with mono- and multiubiquitinated species
detectable(23) . As with tyrosine phosphorylation, the
subunit is the most prominent substrate for this modification, likely
due to the nine intracellular lysines in each
monomer, compared
with two or three for each of the other invariant subunits. Fundamental
to understanding TCR ubiquitination is a determination of the events
that couple receptor engagement to this modification. Using Jurkat (24) , a well characterized human T leukemia cell line, we
address the relationship between receptor occupancy and ubiquitination.
Our findings establish a relationship between receptor ubiquitination
and early signaling events mediated by TCR engagement and the
activation of protein tyrosine kinases.
OKT3 is a
monoclonal antibody directed against the subunit of the CD3
complex of the TCR(29) . Monoclonal anti-phosphotyrosine
antibody 4G10 was a gift from Brian Druker (University of Oregon Health
Sciences Center). Polyclonal rabbit antisera 527, raised against a
peptide corresponding to amino acids 131-143 of human
, was
generated as described(30) . Polyclonal anti-Lck (31) was provided by Lawrence Samelson (National Institutes of
Health). The monoclonal antibody A2B4-2 has been previously
described(32) . Polyclonal rabbit anti-ubiquitin has been
described(23) .
Herbimycin A (Calbiochem, San Diego, CA) was
dissolved in dimethyl sulfoxide. Pervanadate was formed by a 1:1
mixture of 100 mM sodium orthovanadate (Sigma) and 0.035%
HO
and added to cells at 1:50. PMA (Sigma) was
dissolved in ethanol.
Figure 1:
Ubiquitination and
tyrosine phosphorylation. A, Jurkat cells were treated for 18
h with carrier (dimethyl sulfoxide) (two left lanes) or with
herbimycin A at a final concentration of 0.3 µM (right
center lane) or 3.0 µM (far right lane)
prior to incubation at 37 °C for 10 min in the absence (far
left lane) or the presence (three right lanes) of a
saturating amount of OKT3, an antibody directed against the
subunit of the CD3 complex of the TCR (anti-CD3). TCRs from Triton
X-100 soluble cell lysates were immunoprecipitated with a mixture of
OKT3 and a rabbit polyclonal anti-
antiserum (527) and resolved on
12% SDS-PAGE followed by immunoblotting with either anti-ubiquitin (upper panel) or anti-phosphotyrosine (lower panel).
The 50-kDa band present in the unstimulated sample corresponds to the
heavy chain of immunoglobulin. B and C, Jurkat cells
were preincubated at 4 °C for 10 min prior to stimulation at 37
°C as above. Far left lane, no treatment; left center
lane, pervanadate alone; right center lane, anti-CD3; far right lane, anti-CD3 and pervanadate. B represents Triton X-100-soluble whole cell lysates from 1.5
10
cell equivalents resolved on 7% SDS-PAGE and
immunoblotted with anti-phosphotyrosine. In C,
immunoprecipitated TCRs were resolved on 12% gels followed by
immunoblotting with either anti-ubiquitin or anti-phosphotyrosine as
indicated.
The herbimycin A results suggest a
relationship between TCR ubiquitination and TCR-mediated tyrosine
kinase activation. To further assess this relationship, the effects of
pervanadate on TCR ubiquitination were evaluated. Pervanadate increases
global cellular tyrosine phosphorylation by inhibiting protein tyrosine
phosphatases and has been shown to mimic TCR-mediated
signaling(37, 38, 39, 40) . When
Jurkat cells were pretreated with pervanadate, a dramatic increase in
total cellular tyrosine phosphorylation was observed in whole cell
lysates, regardless of TCR engagement with anti-CD3 (Fig. 1B). When TCRs were specifically
immunoprecipitated from pervanadate-treated cells, the subunit of
the TCR was also found to be tyrosine-phosphorylated (Fig. 1C, lower panel). The level of 21-kDa
phospho-
seen with pervanadate alone was substantially greater
than that achieved with anti-CD3 (OKT3) cross-linking, and together
pervanadate and anti-CD3 were synergistic with regard to
phosphorylation. When evaluated for ubiquitination (Fig. 1C, upper panel), pervanadate by itself
resulted in the appearance of ubiquitinated TCRs, although the level of
ubiquitination achieved with pervanadate was consistently less than
that seen with anti-CD3 (Fig. 1C, upper panel,
and data not shown). As with tyrosine phosphorylation, anti-CD3 and
pervanadate together had synergistic effects on ubiquitination. In
multiple experiments, concomitant treatment with pervanadate and
anti-CD3 resulted in increases in ubiquitination from 2- to 8-fold
relative to anti-CD3 alone. In conjunction with the herbimycin A
results, these findings suggest that tyrosine kinase activation plays a
crucial role in TCR ubiquitination and that receptor engagement
facilitates the generation of TCR-ubiquitin conjugates.
