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*
Servei d’Immunologia, Institut d’Investigacions Biomédiques August Pii Sunger, Hospital Clínic, Barcelona, Spain;
Department of Organic Chemistry, University of Barcelona, Barcelona, Spain;
Basel Institute for Immunology, Basel, Switzerland; and
§
Institut National de la Santé et de la Recherche Médicale, U343, Hôpital de l’Archet, Nice, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The signal transduction via surface receptors results in recruitment of critical signal-transducing enzymes from the cytosol to the inner surface of the plasma membrane. Association with protein kinases has emerged in the last few years as a major signaling mechanism used by molecules lacking intrinsic catalytic activity in their cytoplasmic domains (13, 14, 15, 16). CD5 possesses a long cytoplasmic domain (94 amino acids) devoid of any intrinsic catalytic activity. This highly conserved cytoplasmic domain contains 11 serines, 4 threonines, and 4 tyrosines that are potential targets of intracellular protein kinases, and thus suitable for interaction with signaling mediators and phosphorylation. The presence of a double tyrosine-based motif (D-(X)2-Y-(X)11-Y-(X)2-L) is particularly intriguing due to its similarity to the immunoreceptor tyrosine-based activation motif found in accessory molecules that associate with the lymphocyte Ag receptors (17). Tyrosines within that motif are susceptible to phosphorylation by members of the src family of protein-tyrosine kinases associated with the Ag receptor complex. This phosphorylation then serves as a docking place for src homology domain 2-containing proteins. In agreement with this model, CD5 has been found to be tyrosine phosphorylated upon both T and B cell Ag-specific receptor ligation (4, 18, 19) and to associate with p56lck, p59fyn (19), and Zap-70 (20). Nevertheless, information is still lacking concerning the location of residues targeted by these protein-tyrosine kinases, or the functional consequences of their phosphorylation. Similarly, little is known about the targeting of CD5 by serine/threonine kinases. CD5 is found constitutively phosphorylated on serine residues and is hyperphosphorylated after phorbol ester treatment, which suggests that it is a substrate for PKC and/or other serine/threonine kinases (21, 22). The association of an uncharacterized, activation-inducible serine kinase with CD5 has been reported (23). Detailed analysis of human CD5 cytoplasmic sequences reveals several potential phosphorylation sites for serine/threonine kinases. There are five serine residues (S415, S423, S458, S459, and S461) and two threonine residues (T410, T412) in the consensus sequences for casein kinase II (CKII, S/T-[X]2-D/E or S-[X]2-S(P)) (24) and PKC (R-[X]2-S/T or S/T-X-R/K) (25), respectively. However, the involvement of these serine/threonine residues as phosphorylation targets has not been demonstrated to date. Herein we use wild-type and cytoplasmic mutant molecules to show that CKII associates with and is responsible for the constitutive phosphorylation of the CD5 receptor at its C terminus. Additionally, our results argue for a role of CKII-mediated phosphorylation in the signaling events initiated by CD5.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Low m.w. heparin (3000 m.w.) from porcine intestinal mucosa and PMA were purchased from Sigma (St. Louis, MO). The mouse anti-CD5 mAb Cris-1 (IgG2a) was produced in our laboratory by Dr. R. Vilella using PBMC as immunizing cells, and was affinity purified by passing either culture supernatant or ascitic fluid through a 5-ml HiTrap protein G column (Pharmacia, Piscataway, NJ). Affinity-purified Leu1 (IgG2a) mAb was purchased from Becton Dickinson (Mountain View, CA). The F145GF3 mAb was kindly provided by D. Carrière (Sanofi Recherche, Montpellier, France).
Construction of wild-type and cytoplasmic mutant human CD5 molecules
Construction of CD5.WT, CD5.K384stop, and
CD5.H449stop molecules was done as described (12).
CD5.E418stop was obtained after subcloning an
EcoRI/EcoRI fragment from the cDNA clone pT1-2
into M13 mp18 and inserting a new MseI site by
oligonucleotide-directed in vitro mutagenesis (Kunkel’s method) with
the sense oligonucleotide 5'-TCCCATGCTTAAAACCCCACA-3' that
introduces a premature stop codon at E418. CD5.
