From the Division of Clinical Immunology and
Rheumatology, Department of Medicine, University of Alabama at
Birmingham, Birmingham, Alabama 35294 and the
Department of
Biochemistry, University of Western Ontario, London, Ontario
N6A 5C1, Canada
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ABSTRACT |
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The transmembrane protein CD5, expressed on all T
cells and the B1 subset of B cells, modulates antigen receptor-mediated activation. We used the yeast two-hybrid system to identify proteins that interact with its cytoplasmic domain and play a role in CD5 proximal signaling events. We found that the subunit of the serine/threonine kinase casein kinase 2 (CK2) interacts specifically with the cytoplasmic domain of CD5. Co-immunoprecipitation experiments showed activation-independent association of CK2 with CD5 in human and
murine B and T cell lines and murine splenocytes. The interaction of
CK2 holoenzyme with CD5 is mediated by the amino terminus of the
regulatory subunit
. CK2 binds and phosphorylates CD5 at the CK2
motifs flanked by Ser459 and Ser461.
Cross-linking of CD5 leads to the activation of CD5-associated CK2 in a
murine B-lymphoma cell line and a human T-leukemia cell line and is
independent of net recruitment of CK2 to CD5. In contrast, CK2 is not
activated following cross-linking of the B cell receptor complex or the
T cell receptor complex. This direct regulation of CK2 by a cell
surface receptor provides a novel pathway for control of cell
activation that could play a significant role in regulation of
CD5-dependent antigen receptor activation in T and B cells.
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INTRODUCTION |
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CD5 is a 67-kDa glycoprotein that belongs to the cysteine-rich macrophage scavenger receptor family of proteins expressed on all thymocytes and T cells and a subset of B cells, described as B1a B cells (CD5 B cells) (1-4). It is expressed on T cells very early in development, before the expression of the TCR-CD31 complex, and during progressive stages of thymocyte development, the level of CD5 expression increases, suggesting a role in thymocyte biology (5). In cells of B-lineage, the onset of CD5 expression is not well defined, but it is expressed in all Ableson transformed lines, which represent the pre-B stage (6, 7). Proposed counter-receptors for CD5 include the B cell-specific CD72, gp35-37, which is expressed on activated splenocytes and activated T cell clones, and the Ig VH framework region (8-11). The functional significance of these candidates in context with CD5 activation has not been been established.
CD5 is physically associated with the antigen receptor complex in both T and B cells and modulates intracellular signals initiated by both TCR and BCR (12-14). The conserved cytoplasmic domain of CD5 contains four tyrosines and several sites for serine and threonine phosphorylation (15-20). Two of the tyrosines form an imperfect immunoreceptor tyrosine activation motif (21, 22). The serine/threonine sites include four CK2-dependent serine phosphorylation sites and a protein kinase C-dependent threonine phosphorylation site. TCR cross-linking leads to rapid tyrosine phosphorylation followed by serine/threonine phosphorylation of CD5 (14, 23, 24). In contrast, CD5 ligation leads to tyrosine kinase activation and tyrosine phosphorylation of several substrates but only to serine phosphorylation of its own cytoplasmic domain (25, 26). CD5 can associate with p56lck and Zap70, but it is unknown if these tyrosine kinases are involved in tyrosine phosphorylation of CD5 in cells (27, 28).
Mitogenic CD5 antibodies cooperate with antibodies to CD28 to induce
proliferation in mature T cells in the absence of TCR-CD3 stimulation
(29-31). CD5 ligation synergizes with CD3 stimulation to increase
intracellular calcium, interleukin-2 secretion, and interleukin-2
receptor expression (32-35) and is involved in both TCR-dependent and -independent activation of diacylglycerol
production (36). These results suggest that in mature T cells, CD5
functions as a co-stimulatory molecule of T cell activation. In
contrast, CD5 appears to attenuate TCR-CD3-induced signals in
thymocytes (37). Single positive thymocytes from CD5-deficient mice
exhibit enhanced proliferation to TCR-CD3-induced signals, with
hyperphosphorylation of Vav and phospholipase C-, and enhanced
intracellular calcium mobilization. In mature B1a B cells, CD5 appears
to function as a negative regulator of BCR-induced signals (38). The
basis of these opposing effects of CD5 signaling in immature and in mature thymocytes is unclear.