Figure 2:
Dependence of ubiquitination on Lck. A, equal numbers of Jurkat and receptor-matched Lck-deficient
JCaM 1.6.22 cells were incubated in the presence or the absence of
anti-CD3 at 37 °C, as indicated in the figure, prior to lysis and
immunoprecipitation as described for Fig. 1. Immunoblotting was
carried out with either anti-ubiquitin or anti-phosphotyrosine as
indicated. B, anti-Lck immunoblot of Triton X-100-soluble
lysates showing restoration of wild type Lck expression in JLck.H5 and
JLck.B3 stable transfectants of JCaM1.6.22. The positions of wild type
Lck and a band corresponding to an inactive mutant form of this enzyme
expressed in JCaM1.6 (26) are indicated by arrows.
Lysates from 1 10
cells were loaded in each lane. C, TCRs from Jurkat and stable transfectants of JCaM1.6.22
with Lck were evaluated as in A.
As shown (Fig. 3A), although anti-CD3-dependent receptor endocytosis is less pronounced in Lck-deficient JCaM1.6.22 when compared with Jurkat (Fig. 3A, compare a and b with e and f), treatment with PMA bypasses TCR-mediated signaling and results in an intracellular redistribution of receptors comparable with that seen with Jurkat (Fig. 3A, c and g; d and h). Having established that PMA results in receptor redistribution in JCaM1.6.22, the effects of combinations of PMA and anti-CD3 on TCR ubiquitination were evaluated in this cell line. Neither PMA plus anti-CD3 nor PMA alone (Fig. 3B) resulted in any detectable receptor ubiquitination in JCaM1.6. Thus, serine phosphorylation of TCR subunits and receptor redistribution is not sufficient to result in ubiquitination of TCRs. Ionomycin (a calcium ionophore) and PMA, which together activate Jurkat cells in a TCR-independent fashion(45) , also failed to restore ubiquitination in JCaM1.6.22, even when receptors were concomitantly cross-linked with anti-CD3 (not shown). Consistent with the requirement for tyrosine kinase activation for ubiquitination, treatment of wild type Jurkat with PMA and/or ionomycin in the absence of TCR engagement resulted in neither TCR ubiquitination nor tyrosine phosphorylation (not shown).
Figure 3:
Protein kinase C activation fails to
stimulate ubiquitination. A, cell surface expression of TCRs
in Jurkat (a-d) and JCaM 1.6.22 (e-h)
cells in response to combinations of anti-CD3 and PMA (100 ng/ml).
Cells were incubated with the indicated additions for 30 min at 37
°C and then cooled to 4 °C. Samples were incubated with
anti-CD3 (solid lines) or a nonbinding murine antibody
(A2B4-2) (dotted lines, in upper two panels only), followed by incubation with fluorescein
isothiocyanate-labeled goat anti-mouse F(ab`) (Southern
Biotechnology Associates, Birmingham, AL) and analysis by FACScan
(Becton Dickinson, Mountain View, CA). B, cells were incubated
at 4 °C with the indicated additions prior to stimulation at 37
°C followed by lysis, immunoprecipitation, SDS-PAGE, and
immunoblotting with anti-ubiquitin.