E418-L444 was
constructed by subjecting pT1-2 to inverse PCR mutagenesis with the
sense 5'-GAAGGGGTTCTGCATCGCTCCTCC-3' and the antisense
5'-AGCATGGGATCGGACGGTTGCCGT-3' oligonucleotides. Inverse PCR
mutagenesis with the sense 5'-GACGGTGACTATGATCTGCATGGG-3' and
antisense 5'-GGCGGAGTTGTCAGGCTGCATGGA-3' oligonucleotides was also
used to generate S459
A and S461G substitutions (CD5.S459A,S461G),
which in turn resulted in the generation of new restriction sites for
EagI and HphI, respectively. The CD5.Y463A
construction was also done by inverse PCR mutagenesis with the sense
5'-CTGATCTGCATGGGGCTCAGAGGCT-3' and the antisense
5'-CGTCACTGTCGGAGGAGTTGTCAGGC-3' oligonucleotides. All
oligonucleotide-directed changes were checked by double-stranded DNA
sequencing (SequiTherm cycle sequencing kit; Epicentre Technologies,
Madison, WI). Plasmid constructions were purified by cesium-chloride
density gradients.
Radiolabeling and stimulation conditions of cell transfectants
The Jurkat cells used in this study were obtained from the European Collection of Animal Cell Cultures (Salisbury, U.K.). The CD5-deficient 2G5 Jurkat clone was obtained in our laboratory by cell-sorting selection and further limiting dilution cloning of CD5-dull populations derived from wild-type Jurkat cells (12). 2G5 cells remained negative for CD5 expression after more than 1 mo of continuous culture, as well as after several days of PMA exposure (5–10 ng/ml), a well-known up-regulator of CD5 expression in Jurkat cells (26). CD5-specific mRNA levels were not detected by Northern blot in PMA-treated and untreated 2G5 cells compared with wild-type Jurkat cells. The phenotypic analysis of 2G5 cells was CD2+, CD3+, CD4+, CD5-, CD8-, CD18+, CD45+, and HLA class I+. The 2G5 cells presented intracellular Ca2+ mobilization responses following anti-CD3 treatment identical to wild-type Jurkat cells, in both magnitude and duration. Transfection conditions and screening of drug-resistant colonies were performed as previously reported (12). Cell transfectants were metabolically labeled with 32P, as described elsewhere (22). When needed, cells were preincubated for 30 min in the presence of heparin just before 32P labeling, without detecting significant losses in cell viability.
Immunoprecipitation and Western blot analysis
For phosphorylation analysis, immunoprecipitation was performed as previously described (22). Nonreducing conditions were preferred to enhance detection by the anti-CD5 mAbs used for Western blotting and to minimize the interference of Ig heavy chains during CD5 detection. Nitrocellulose membranes were blocked for 30 min at 37°C with 5% nonfat dry milk powder in PBS, and then incubated at room temperature for 1–2 h with Leu1 mAb in blocking solution. After extensive washing in PBS/0.25% gelatin, the membrane was incubated with a 1/400 dilution of peroxidase-labeled goat anti-mouse Ig serum (Dako, Carpenteria, CA) in blocking solution. Ag-bound Ab was detected by chemoluminescence (ECL; Amersham, Arlington Heights, IL).
Peptide chemistry and in vitro CKII assays
Peptides reproducing the 451–459 and 456–464 regions of CD5
(SSMQPDNSS and DNSSDSDYD, respectively), analogues of the latter
(DNSSDADYD, DNSADADYD, DNSADSDYD, and DNAADADYD), and the control
peptide DDDSDDD were synthesized as C-terminal carboxamides, with three
additional Arg residues at the N terminus to ensure proper binding to
P81 paper. The synthetic chemistry was based on the Fmoc/t-butyl
protecting scheme (27) and was performed on resins that had been made
functional by addition of the 5-(4-(9-fluorenylmethyl oxycarbonil
aminomethyl valeric acid (PAL) linker (28). Following cleavage
of the peptide resins with trifluoroacetic acid/water (95:5; 2 h),
the free peptides showed sufficient purity (>90%) by HPLC and were
satisfactorily characterized by amino acid analysis and matrix-assisted
laser desorption ionization–time of flight (MALDI-TOF) mass
spectrometry (Lasermat 2000; Finnigan-MAT, San Jose, CA). The control
RRREEETEEE peptide was purchased from Promega (Madison, WI). The in
vitro CKII-mediated phosphorylation of synthetic peptides was conducted
at 30°C for 10 min in a final volume of 40 µl that contained 20
µg peptides, 25 ng purified sea star CKII (Upstate Biotechnology,
Lake Placid, NY), 20 mM MOPS, pH 7.2, 25 mM ß-glycerol
phosphate, 5 mM EGTA, 1 mM orthovanadate, 1 mM DTT, 18.75 mM
MgCl2, 125 µM ATP, 4 mg/ml BSA, and 600 µM
[
-32P]ATP (7000 Ci/mmol; ICN, Costa Mesa, CA).
