(Received for publication, November 27, 1995; and in revised form, February 13, 1996)
From the
Several cell surface receptors including the T cell receptor
(TCR) are phosphorylated and down-regulated following activation of
protein kinases. We have recently shown that both phosphorylation of
Ser-126 and the presence of the di-leucine sequence Leu-131 and Leu-132
in CD3 are required for protein kinase C (PKC)-mediated TCR
down-regulation. To identify additional residues required for
PKC-mediated phosphorylation of CD3
and for TCR down-regulation,
an alanine scanning of CD3
was done. Mutations of Arg-124,
Ser-126, Lys-128, and Gln-129 inhibited both phosphorylation and TCR
down-regulation, whereas mutation of Asp-127 only inhibited
down-regulation. Further analyses demonstrated a discrepancy between
the ability to be phosphorylated on CD3
and to down-regulate the
TCR in several transfectants. Phosphorylation was not as strictly
dependent on the nature and position of the phosphoacceptor group and
basic residues as were the subsequent steps involved in TCR
down-regulation. Our results suggest that PKC-mediated TCR
down-regulation may be regarded as a two-step process. 1) Recognition
and phosphorylation of CD3
by PKC. In this process Arg-124,
Ser-126, Lys-128, and Gln-129 are important. 2) Recognition of
phosphorylated CD3
by molecules involved in receptor
internalization. In this process Ser(P)-126, Asp-127, Leu-131, and
Leu-132 are important.
The T cell receptor (TCR) ()is a mitogenic receptor
coupled to protein tyrosine kinases(1, 2) . Following
stimulation of the TCR, signals that lead to T cell activation and
proliferation are transmitted across the T cell membrane (2, 3) . Both TCR down-regulation and antigen
unresponsiveness can be induced following stimulation with specific
anti-TCR antibodies(4, 5, 6) , with
supraoptimal doses of antigen(7, 8) , or with phorbol
esters(9, 10) . Physiological stimulation of the TCR
leads to serine phosphorylation of CD3
(11) , and recently,
it was found that T cell activation correlates with the degree of TCR
down-regulation(12) . Thus, down-regulation of the TCR appears
to play a physiologically important role in the regulation of T cell
function.
The TCR is a multimeric receptor composed of four dimers:
the clonotypic Ti heterodimer, generally Ti, the
CD3
and CD3
dimers, and the
homodimer(13, 14, 15, 16, 17) .
The disulfide-linked Ti
heterodimer is responsible for ligand
recognition(18, 19) , and the activation signals are
delivered through the associated CD3 and
chains (reviewed in (20) ). After TCR stimulation, a number of cellular proteins
including phospholipase C
1 become phosphorylated on tyrosine
residues(21, 22, 23) . Tyrosine
phosphorylation of phospholipase C
1 augments its enzymatic
activity, resulting in the production of inositol 1,4,5-trisphosphate
and diacylglycerol(24) . These second messengers are
responsible for an increase in intracellular Ca
concentration and an activation of protein kinase C
(PKC)(25, 26) . Among other substrates, the activated
PKC phosphorylates the CD3
chain(27, 28) .
Furthermore, activation of PKC leads to internalization and
down-regulation of the TCR at the cell
surface(9, 10, 11, 27, 29, 30) ,
and we have recently shown that in addition to the di-leucine sequence
Leu-131 and Leu-132(31) , phosphorylation of Ser-126 in the
cytoplasmic tail of CD3
is required for PKC-mediated
down-regulation of the TCR (32, 33) (amino acid
numbering of CD3
according to (34) ). The role of the
amino acids that surround Ser-126 as substrate specificity determinants
for PKC and their role in the molecular mechanisms involved in TCR
down-regulation following Ser-126 phosphorylation are unknown.
Based
on the amino acid sequences that render synthetic peptides optimal
substrates for protein kinases and on the sequence features that
surround known phosphorylation sites on protein substrates, consensus
sequences have been proposed as substrate specificity determinants for
protein kinases(35, 36, 37, 38) .
PKC requires basic amino acids near the phosphoacceptor group of the
substrate, and phosphoacceptor groups surrounded by basic amino acids
at both the N- and C-terminal seem to be better substrates for
PKC, -
, and -
than phosphoacceptor groups surrounded by
basic amino acids at only the N- or C-terminal side(37) . Most
studies describing substrate specificity determinants for PKC are based
on in vitro analyses of synthetic
peptides(39, 40, 41, 42, 43, 44) .