To define the molecules that may interact with CD5 and play a role in
CD5-proximal signaling, we used the yeast two-hybrid system. The entire
94-amino acid cytoplasmic domain of human CD5, Y378-L471, was fused to
the GAL4 binding domain (BD) and used as a "bait" to screen an GAL4
activation domain (AD) cDNA library prepared from Epstein-Barr
virus-transformed human peripheral blood lymphocytes. Using this
approach, we show that casein kinase 2 (CK2), a serine/threonine
kinase, interacts specifically with the cytoplasmic domain of CD5. The
interaction of CK2 with CD5 is constitutive in human and mouse cell
lines and murine splenocytes and is mediated by the regulatory subunit of the tetrameric CK2 holoenzyme. We have mapped the CK2
binding and phosphorylation sites on CD5 to the two carboxyl-terminal
CK2 motifs and have demonstrated CD5-dependent activation
of CK2 in both B and T cell lines. This recruitment of a novel
signaling pathway by CD5 is likely to have significant implications for
CD5-dependent regulation of TCR- and BCR-induced
activation.
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EXPERIMENTAL PROCEDURES |
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Cell Lines, Tissue, and Reagents-- Murine B-lymphoma cell lines CH12 (gift from Dr. John. F. Kearney) and NYC31 (gift from Dr. Hans-Martin Jäck), and the human T-leukemia cell line Jurkat (gift from Dr. Louis B. Justement) were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum (Life Technologies, Inc.). Murine spleens were obtained from 6-8-week-old Balb/c mice (The Jackson Laboratory, Bar Harbor, ME). Anti-mouse CD5 mAb (clone 53-7.3), anti-human CD3 (clone UCHT1), and anti-human CD19 (clone HIB19) were obtained from Pharmingen (San Diego, CA). Polyclonal anti-CD5 rabbit serum to the conserved peptide sequence, TASHVDNEYSQPPR, in the CD5 cytoplasmic domain was a gift from Drs. Greg Appleyard and Bruce Wilkie (39). Goat anti-rabbit µ, F(ab')2 fraction, and peroxidase-conjugated goat anti-rabbit IgG were from Jackson ImmunoResearch Laboratories (West Grove, PA). Protein A-agarose and protein G-agarose were obtained from Life Technologies, Inc., and SuperSignal chemiluminescence substrate was obtained from Pierce. Anti-mouse CD5 was conjugated to agarose using the EDC kit from Pierce. The CD5-derived peptide DNSSDSYDLHGAQRL, containing the CD5 cytoplasmic domain residues 456-471, was obtained from Bio Synthesis (Lewisville, TX), and the standard CK2 substrate peptide RRREEETEEE was obtained from Research Genetics (Huntsville, AL). Purified CK2 was obtained from Boehringer Mannheim. Sepharose 4B was purchased from Amersham Pharmacia Biotech.
Yeast Two-hybrid Screen-- To generate the GAL4 binding domain-CD5 cytoplasmic domain (BD-CD5) fusion, we amplified by polymerase chain reaction the cDNA representing the 94-amino acid cytoplasmic domain (Tyr378-Leu471) from the CD5 cDNA clone, pT2-2 (15) using sense 5'-GCGTCGGACCCTACAAGAAGTAGTGAAG and antisense 5'-AACTGCAGGGGCGGCCGAGCTGTTGTG-3' primers and cloned the product into the pGBT9 vector (CLONTECH). After determining the accuracy of the nucleotide sequence by fluorescent dye terminator sequencing (ABI, Foster City, CA), the construct was transformed into the HF7c yeast strain as suggested by the manufacturer (CLONTECH MatchmakerTM). The BD-CD5 was screened for nonspecific activation of the GAL4 promoter directly and in association with a co-transformed irrelevant AD-cDNA construct provided. The BD-CD5 construct was used to screen an AD-cDNA library made with mRNA from Epstein-Barr virus-transformed pooled human peripheral blood lymphocytes in the pACT AD vector (40). Colonies positive for growth on histidine-deficient plates were screened for LacZ expression using the filter assay on Whatman 5 filters.