A 25-µl aliquot of the phosphorylation reaction was spotted on a
2-cm2 piece of P81 paper (Whatman, Clifton, NJ), washed
extensively with 0.75% phosphoric acid, once with acetone, and dried.
Scintillation mixture was then added (Optiphase HiSafe II; LKB
Instruments, Gaithersburg, MD) and assayed for radioactivity in a beta
counter (LKB).
Yeast two-hybrid library screening and direct interaction tests
The cDNA-encoding wild-type and mutant CD5 cytoplasmic domain were PCR amplified from the above-mentioned constructs and fused to the GAL4 DNA-binding domain by insertion into the SalI and NotI sites of pPC62 (29). A directionally cloned mouse B cell cDNA library was constructed with the ZAP-cDNA Synthesis kit (Stratagene, La Jolla, CA) from the IgG2a-expressing K46 murine B cell lymphoma (half of the cells resting and the other half stimulated with anti-Ig for various times) and cloned in pPC86 (29) as a fusion with the GAL4 activation domain. The cDNA library was cotransformed along with the wild-type human CD5 cytoplasmic tail as a bait into the yeast strain HF7c (30) using the lithium acetate procedure (31). Positive clones were isolated by growth on medium lacking histidine and tested for activity of the LacZ-reporter gene in filter assays (32). The pPC86 plasmids from His+LacZ+ clones were sequenced and retrotransformed alone and with pPC62 either empty or containing the cytoplasmic domain of human CD5, CD50, or CD148 lymphocyte surface receptors. For direct interaction tests, HF7c was cotransformed with CD5 cytoplasmic tail mutant constructs cloned into pPC62 and the CKIIß full-length and deletion mutants cloned in pPC86. Transformants were then handled as described above.
Coprecipitation of in vitro translated CKIIß with GST-CD5 fusion protein
The cytoplasmic domain of CD5 (from H424 to L471) was amplified by PCR and cloned into the SalI-NotI sites of pGEX-4T (Pharmacia) to generate a glutathione S-transferase (GST) fusion protein (GST-CD5). Expression of GST fusion proteins and their immobilization to glutathione-Sepharose 4B beads were conducted following manufacturer’s instructions. The amount of fusion protein was estimated by Coomassie blue staining of SDS-PAGE gels. The TNT T7 quick-coupled transcription/translation system (Promega) was used for in vitro translation of 35S-labeled full-length CKIIß. For that purpose, we used a PCR product resulting from the amplification of pPC86-CKIIß with the sense 5'-AATTAATACGACTCACTATAGGGAGCCACCATGAGTAGCTCTGAGGAGGT-3' and antisense 5'-GAGCTCGACGTCTTACTTACTTAGC-3' oligonucleotides. The translated protein was incubated overnight with immobilized GST alone or GST-CD5. Bound protein was separated by SDS/12% PAGE, and the gel was dried and autoradiographed.
TLC analysis of membrane lipids
Jurkat cells were isotopically labeled overnight by incubation in HG buffer (137 mM NaCl, 2.7 mM KCl, 1 mM Na2HPO4, 2.5 mM glucose, 20 mM HEPES, 0.1% BSA, 1 mM MgCl2, and 1 mM CaCl2, pH 7.4) with [3H]palmitic acid, at 2–4 µCi/ml. After two HG washes, aliquots of 2 x 106 cells were resuspended in 0.5 ml HG buffer and stimulated for the indicated times. Cells were sedimented at 4°C (10,000 x g for 10 s) at the end of the incubation period, and lipids were extracted from the cell pellets, as previously described (33). DAG was separated from triglycerides, cholesterol esters, and phospholipids on LK6D Silicagel plates (Whatman) with n-hexane/diethylether/formic acid (80:20:3). Standards were visualized by iodine staining. Radioactive measurements were performed on an automatic linear thin-layer radiochromatography scanner (Berthold, Nashua, NH).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As a first approach to the mapping of the CD5
phosphorylation sites, we generated a series of cytoplasmic tail
mutants lacking either 23 (CD5.H449stop), 54
(CD5.E418stop), or 88 (CD5.K384stop) residues
from its C terminus, as well as 27 residues (CD5.E418-L444) from the
central cytoplasmic region (Fig. 1
).