Such peptides are powerful investigative tools, but their small size
and random conformation may limit their ability to mimic the
intracellular proteins they are intended to model. Furthermore, in
vivo experiments are required to analyze the consequences of
substrate phosphorylation.
The aim of this study was to identify
amino acids in the cytoplasmic tail of CD3 required in vivo for PKC-mediated phosphorylation of CD3
and for
down-regulation of the TCR.
Figure 1:
Alanine scanning of CD3 from
Arg-121 to Thr-130. A, schematic representation of the amino
acid sequences in the cytoplasmic tails of the CD3
chains
expressed in the indicated cell lines. B, cells were incubated
with different concentrations of PDB for 1 h, and TCR down-regulation
was determined by staining with anti-CD3 monoclonal antibodies and flow
cytometry comparing MFI of PDB-treated cells with MFI of untreated
cells. Cell lines that showed a reduced TCR down-regulation are shown
with an asterisk. JGN-A125A is identical to
JGN-WT.
Figure 2:
A
basic residue at position 124 renders CD3 an optimal substrate for
PKC, whereas an acidic residue at this position acts as a negative
substrate determinant. A, schematic representation of the
amino acid sequences in the cytoplasmic tails of the CD3
chains
expressed in the indicated cell lines and a summation of the results
from the CD3
phosphorylation and TCR down-regulation analyses.
CD3
phosphorylation was scored in a semiquantitative way as
follows: +++, clearly phosphorylated at 22.5 nM PDB; ++, weakly phosphorylated at 22.5 nM PDB
but clearly phosphorylated at 225 nM PDB; +, not
phosphorylated at 22.5 nM PDB and weakly phosphorylated at 225
nM PDB; -, neither phosphorylated at 22.5 nor 225 nM PDB. TCR down-regulation was scored according to the percent
anti-CD3 binding following incubation with PDB (110 nM) for 1
h: +++, 0-40% anti-CD3 binding; ++,
40-60% anti-CD3 binding; +, 60-80% anti-CD3 binding;
(+), 80-95% anti-CD3 binding; -, >95% anti-CD3
binding. B, cells were incubated with different concentrations
of PDB for 1 h, and TCR down-regulation was determined by staining with
anti-CD3 monoclonal antibodies and flow cytometry comparing MFI of
PDB-treated cells with MFI of untreated cells. C, phosphorylation analyses of CD3
from JGN-WT (lanes 1 and 4), JGN-R124A (lanes 2 and 5), and
JGN-R124E (lanes 3 and 6) cells. D, FACS
histograms of untreated cells (white) and cells treated with
PDB (110 nM) (black) for 1 h. The cell line and the
percent anti-CD3 binding following PDB treatment are given in the upper left corner of each histogram. The ordinate gives the relative cell number. The abscissa gives the
fluorescence intensity in a logarithmic scale in arbitrary units. Mean
fluorescence intensity of the cell lines stained with irrelevant
monoclonal antibodies varied between two and five arbitrary units (data
not shown).
To directly examine the
role of the different mutations in PKC-mediated CD3
phosphorylation, cells were loaded with
P and incubated
with PDB for 10 min. Following cell lysis and immunoprecipitation, the
samples were run on SDS-PAGE and analyzed by autoradiography. JGN-WT
and JGN-R124K CD3
clearly became phosphorylated following
incubation with 22.5 nM PDB, whereas JGN-R124A CD3
became
weakly phosphorylated and JGN-R124E CD3
did not become
phosphorylated at this PDB concentration (Fig. 2C, and
data not shown). Increasing the concentration of PDB to 225 nM resulted in an increased phosphorylation of both the JGN-WT,
JGN-R124K, and JGN-R124A CD3
chains, and a weak phosphorylation of
the JGN-R124E CD3
became apparent (Fig. 2C, and
data not shown).