Mapping Studies and Constructs--
The generation of AD-CK2,
AD-CK2
', AD-CK2
2-215, AD-CK2
2-132, and AD-CK2
133-215
constructs has been described previously (41, 42). To generate BD-CD5
deletion mutants, we used the Seamless Cloning kit from Stratagene.
This system makes use of the type II restriction enzyme EamI
to generate restriction-site independent deletions. The six
primers used to generate all the deletions are as follows: 1)
5'-TCCTCTTCTGACAACCCCACAGCCTCC-3', 2)
5'-AGCTCTTCAGTCTCGGACGGTTGCCGT-3', 3)
5'-TCCTCTTCGGACAACGAATACAGCCAA-3', 4)
5'-CACTCTTCAGTCGGCTGTGGGGTTCTC-3', 5)
5'-TCCTCTTCTGACTATGATCTGCATGGG-3', and 6)
5'-GTCTCTTCAGTCGTTGTCAGGCTGCAT-3'. The BD-CD5
415-417 construct was
generated using primers 1 and 2, the BD-CD5
423-425 construct used
primers 3 and 4, the BD-CD5
415-425 construct used primers 2 and 3, the BD-CD5
415-461 construct used primers 2 and 5, the BD-CD5
425-461 construct used primers 4 and 5, and the
BD-CD5
458-461 construct used primers 5 and 6. Site-specific
mutagenesis for constructing S459G and S461G single and double mutants
of BD-CD5 was performed using the QuikChange mutagenesis kit from
Stratagene. The primers used were 5'-CAGCCTGACAACTCCGGCGACAGTGACTAT-3'
(sense) and 5'-ATAGTCACTGTCGCCGGAGTTGTCAGGCTG-3' (antisense) for the
S459G mutant, 5'-GACAACTCCTCCGACGGTGACTATGATCTG-3' (sense) and
5'-CAGATCATAGTCACCGTCGGAGGAGTTGTC-3' (antisense) for the S461G mutant,
and 5'- CCTGACAACTCCGGCGACGGTGACTATGATCTG-3' (sense) and
5'-CAGATCATAGTCACCGTCGCCGGAGTTGTCAGGCTG-3' (antisense) for the
S459G,S461G double mutant. To generate the pThioHis-CD5 fusion protein,
the CD5 cytoplasmic domain cDNA was amplified using sense primer
5'-ATCGAATTCTACAAGAAGCTAGTGAAG-3' and the BD-CD5 antisense primer
and cloned in frame into the pThioHisA vector (Invitrogen, Carlsbad,
CA). Site-specific mutants S459A,S461A (single and double mutants) were
constructed as described above for BD-CD5 Ser
Gly mutants except
that the codons were changed to reflect Ser
Ala mutations. The
absence of polymerase chain reaction-introduced artifacts and the
presence of desired nucleotide changes were established by
bidirectional nucleotide sequencing using dye terminator chemistry.
Immunoprecipitation and Western Blot Analysis--
Cells (2 × 107) were lysed in 0.5 ml of lysis buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% v/v
Nonidet P-40, and protease and phosphatase inhibitors (44). The
cellular debris was removed by centrifugation, and the lysate was
precleared with protein A-agarose and Sepharose 4B. Lysates prepared
from CH12, NYC31, or spleen cells were immunoprecipitated with agarose
conjugated anti-mouse CD5 or agarose-conjugated rat IgG2a, and lysates
from Jurkat cells were incubated with anti-human CD5 or anti-mouse CD19
(IgG1 isotype control) followed by precipitation with protein G-agarose. The immunoprecipitates were analyzed by Western blot analysis using rabbit antiserum to CK2 followed by
peroxidase-conjugated goat anti-rabbit IgG and SuperSignal
chemiluminescence substrate.