These mutant molecules, as well as the wild type (CD5.WT), were
transfected into the previously reported CD5- Jurkat 2G5
cell clone (12). 32P-labeled 2G5 transfectants were
subjected to phosphorylation analysis under basal conditions or
following stimulation with PMA (50 ng/ml, 30 min), a potent PKC
activator known to hyperphosphorylate CD5 (22). CD5 was
immunoprecipitated from cell lysates, resolved by SDS-PAGE, and
autoradiographed to show relative phosphorylation level. As expected,
CD5.WT was constitutively phosphorylated and hyperphosphorylated after
PMA stimulation (Fig. 2). This
hyperphosphorylation brings about the appearance of slow-migrating CD5
forms (22). On the contrary, CD5.K384stop, which is devoid
of most of the cytoplasmic tail, was not phosphorylated in either
unstimulated or PMA-stimulated cells. Interestingly,
CD5.H449stop was hyperphosphorylated following PMA
stimulation, but showed a significant drop in the phosphorylation level
in unstimulated cells with respect to CD5.WT. This indicates that a
major constitutive phosphorylation site(s) resides on the C-terminal
region and that the inducible phosphorylation site(s) resides N
terminal to H449. The low remaining constitutive phosphorylation signal
in CD5.H449stop reflects the partially activated state of
Jurkat 2G5 cells, as this band corresponds in m.w. to the
hyperphosphorylated form (22).
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E418-L444 mutants (Fig. 2
).
CD5.E418stop showed complete abrogation of phosphorylation
in unstimulated cells and a strong reduction in the level of
PMA-induced hyperphosphorylation. Additionally, CD5.E418-L444 was
phosphorylated in unstimulated cells and showed PMA-stimulated
hyperphosphorylation. This confirmed the existence of a major
constitutive phosphorylation site(s) at the C terminus of CD5. All of
the above-mentioned cytoplasmic tail mutants were also stably
transfected on murine NSO myeloma cells and 9.4 hybridoma T cells, and
we obtained identical results to those reported for human Jurkat 2G5
cells (data not shown). This implies a highly conserved kinase(s) in
the constitutive (and PMA-inducible) phosphorylation of CD5. A C-terminal serine cluster is the substrate for CKII and accounts for the constitutive phosphorylation of CD5
The C-terminal region deleted in CD5.H449stop contains
consensus sites for CKII at positions S459 and S461
(S/T-(X)2-D/E) and at position S458
(S-(X)2-S(P)) (24), the latter being only targetable after
phosphorylation of S461. In addition to these, two serines (S451 and
S452) and one tyrosine (Y463) are also present, which do not conform to
any known consensus kinase site. To explore the possibility that CKII
accounts for constitutive CD5 phosphorylation, we performed in vivo
CKII inhibition experiments using heparin, a potent in vitro CKII
inhibitor (34). High doses (5–50 mM) of low m.w. heparin (3000 mw)
were used to ensure heparin entry into cells. Under these conditions,
we observed a dose-dependent inhibition of the in vivo constitutive CD5
phosphorylation (Fig. 3A),
which supports involvement of CKII in the constitutive phosphorylation
of CD5. The specificity of the heparin effects was controlled by
analyzing the phosphorylation of CD5.E418stop, which
contains PKC but not CKII consensus sites. As shown by Fig. 3B, the PMA-induced phosphorylation of
CD5.E418stop was resistant to heparin, but not to the
potent PKC-inhibitor staurosporine.