Figure 3:
Analyses of the phosphoacceptor group. A, schematic representation of the amino acid sequences in the
cytoplasmic tails of the CD3 chains expressed in the indicated
cell lines and a summation of the results from the CD3
phosphorylation and TCR down-regulation analyses. CD3
phosphorylation and TCR down-regulation were scored as described in the
legend to Fig. 2. B, cells were incubated with
different concentrations of PDB for 1 h, and TCR down-regulation was
determined by staining with anti-CD3 monoclonal antibodies and flow
cytometry comparing MFI of PDB-treated cells with MFI of untreated
cells. C, phosphorylation analyses of CD3
from JGN-WT (lanes 1 and 5), JGN-S126T (lanes 2 and 6), JGN-125SA (lanes 3 and 7), and JGN-126DS (lanes 4 and 8) cells. D, FACS histograms as
described in the legend to Fig. 2.
Next
we wanted to study how critical the exact position of the
phosphoacceptor group was for PKC-mediated phosphorylation and TCR
down-regulation. Accordingly, JGN cells were transfected with
constructs in which the serine was transposed either one position
N-terminal (Fig. 3A, construct 125SA) or one position
C-terminal (Fig. 3A, construct 126DS). As in JGN-S126T
cells, a discrepancy between the ability to function as a PKC substrate
and the ability to function in TCR down-regulation was observed for
both of the mutated CD3 chains (Fig. 3). 125SA- and
126DS-CD3
clearly became phosphorylated following PKC activation,
although not as efficiently as WT-CD3
. However, down-regulation of
the TCR was completely abolished or severely inhibited in JGN-125SA and
JGN-126DS cells, respectively.
Figure 4:
Role of the acidic and the basic residue
C-terminal to Ser-126. A, schematic representation of the
amino acid sequences in the cytoplasmic tails of the CD3 chains
expressed in the indicated cell lines and a summation of the results
from the CD3
phosphorylation and TCR down-regulation analyses.
CD3
phosphorylation and TCR down-regulation were scored as
described in the legend to Fig. 2. B, cells were
incubated with different concentrations of PDB for 1 h, and TCR
down-regulation was determined by staining with anti-CD3 monoclonal
antibodies and flow cytometry comparing MFI of PDB-treated cells with
MFI of untreated cells. C, phosphorylation analyses of
CD3
from JGN-WT (lane 1), JGN-D127A (lane 2),
JGN-K128A (lane 3), JGN-128QK (lane 4), and JGN-127KD (lane 5) cells. D, FACS histograms as described in
the legend to Fig. 2.
How critical the exact position of the basic
residue C-terminal to the Ser-126 phosphoacceptor was for PKC-mediated
phosphorylation of CD3 and TCR down-regulation was studied using
the constructs 127KD and 128QK, in which the lysine was transposed one
residue N- and C-terminal, respectively (Fig. 4A). TCR
down-regulation following PKC activation was completely abolished in
JGN-127KD cells and severely inhibited in JGN-128QK cells (Fig. 4, B and D). However,
P
analyses showed that both 127KD- and 128QK-CD3
became
phosphorylated following incubation with 225 nM PDB, although
to a lesser degree than wild-type CD3
(Fig. 4C).
These results demonstrated that a basic residue positioned either one,
two, or three residues C-terminal to Ser-126 acted as a substrate
determinant for PKC. Furthermore, the finding that TCR down-regulation
in JGN-127KD cells was completely abolished, although phosphorylation
was intact, indicated that the mechanisms involved in TCR
down-regulation following Ser-126 phosphorylation was highly dependent
on the position of the acidic aspartic acid.
Figure 5:
A polar amino acid three residues
C-terminal to Ser-126 optimizes CD3 as a substrate for PKC. A, schematic representation of the amino acid sequences in the
cytoplasmic tails of the CD3
chains expressed in the indicated
cell lines and a summation of the results from the CD3
phosphorylation and TCR down-regulation analyses. CD3
phosphorylation and TCR down-regulation were scored as described in the
legend to Fig. 2. B, cells were incubated with
different concentrations of PDB for 1 h, and TCR down-regulation was
determined by staining with anti-CD3 monoclonal antibodies and flow
cytometry comparing MFI of PDB-treated cells with MFI of untreated
cells. C, phosphorylation analyses of CD3
from JGN-WT (lanes 1 and 3) and JGN-Q129A (lanes 2 and 4) cells. D, FACS histograms as described in the
legend to Fig. 2.