In Vitro Kinase Assay--
Ten µg of the CK2 standard
substrate peptide RRREEETEEE (45) or synthetic CD5-derived peptide were
incubated in 25 µl of kinase buffer (100 mM Tris-HCl, pH
8.0, 100 mM NaCl, 50 mM KCl, 20 mM
MgCl2, and 100 µM sodium orthovanadate). The
reaction was initiated by addition of 10 µCi of
[-32P]ATP (Amersham Pharmacia Biotech) and 5U CK2
(Boehringer Mannheim) and incubated at 37 °C for 10 min. The
reaction mixture was applied to a P-81 filter disc (Whatman) and washed
extensively with 75 mM phosphoric acid, and radioactivity
was determined in a liquid scintillation counter (Beckman). For some
experiments, pThioHisCD5 fusion proteins were used as the substrate in
the in vitro kinase assay.
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RESULTS |
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CK2 Associates with CD5 Cytoplasmic Domain--
We used the
yeast two-hybrid system to identify proteins that interact with the
cytoplasmic domain of CD5. We fused the entire 94 amino acid
cytoplasmic domain of human CD5 to the GAL4 BD to generate BD-CD5 and
used it as a bait to screen an AD-cDNA library made from human
peripheral blood lymphocytes. From a screen of 6 × 106 co-transformants, we obtained 536 yeast colonies that
grew on histidine-deficient plates, of which 510 were positive for LacZ expression by filter assay. We determined the nucleotide sequence of 10 randomly selected AD-cDNA isolated from
His+LacZ+ yeast colonies, and a BLAST analysis
showed that 6 of the 10 were identical to the
subunit of human CK2
(Fig. 1). In each of the six AD-CK2
clones, the in frame fusion with the GAL4-AD occurred in the
5'-untranslated (UT) region, and five of these six clearly represented
independent clones because they had different lengths of 5'-UT amino
acids. The interaction of AD-CK2
was specific to CD5 cytoplasmic
domain as BD-lamin C or BD alone did not interact with CK2
(Table
I). A polymerase chain reaction-based
assay using primers that specifically amplify a 300-base pair fragment within the coding region of CK2
and performed directly on yeasts derived from growth-positive yeast colonies revealed that of the remaining 500 colonies, 245 (48%) were AD-CK2
.
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Interaction of CK2 with CD5 in Mammalian Cells--
To determine
whether CK2 associates with CD5 in mammalian cells, we performed
co-immunoprecipitation experiments. 1% Nonidet P-40 lysates from
2 × 107 murine B-lymphoma cell lines CH12 and NYC31,
murine splenocytes, or the Jurkat human T cell line were
immunoprecipitated with anti-CD5 or control mAb. Western blots of these
immunoprecipitates probed anti-CK2 antibody showed that CK2
co-immunoprecipitated readily with CD5 specifically in each of these
tissues (Fig. 2). As determined by
comparison to whole cell lysates, the amount of CK2 associated with CD5
was less than 1% of total CK2, a very abundant cellular kinase (data
not shown).
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The Interaction of CK2 with CD5 Is Mediated by the Subunit--
We did not isolate any clone in the yeast two-hybrid
screen that contained either of the two catalytic subunits of CK2,
, or
'. Due to the divergence of sequence homology in human and yeast
CK2
, it seemed unlikely that human CK2
could complex with yeast
CK2
to mediate the restoration of the GAL4 transcriptional activator. Therefore, the yeast two- hybrid screen suggested that CK2
may interact with CD5 via its regulatory
subunit. However, to
directly test whether the interaction of CK2 with CD5 is mediated by
its catalytic domains, we tested the ability of BD-CD5 to interact with
AD-CK2
or AD-CK2
' in the yeast two-hybrid assay along with the
AD-CK2
clone 15-15 obtained from our library screen. We found that
only co-transformants of BD-CD5 and AD-CK2
(clone 15-15) grew on
histidine-deficient plates and expressed LacZ. AD-CK2
and AD-CK2
'
did not interact with BD-CD5 (Table II).