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). Peptides reproducing two contiguous
and partly overlapping serine-rich sequences within the C terminus of
CD5 (from S451 to D464) were assayed in vitro as phosphorylation
substrates for purified CKII. Our results showed that, while the
SSMQPDNSS peptide was not phosphorylated above background (CKII
peptide), the DNSSDSDYD peptide showed a 300- to 400-fold increase over
background. The positive control EEETEEE and DDDSDDD peptides resulted
in 100- to 150-fold increase in radioactivity incorporation over
background. Interestingly, the DNSSDSDYD peptide showed two- to
threefold higher phosphorylation levels compared with DDDSDDD,
indicating the existence of two or more phosphorylatable residues. To
prove the specificity of our in vitro CKII assay, as well as to confirm
that all three serines present in the DNSSDSDYD peptide are CKII
substrates, we also assayed peptide variants containing SA
substitutions. The substitution of all three or the two most C-terminal
serines (DNAADADYD and
DNSADADYD, respectively) completely abrogated
the incorporation of radioactivity into peptides, indicating that S459
and S461 are major substrates for CKII. Mutation of S461 alone
(DNSSDADYD) confirmed these results by reducing the
signal down to levels similar to positive control peptides. Replacement
of only S459 (DNSADSDYD) gave an optimal signal compatible
with S458 also being a good CKII substrate. The increased signal level
compared with the wild-type peptide (DNSSDSDYD) may reflect differences
in the Vmax/Km of the peptides used. The fact that
DNSADADYD was not a good substrate suggests
that S458 phosphorylation requires prior phosphorylation of S461.
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A
and S461G substitutions (CD5.S459A,S461G) (Fig. 1). These
substitutions result in disruption of the CKII consensus sites not only
at S459 and S461, but also at S458. The in vivo phosphorylation
analysis of CD5.S459,S461G showed almost complete abrogation of the
constitutive phosphorylation, similar to that of
CD5.H449stop (Fig. 5). Thus,
by introducing two single amino acid substitutions (disrupting three
CKII consensus sites of CD5), we obtained the same effects as deleting
the 23 C-terminal residues. This confirms that the major constitutive
phosphorylation site is formed by the cluster of three C-terminal
serines matching CKII consensus sites (S458, S459, and S461), and that
S451 and S452 (and other serines in the cytoplasmic domain) do not
contribute to constitutive phosphorylation of CD5.
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To identify proteins that interact with the cytoplasmic domain of
human CD5, we employed the two-hybrid cloning procedure. The complete
CD5 cytoplasmic region fused to the GAL4 DNA-binding domain was used as
a bait to screen a mouse B cell cDNA library in which the cDNA was
fused to the GAL4 transcriptional activation domain. From the
106 cotransformants screened, seven clones showed the
His+LacZ+ phenotype. The sequence analysis of
one of these clones revealed that it contained a 0.8-kb insert encoding
for full-length murine CKII ß subunit cDNA, which is 100% identical
to its human counterpart, at protein level. That clone interacted
specifically with the cytoplasmic domain of CD5, since no interaction
was detected with the GAL4 DNA-binding domain alone or fused to
unrelated proteins (the cytoplasmic domains from CD50/ICAM-3 and
CD148/HPTP
). This ruled out the possibility that CKIIß contains
latent transcriptional activity or that it interacts nonspecifically
with other proteins.
Additional validation of the CD5/CKIIß interaction was achieved by in
vitro coprecipitation experiments. The CKIIß subunit was translated
in vitro by using a reticulocyte lysate system and then incubated with
equivalent amounts of immobilized GST or GST-CD5 fusion protein (as
tested by Coomassie blue staining of SDS-PAGE gels). As shown in Fig. 6, the GST-CD5 fusion protein, composed
of the C-terminal half (47 amino acids) of the CD5 cytoplasmic region,
bound significant amounts of CKIIß compared with the GST control
protein (10-fold greater). This confirmed the yeast two-hybrid results,
i.e., that CKIIß specifically associates with the CD5 cytoplasmic
region.
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). None
of the CKIIß deletion mutants analyzed (CKIIß.R150stop
and CKIIß.E73stop) revealed interaction with full-length
CD5 cytoplasmic tail (Fig. 7B), suggesting that the most
C-terminal region of CKIIß is involved in the binding to CD5.