Figure 6:
TCR internalization is dependent on
Ser-126 phosphorylation that can be partially mimicked by introduction
of a negatively charged amino acid. A, schematic
representation of the amino acid sequences in the cytoplasmic tails of
the CD3 chains expressed in the indicated cell lines and a
summation of the results from the CD3
phosphorylation and TCR
internalization analyses. CD3
phosphorylation and PDB-induced TCR
internalization were scored as described in the legend to Fig. 2. B, cells were incubated with different
concentrations of PDB for 1 h, and TCR down-regulation was determined
by staining with anti-CD3 monoclonal antibodies and flow cytometry
comparing MFI of PDB-treated cells with MFI of untreated cells. C, cells were incubated with
I-labeled Fab fragments of
the monoclonal antibody F101.01 for the periods indicated. The
acid-sensitive and the acid-resistant
I-labeled Fab
fragments were subsequently measured. The percentage of acid-resistant
I to total
I was calculated and taken as
the percentage of TCR internalized.
The present study demonstrated that, although not absolutely
required, a basic amino acid (Arg-124) two residues N-terminal to the
phosphoacceptor group (Ser-126) was optimal in rendering CD3 a PKC
substrate in vivo. Studies of synthetic peptides have
suggested that in some instances PKC prefers arginine to lysine as
substrate determinant(40) . However, we could not detect any
change in the level of CD3
phosphorylation or TCR down-regulation
by substitution of lysine for Arg-124, indicating that it is their
common characteristic in form of a positive charge that makes arginine
and lysine PKC substrate determinants. Interestingly, substitution of
the acidic amino acid glutamic acid for Arg-124 had a stronger
inhibitory effect on CD3
phosphorylation and TCR down-regulation
than substitution of an apolar amino acid for Arg-124. This showed that
an acidic amino acid located two residues N-terminal to the
phosphoacceptor group acted as a negative substrate determinant for
PKC. Furthermore, it indicated that Arg-124 is involved in
electrostatic contacts with acidic amino acids in the catalytic domain
of PKC. This agrees with results obtained by model building of PKC in
which Lys-23 (corresponding to CD3
Arg-124) of the PKC
pseudosubstrate makes electrostatic contacts with Asp-470 and Glu-533
in the catalytic domain of PKC(50) . Thus, the consequence of
the R124E mutation would be a local electrostatic repulsion in contrast
to an electrostatic attraction. To our knowledge, this is the first
description of the existence of a negative substrate determinant for
PKC in vivo. That the inhibitory effect of R124A could be
compensated for by increasing the concentration of PDB suggested that,
although important for optimal CD3
phosphorylation, Arg-124 was
not required for the subsequent steps in TCR internalization.
PKC
phosphorylates substrates at either serine or threonine. It is still
not clear whether PKC prefers serine to threonine although two
experiments have pointed in that direction(40, 51) .
It was therefore of interest to test the effect of the substitution of
threonine for Ser-126. From the P analyses it was found
that S126T-CD3
was as good a PKC substrate as WT-CD3
.
However, TCR down-regulation was significantly inhibited in JGN-S126T
cells. This indicated that threonine is as well accepted a
phosphoacceptor as serine by PKC but that the mechanisms involved in
TCR down-regulation following CD3
phosphorylation are critically
dependent on the nature of the phosphoacceptor group. Similarly, PKC
allowed a certain variability in the position of the phosphoacceptor
group, which agrees with the proposed broad definition of the PKC
consensus sequence(38) . Although not as efficient as
WT-CD3
, both 125SA- and 126DS-CD3
were substrates for PKC.
However, TCR down-regulation was completely abolished or severely
inhibited in JGN-125SA and JGN-126DS cells, respectively. This
indicated that the position of the phosphoacceptor group is very
critical for the mechanisms involved in TCR down-regulation following
CD3
phosphorylation. Furthermore, these mechanisms are, in
contrast to PKC-mediated phosphorylation, highly dependent on the
presence of a negatively charged amino acid (Asp-127) just C-terminal
to the phosphoacceptor group. In agreement with this a role for acidic
residues in endocytic targeting of receptors with di-leucine-based
motifs has very recently been described(52) .