The lack of interaction between BD-CD5 and either AD-CK2
or
AD-CK2
' is unlikely to be a construct artifact, because these
constructs have been shown previously to be functional and have the
capability to interact with CK2
(41, 42). Based on these data, we
conclude that the interaction of CK2 to CD5 is mediated by the
subunit.
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The Interaction of CK2 with CD5 Is Mediated by Its Amino
Terminus--
The observation that each of the six completely
sequenced AD-CK2
clones isolated from library were in frame fusions
at the 5'-UT region suggested to us that CK2
might be interacting
with CD5 via its amino terminus. To test this possibility directly, we
compared the ability of the full-length AD-CK2
clone 15-15, which
has an in frame fusion in the 5'-UT region, the full-length clone
AD-CK2
2-215, which lacks a "linker" region, and deletion constructs of AD-CK2
constructs to interact with BD-CD5 (Fig. 3). Yeast containing BD-CD5 and AD clone
15-15, AD-CK2
2-215, or AD-CK2
2-132, but not AD-CK2
133-215,
grew in the absence of histidine and expressed LacZ (Fig. 3).
Interestingly, the growth on histidine-deficient plates of yeast
containing BD-CD5 and AD clone 15-15 was most rapid. In the LacZ assay,
this co-transformant also developed the most intense blue color in the
shortest time (30 min versus 3 h) compared with
co-transformants containing AD-CK2
2-215 and AD-CK2
2-132. This
observation suggests that the "linker" contributed by the 5'-UT
region facilitated the interaction between AD-CK2
fusion and BD-CD5,
most probably by making the amino terminus more accessible. From these
results, we conclude that the amino terminus of CK2
mediates the
interaction with CD5 cytoplasmic tail.
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Mapping of CK2 Binding Site on CD5--
The cytoplasmic tail of
CD5 has four putative serine phosphorylation sites, Ser415,
Ser423, Ser459, and Ser461, all of
which have the consensus motif ((S/T)XX(D/E)) for
phosphorylation by CK2 (47, 48). To determine which of these motifs are
involved in interaction with CK2, we generated a panel of BD-CD5
deletion constructs in which we had deleted one or more of these motifs and tested for their ability to interact with AD-CK2
in the yeast two-hybrid assay (Fig. 4A). We
found that deletion of the motif at Ser415 and
Ser423 independently or together did not affect the ability
of CK2
to interact with CD5. In contrast, the interaction of CK2
with CD5 was completely absent in the three constructs that included Ser459 and Ser461 and the non-CK2 site
Ser458. These data show that the interaction of CK2
with
CD5 is limited to the two overlapping CK2 motifs that include
Ser459 and Ser461
(458SSDSDYD464).
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CK2 Phosphorylates CD5 at Ser459 and Ser461-- To determine whether CK2 can phosphorylate CD5, we performed an in vitro kinase assay with purified CK2 using a 16-amino acid synthetic CD5-peptide (456DNSSDSDYDLHGAQRL471) that included Ser459 and Ser461 CK2 motifs and compared its ability to be phosphorylated with CK2 standard substrate peptide (RRREEETEEE) (45). After normalizing for equivalent moles of peptide, we determined that the 32P incorporation in CD5-peptide was approximately 1.5-fold greater than CK2 control peptide (Fig. 5A). Because there is only one available phosphorylation site in the CK2 standard peptide, the greater phosphorylation may indicate that both the CK2 sites in CD5-peptide are phosphorylated.