Full-length CKIIß interacted strongly with the deletion mutant
CD5.E418-L444, yet complete abrogation of the interaction was
achieved by the CD5.E418stop and CD5.L444stop
truncations (Fig. 7A), indicating the relevance of the
C-terminal region of CD5 for CKIIß binding. To narrow down this
region, we examined additional constructions. The
CD5.H449stop truncation led to a significant but incomplete
abrogation of the interaction, which was not improved by elongation to
S457 (see CD5.S458stop results, Fig. 7A). A
similar attenuated interaction was observed with CD5.S459A,S461G and
CD5.S459A,460-L465 mutants (Fig. 7A). The single Y463A
substitution (CD5.Y463A) had no effect on the interaction (Fig. 7A). These results indicate that the extreme C-terminal
domain of CD5 (from L448 to L471), encompassing the constitutive
phosphorylation sites, is essential for CKII binding, and the CKII
phosphorylation sites (S459 and S461) are both relevant, but not
essential for the binding of CKIIß to CD5.
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We have reported recently that the C-terminal cytoplasmic
region of CD5 (from H449 to L471) harbors key elements for the
activation of a PC-PLC (12). Therefore, we performed structure-function
analyses involving the C-terminal CKII phosphorylation sites. Wild-type
and cytoplasmic tail mutants (CD5.H449stop,
CD5.S459A,S461G, and CD5.Y463A) stably expressed on 2G5 cells were
assayed for their ability to mediate PC-PLC-mediated DAG release
following stimulation with anti-CD5 mAbs. The transfectants used in
this study showed similar CD3 and CD5 cell surface expression levels
and similar CD3-mediated Ca2+ mobilization responses. As
shown in Fig. 8, CD5.WT elicited a rapid
(peaked at 2 min) and transient (declined to basal levels at 5 min) DAG
release. The CD5.S459A,S461G mutant was defective in signaling PC-PLC
activation, in a way similar to that found for the
CD5.H449stop. In contrast, CD5.Y463A retained its ability
to signal PC-PLC activity. These findings illustrate the functional
relevance of constitutive CKII-mediated phosphorylation of CD5 to
PC-PLC activation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(37). The second, identified as CKII, associates with the
C-terminal region and is responsible for constitutive serine
phosphorylation of CD5. Importantly, preliminary evidence also
indicates that this constitutive phosphorylation may be a critical
element for the generation of lipid second messengers through CD5.
CKII is a ubiquitous, pleiotropic, and highly conserved
protein-serine/threonine kinase that is central to cell regulation
(34). CKII exists as a tetramer (
2ß2),
with catalytic subunits and regulatory ß subunits. CKII
phosphorylates and regulates the function of several important cellular
proteins, including transcription factors (38, 39, 40, 41), cytoskeletal
proteins (42, 43), and membrane receptors (44, 45). In this study, we
demonstrate that the major constitutive phosphorylation site of CD5 is
formed by a cluster of serine residues at its C terminus that represent
classic CKII consensus phosphorylation motifs and can be phosphorylated
in vitro by CKII (S458, S459, and S461). In addition, constitutive
phosphorylation of wild-type CD5 was abolished by the CKII inhibitor
heparin. These results strongly argue that CKII (or a CKII-like kinase)
is involved in the constitutive phosphorylation of CD5. The
identification of the C-terminal serines as the constitutive
phosphorylation sites is supported by the observations that the
C-terminal cytoplasmic deletion, from H449 to L471, abrogates the CD5
constitutive phosphorylation and identical results are obtained by
simultaneous substitution of S459A and S461G. The fact that CKII
is highly active in the absence of a second messenger argues for a role
for CKII in CD5 constitutive phosphorylation. The yeast two-hybrid
system approach demonstrates that the specific association requires the
extreme C-terminal domain of CD5 (residues L444 to L471). This
interaction was further confirmed by precipitation of in vitro
translated CKIIß subunit by a GST-CD5 fusion protein. The CD5
C-terminal serines targeted by CKII are relevant, but not essential for
CKIIß binding, in a similar way to that described for CKIIß
interaction with the growth suppressor protein p53 (46). Interestingly,
all of the CD5 residues involved in the CKIIß interaction (L444 to
L471) are highly conserved among mammalian (37) and nonmammalian (47)
species and are encoded by a single exon (exon 10, our unpublished
data), thus arguing for a relevant functional role for this region. At
present, we have been unable to unequivocally show that the majority of
CD5 surface molecules on T cells stably associate with heterotetrameric
active CKII. This is not surprising, since the majority of cellular
CKII localizes to the nucleus. Nevertheless, in-gel kinase assays using
either dephosphorylated -casein or phosvitin as CKII substrates show
a minor increase in the phosphorylation of 44–42-kDa bands (catalytic
CKII,' subunits) associated to CD5 immunoprecipitates with
respect to controls (data not shown). This experiment has been
reproduced several times. Similarly, Western blot analysis did not
reveal significantly higher amounts of CKII subunit associated with
CD5 immunoprecipitates. Therefore, it is possible that the interaction
is an artifact of the yeast two-hybrid and the in vitro translation
systems, in which both proteins are overexpressed. Alternatively, a
rather transient association of CKII with CD5 is also possible. This
transient association could take place either during CD5 biosynthesis
(by rough endoplasmic reticulum-resident CKII) (45) or once exported to
the membrane.