The existence
of a basic amino acid C-terminal to Ser-126 was absolutely required for
PKC-mediated phosphorylation of CD3. The nature of the basic amino
acid (lysine/arginine) did not affect the ability of CD3
to be a
substrate for PKC and to function in down-regulation of the TCR. The
position of the basic amino acid two residues C-terminal to Ser-126 was
optimal for recognition and phosphorylation by PKC, but basic amino
acids positioned either one or three residues C-terminal to Ser-126
also functioned as substrate determinants. Again a discrepancy between
the ability to function as a substrate determinant and the ability to
function in the mechanisms involved in TCR down-regulation following
Ser-126 phosphorylation was observed. This was most clearly seen in
JGN-127KD cells in which CD3
definitely became phosphorylated, but
TCR down-regulation was completely abolished. This further supported
the observation that the mechanisms involved in down-regulation of the
TCR following Ser-126 phosphorylation are dependent on an acidic amino
acid right C-terminal to the acidic phosphoryl group.
The
observation that a polar amino acid (Gln-129) three residues C-terminal
to the phosphoacceptor group was a substrate determinant for PKC was at
first sight surprising because previously only basic amino acids have
been described as substrate determinants for
PKC(36, 38) . However, the pseudosubstrate site of
PKC, -
, and -
all contain a glutamine three residues
C-terminal to the phosphoacceptor substitute
alanine(53, 54, 55) . Taken with our results,
this indicated that a polar amino acid three residues C-terminal to the
phosphoacceptor group contributes to the binding efficiency of
(pseudo)substrates to PKC in vivo. Interestingly, substitution
of alanine for Gln-129 did not seem to affect the mechanisms involved
in TCR down-regulation following Ser-126 phosphorylation as the
inhibitory effect of this substitution on TCR down-regulation could be
compensated for by increasing the concentrations of PDB.
From the results described above, it can be concluded that substrate recognition by PKC in vivo is not strictly dependent on a precisely defined consensus sequence but that PKC can recognize and phosphorylate a broad range of related substrates, as also described for the cyclic AMP-dependent protein kinase (reviewed in (56) ). However, the molecular mechanisms involved in the subsequent steps in TCR internalization seem to be much more critically dependent on the nature of the phosphoacceptor group and the amino acids that surround it.
The molecular mechanisms behind PKC-mediated TCR down-regulation may
be divided in two steps. 1) Recognition of CD3 as a substrate for
PKC with subsequent phosphorylation of Ser-126. In this process
Arg-124, Ser-126, Lys-128, and Gln-129 seem to be important. 2)
Recognition of phosphorylated CD3
by molecules involved in
receptor internalization. In this process Ser(P)-126, Asp-127, Leu-131,
and Leu-132 seem to be important. Whether the pSD-LL motif is
recognized as one large motif or phosphorylation of Ser-126 causes a
conformational change that exposes the D-LL motif remains to be
determined.
Di-leucine or leucine-isoleucine sorting signals have
been identified in other molecules, e.g. the mannose
6-phosphate/insulin-like growth factor-II
receptor(57, 58) , the cation-dependent
mannose-6-phosphate receptor(59) , the lysosomal integral
membrane protein II(60) , CD4(61, 62) , and
the invariant chain(63, 64) . Interestingly, in these
molecules an acidic amino acid or a serine is found four and five
residues N-terminal to the di-leucine/leucine-isoleucine sequence,
exactly corresponding to the position of Ser-126, Asp-127, and
Leu-131/132 in CD3. This supports the hypothesis that molecules
involved in sorting/internalization of receptors with di-leucine-based
motifs recognize a motif composed of both acidic amino acids and the
di-leucine/leucine-isoleucine sequence. That the spontaneous TCR
internalization rate in JGN-S126E cells was higher than that of JGN-WT
cells further indicated that the electrostatic effects of Ser-126
phosphorylation play an important role for recognition of the motif by
molecules involved in receptor sorting/internalization. The existence
of an internalization motif that becomes activated by protein
kinase-mediated phosphorylation would afford cells a means to couple
receptor-mediated activation with receptor desensitization.
Alternatively, for T cells where ligands are scarce, down-regulation of
activated receptors could allow additional receptors to be activated,
thereby intensifying cell activation, as recently
suggested(12) .