Figure 4:
Dependence of ubiquitination on CD45
expression. A, TCRs from Jurkat cells or a CD45-deficient
variant (J45.01) were immunoblotted with anti-ubiquitin after
stimulation at 37 °C as described for Fig. 1. B,
comparison of Jurkat and J45.01 to a CD45 positive transfectant of
J45.01 (J45.LB3.3). Immunoblotting was carried out with either
anti-ubiquitin (upper panel) or anti- (lower
panel).
This study establishes that TCR ubiquitination requires intact coupling to tyrosine kinase activation and is not simply the consequence of the recognition of aggregated TCR cytoplasmic domains by E2/E3 enzymes. This requirement is not bypassed by stimulating serine phosphorylation and internalization of engaged receptors. Pervanadate, which results in an acute increase in tyrosine phosphorylation, is sufficient to result in detectable levels of TCR ubiquitination, even in the absence of receptor ligation. Thus, an acute increase in tyrosine phosphorylation, independent of receptor occupancy, appears to be sufficient to result in ubiquitination. However, the finding that the level of ubiquitination seen with pervanadate is consistently less than that found with receptor occupancy suggests that specific signals generated in response to TCR engagement may be important in stimulating a maximal level of TCR ubiquitination. These signals may be a direct manifestation of TCR oligomerization or perhaps reflect a different temporal order or pattern of phosphorylation induced when tyrosine kinases are activated by TCR ligation.
The platelet-derived growth factor receptor and c-kit (the stem cell factor receptor) are tyrosine kinase-containing members of the growth factor family of receptors that are ubiquitinated in response to their cognate ligands(48, 49) . In the case of platelet-derived growth factor receptor, mutation of autophosphorylation sites correlates with decreased ligand-dependent ubiquitination(50) , and for c-kit, the tyrosine kinase inhibitor genestein results in decreased ligand-dependent ubiquitination(49) . Several other mammalian transmembrane receptors that either signal by coupling to tyrosine kinase activation (51) or that contain intrinsic tyrosine kinase activity (52) are ubiquitinated in an occupancy-dependent manner. Taken together with our findings, these observations suggest that tyrosine phosphorylation likely plays an important role in ligand-dependent ubiquitination of a number of mammalian transmembrane receptors.
One means by which receptor ubiquitination might occur in response to tyrosine kinase activation is by the phosphorylation of E2/E3 enzymes with resultant changes in their associations and/or activities. In fact, in one case, the in vitro tyrosine phosphorylation of an E2 was found to correlate with enhanced ubiquitination(53) . Alternatively, receptors might be ubiquitinated by E2/E3 enzymes that contain sites that bind phosphotyrosine, such as is seen with SH2 domains, although no enzymes fitting this description have thus far been identified.
The function of ubiquitination in the biology of transmembrane receptors remains to be elucidated. It has generally been assumed that transmembrane receptors are degraded in lysosomes. However, to be exposed to lysosomal proteases, intracytosolic receptor domains would first need to be engulfed in autophagocytic vesicles. Alternatively, the intracytosolic domains of receptors could be degraded, at least in part, in a ubiquitin-dependent fashion in the 26 S proteasome. This could be viewed as a protective mechanism, insuring the degradation of the signaling domains of activated receptors. Although results obtained with the platelet-derived growth factor receptor are suggestive(50) , for no transmembrane receptor has a causal relationship between ligand-induced ubiquitination and proteasomal degradation been established. The recent finding of herbimycin A-induced ubiquitination and proteasomal degradation of the insulin-like growth factor receptor, while not addressing the issue of ligand-dependent ubiquitination, demonstrates that transmembrane receptors may, in fact, be degraded by proteasomes (54) .
Regardless of the fate of ubiquitinated receptors, the steric effects of ubiquitin moieties branching off of the intracytoplasmic tails of receptors would be expected to impact negatively on intracellular associations with signaling molecules and between receptors. For the TCR, Fyn, ZAP-70, and CD45 are among the TCR-associated proteins that could be affected by ubiquitination. Thus, whether or not ubiquitination is a major factor in ligand-dependent receptor degradation, it is likely that this modification represents a means of modulating the function of activated transmembrane receptors.