Constitutive phosphorylation of CD5 also seems to be of functional
relevance to cell signaling. We have reported previously that the
C-terminal region, including the CKII sites (from H449 to L471), is
important in CD5-mediated PC-PLC activation (12). This work shows that
a S459A and S461G double substitution abrogates CD5 constitutive
CKII-mediated phosphorylation and renders the molecule defective in
signaling PC-PLC activation. This abrogation is selective, since CD5
molecules lacking the C-terminal cytoplasmic region induce tyrosine
phosphorylation of intracellular proteins (our unpublished data). This
selectivity is also seen by a Y463A substitution that has no such
deleterious effect on CD5-mediated PC-PLC activation. Recent data from
our laboratory indicate that acidic sphingomyelinase (A-SMase)
activation is secondary to CD5-mediated PC-PLC activation and fully
confirm the above-mentioned results (Simarro et al., in preparation).
These data show the harmlessness of Y463 substitution as well as the
deleterious effect of S459 and S461 substitutions on A-SMase
activation. Interestingly, our data, together with the reported failure
to activate phosphoinositide 3-kinase with anti-CD5 mAbs in T cells
(48), disagree with the reported binding of phosphoinositide 3-kinase
to a tyrosine-phosphorylated CD5 peptide (SDSDYDLHGA) containing two of
the serine residues involved in the constitutive phosphorylation of CD5
(49). The conflicting results warrant reexploring the binding
properties of the peptide SDSDYDLHGA in its serine-phosphorylated form,
which would more accurately represent the situation in vivo.
In conclusion, this study shows that a highly conserved C-terminal cytoplasmic region of CD5 associates with and is a substrate for CKII. The identification of the serine residues involved in the constitutive phosphorylation of CD5 as presumably relevant to the generation of lipid second messengers provides a significant advance in our understanding of the CD5 signal-transduction pathway. The molecular interactions linking constitutively phosphorylated CD5 with the PC-PLC/A-SMase pathway (either directly or indirectly) still await further elucidation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Francisco Lozano, Servei d’Immunologia, Hospital Clínic, Villarroel 170, 08036 Barcelona, Spain. E-mail address:
3 Abbreviations used in this paper: PKC, protein kinase C; A-SMase, acidic sphingomyelinase; CKII, casein kinase II; DAG, diacylglycerol; GST, glutathione S-transferase; PC-PLC, phosphatidylcholine-specific phospholipase C.