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Cross-linking of CD5 Activates CK2-- We tested the possibility that CD5 may function as a regulator of CK2 activity because CK2 interacts with CD5 constitutively via its regulatory subunit in the absence of demonstrable phosphorylation. Using CK2 standard peptide as substrate, we determined the CK2 activity in CD5 immunoprecipitates from the B lymphoma line, CH12, following stimulation with anti-CD5 mAb or control antibody. We found that CD5-associated CK2 activity increased 9-fold following stimulation with anti-CD5 antibody compared with CK2 activity from control antibody-treated cells (Fig. 6A). The activation of CD5-associated CK2 was not due to net recruitment of CK2 to CD5 because the amount of CK2 protein co-immunoprecipitated was the same in anti-CD5 stimulated and control antibody-treated cells (Fig. 6B). The CK2 activity in control antibody immunoprecipitates was not altered by CD5 stimulation and did not differ from that in anti-CD5 immunoprecipitates of control antibody-treated cells (Fig. 6A). Activation of CK2 was not limited to B-lineage cells because CK2 activity in anti-CD5 immunoprecipitates from the human T-leukemia cell line Jurkat was increased by approximately 10-fold following cross-linking of CD5 compared with control antibody-treated cells (Fig. 6C). This effect in Jurkat cells was also not due to net recruitment of CK2 to CD5 (Fig. 6D).
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CK2 Activation Is Specific to CD5 Cross-linking--
CD5
associates with the antigen receptor in both T and B cells, and
therefore it is possible that the activation of CK2 is mediated by
associated BCR or TCR molecules. To determine whether the activation of
CK2 is specific to CD5 stimulation, CH12 cells were stimulated with
anti-CD5, anti-µ, or control antibody, and the CK2 activity was
determined in anti-CD5 immunoprecipitates. We observed that CK2
activity was enhanced only in immunoprecipitates from anti-CD5
stimulated cells (Fig. 8). The CK2
activity in anti-CD5 immunoprecipitates from anti-µ stimulated cells
was not different from control antibody treated cells. To determine
whether the lack of observable CK2 activation following anti-µ
stimulation can be explained by decreased association of CK2 with CD5,
we immunoprecipitated CD5 from anti-µ stimulated cells and compared the amount of co-immunoprecipitated CK2 with that co-immunoprecipitated with CD5 from anti-CD5 stimulated cells. The amount of CK2 associated with CD5 was same in anti-CD5 stimulated and anti-µ stimulated cells,
showing that the lack of CK2 activation following BCR cross-linking was
not due to net decrease in CK2 associated with CD5 (Fig.
6B). Similarly, TCR cross-linking of Jurkat cells with
anti-CD3 antibody did not activate CD5-associated CK2, whereas CD5
stimulation did (Fig. 8B). The lack of CK2 activation
following TCR cross-linking was also not due to change in net CK2
association with CD5 (Fig. 6D). Taken together, these data
show that the activation of CK2 is specific to CD5 stimulation.
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DISCUSSION |
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In this study, we have shown that the serine/threonine kinase CK2 interacts specifically with CD5. The direct association of CK2 with a cell surface receptor is particularly intriguing because this kinase is involved in regulating intermediate to distal events of signaling in the cytosol and nucleus (50, 51). This report, the first to demonstrate the localization of CK2 to the cell membrane in association with a cell surface receptor, suggests that CK2 may also play a role in the regulation of membrane proximal signaling events.
The holoenzyme CK2 consists of catalytic subunits and
' and a
regulatory subunit,
, in the tetrameric configuration
2
2,
'
2, or
'2
2 (51). The
and
' subunits are highly
homologous to each other but are products of different genes (50). The kinase is a major regulator of cell growth, cell division, and signal
transduction pathways, and the wide range of substrates phosphorylated
by CK2 includes transcription factors, protein synthesis factors,
nucleic acid synthesis proteins, polymerases, and signal transduction
proteins. The conservation of CK2 through phylogeny suggests that it is
a critical enzyme in cell regulation, and indeed, the disruption of the
catalytic subunits in Saccharomyces cerevisiae confers
lethality (52).