Received for publication March 16, 1998. Accepted for publication August 7, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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chain. J. Exp. Med. 177:1093. chain/CD3 and protein-tyrosine kinases p56lck and p59fyn. Proc. Natl. Acad. Sci. USA 89:9311. molecules in human CD3+ thymocytes. J. Immunol. 159:3739.[Abstract]
. Eur. J. Immunol. 28:2167.[Medline]
Bß regulates its functional interaction with the c-rel proto-oncogene product. Mol. Cell. Biol. 16:5974.[Abstract]
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H. Gary-Gouy, A. Sainz-Perez, J.-B. Marteau, A. Marfaing-Koka, J. Delic, H. Merle-Beral, P. Galanaud, and A. Dalloul Natural Phosphorylation of CD5 in Chronic Lymphocytic Leukemia B Cells and Analysis of CD5-Regulated Genes in a B Cell Line Suggest a Role for CD5 in Malignant Phenotype J. Immunol., October 1, 2007; 179(7): 4335 - 4344. [Abstract] [Full Text] [PDF]
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R. C. Axtell, L. Xu, S. R. Barnum, and C. Raman CD5-CK2 Binding/Activation-Deficient Mice Are Resistant to Experimental Autoimmune Encephalomyelitis: Protection Is Associated with Diminished Populations of IL-17-Expressing T Cells in the Central Nervous System J. Immunol., December 15, 2006; 177(12): 8542 - 8549. [Abstract] [Full Text] [PDF]
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I. Gimferrer, A. Ibanez, M. Farnos, M.-R. Sarrias, R. Fenutria, S. Rosello, P. Zimmermann, G. David, J. Vives, C. Serra-Pages, et al. The Lymphocyte Receptor CD6 Interacts with Syntenin-1, a Scaffolding Protein Containing PDZ Domains J. Immunol., August 1, 2005; 175(3): 1406 - 1414. [Abstract] [Full Text] [PDF]
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M. Arman, J. Calvo, M. E. Trojanowska, P. N. Cockerill, M. Santana, M. Lopez-Cabrera, J. Vives, and F. Lozano Transcriptional Regulation of Human CD5: Important Role of Ets Transcription Factors in CD5 Expression in T Cells J. Immunol., June 15, 2004; 172(12): 7519 - 7529. [Abstract] [Full Text] [PDF]
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 A. Mizoguchi, E. Mizoguchi, Y. P. d. Jong, H. Takedatsu, F. I. Preffer, C. Terhorst, and A. K. Bhan Role of the CD5 molecule on TCR {gamma}{delta} T cell-mediated immune functions: development of germinal centers and chronic intestinal inflammation Int. Immunol., January 1, 2003; 15(1): 97 - 108. [Abstract] [Full Text] [PDF]
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X. Lu, R. C. Axtell, J. F. Collawn, A. Gibson, L. B. Justement, and C. Raman AP2 Adaptor Complex-Dependent Internalization of CD5: Differential Regulation in T and B Cells J. Immunol., June 1, 2002; 168(11): 5612 - 5620. [Abstract] [Full Text] [PDF]
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K. M. Dennehy, W. F. Ferris, H. Veenstra, L. A. Zuckerman, N. Killeen, and A. D. Beyers Determination of the tyrosine phosphorylation sites in the T cell transmembrane glycoprotein CD5 Int. Immunol., February 1, 2001; 13(2): 149 - 156. [Abstract] [Full Text] [PDF]
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J. M. Vila, J. Calvo, L. Places, O. Padilla, M. Arman, I. Gimferrer, C. Aussel, J. Vives, and F. Lozano Role of Two Conserved Cytoplasmic Threonine Residues (T410 and T412) in CD5 Signaling J. Immunol., January 1, 2001; 166(1): 396 - 402. [Abstract] [Full Text] [PDF]
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 H. Gary-Gouy, P. Bruhns, C. Schmitt, A. Dalloul, M. Daeron, and G. Bismuth The Pseudo-immunoreceptor Tyrosine-based Activation Motif of CD5 Mediates Its Inhibitory Action on B-cell Receptor Signaling J. Biol. Chem., January 7, 2000; 275(1): 548 - 556. [Abstract] [Full Text] [PDF]
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C. Pena-Rossi, L. A. Zuckerman, J. Strong, J. Kwan, W. Ferris, S. Chan, A. Tarakhovsky, A. D. Beyers, and N. Killeen Negative Regulation of CD4 Lineage Development and Responses by CD5 J. Immunol., December 15, 1999; 163(12): 6494 - 6501. [Abstract] [Full Text] [PDF]
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M. Simarro, J. Calvo, J. M. Vila, L. Places, O. Padilla, J. Alberola-Ila, J. Vives, and F. Lozano Signaling Through CD5 Involves Acidic Sphingomyelinase, Protein Kinase C-{zeta}, Mitogen-Activated Protein Kinase Kinase, and c-Jun NH2-Terminal Kinase J. Immunol., May 1, 1999; 162(9): 5149 - 5155. [Abstract] [Full Text] [PDF]
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) or
stimulated with 10 µg/ml of the anti-CD5 F145GF3 mAb (
) for
the indicated times. Following cell extraction, phospholipids were
analyzed by TLC, and the radioactivity incorporated into DAG was
measured. Values are expressed as cpm ± SD.