CK2 can interact with its substrates either in its holoenzyme
form nucleus (50, 51) or as individual subunits, as illustrated by the
interaction of CK2 with PP2A and the interaction of CK2
with the
serine/threonine kinase Mos (53, 54). Given the association of CK2
with the CD5 cytoplasmic domain in the yeast two-hybrid assay, we can
conclude the holoenzyme form of CK2 interacts with the cytoplasmic
domain of CD5 in intact cells based on the co-immunoprecipitation of
CK2
with CD5 and the presence of CK2 kinase activity in CD5. The
interaction, however, is mediated by the regulatory
subunit as
neither the
nor the
' catalytic subunits associates with CD5
directly. Our mapping experiments localize the site of interaction with
CD5 to the amino terminus of CK2
, as was found with the nucleolar
protein Nopp140 (55). This presumably allows the carboxyl terminus to
be available for interaction with the catalytic subunits (42). Our data
do suggest the possibility that CK2
can interact with CD5 in the
absence of
/
'. If CD5 does interact with free CK2
in
vivo, it will be most likely under conditions where this subunit
is in excess, which may occur in some neoplastic cells (56).
Alternatively, substrates such as the serine/threonine kinase Mos that
interact exclusively with the CK2
subunit at the carboxyl terminus
of CK2
might compete with the catalytic
/
'subunits to form
novel multisubunit complexes with kinase activities (54). At present,
there is no evidence for this intriguing possibility in live cells.
We have identified that of the four CK2 motifs in CD5 cytoplasmic
domain, the kinase interacted with and phosphorylated the two distal
motifs, Ser459 and Ser461. A recent report
indicated that Ser459 and Ser461 are
phosphorylated on CD5 (36), and our data suggest that the kinase
responsible is CK2. It is notable that the phosphorylation was very
specific to Ser459 and Ser461, because
Ser458 was not phosphorylated by CK2. The continued, albeit
reduced, interaction of CK2 with the CD5 Ser459
Gly
and Ser461
Gly double mutant indicates that CK2 binding
may be influenced by but is not absolutely dependent on
phosphorylation. In that context, it is interesting to note that the
CK2 phosphorylation site and binding site in the CD5 cytoplasmic domain
are the same, in spite the of the fact that the interaction is mediated
by the CK2
and not by the catalytic subunits CK2
/
'. Because
the crystal structure for CK2 holoenzyme is not known, we can only
speculate that CK2
interacts with residues proximal to
Ser459 and Ser461 but not directly with them,
in a configuration that allows these sites to be available for
phosphorylation by CK2
, as supported by the observation that
Ser459 and Ser461 are not absolutely required
for binding.
The constitutive association of CK2 with CD5 in cell lines and primary cells and its ability to associate in a phosphorylation-independent manner suggested to us that CD5 may function as a regulator of CK2 activity. In fact, our data indicate that the CK2 associated with CD5 is relatively inactive in unstimulated cells and is activated 9-10-fold in the absence of net recruitment following ligation of CD5. This activation is very specific to CD5 stimulation because ligation of TCR or BCR did not cause this effect, even though CD5 clearly associates with these receptor complexes (12-14). This observation is particularly notable because the mechanisms that regulate CK2 under physiological conditions are poorly understood (50, 51). Although other stimulators of CK2 activity have been reported, those data have not been consistent. The ability to separate activated CK2 from inactive CK2 in the form of complexes with CD5 will be beneficial for studies to define the mechanism that regulates the kinase activity of CK2.
Another potential mechanism of CD5-dependent regulation of
CK2 may be based on the association and dissociation of and
subunits of CK2. The
subunit has a cyclin-like "destruction box" in its amino terminus, which may be involved in
ubiquitin-mediated proteolysis by the proteasome pathway (57).
Therefore, the binding of
to CD5 may protect it from this
degradation pathway. This may have specific relevance in neoplastic
cells that have elevated levels of CK2 and express excess
in
relation to the
/
'catalytic subunits (58). Interestingly,
neoplastic cells also express higher level of CD5.
The association with and activation of CK2 by CD5 is likely to have
significant implication on the regulation of TCR- and BCR-induced
activation. We hypothesize that CK2, following activation by CD5,
translocates and phosphorylates molecules associated with the antigen
receptors in both T and B cells. The effect of this on TCR/BCR
signaling will depend on the substrate, because phosphorylation of a
substrate by CK2 can lead to its positive or negative regulation (46,
59-65). In support of this hypothesis, Simarro et al. (36) have recently reported that the integrity of distal region of CD5
cytoplasmic domain, the site of CK2 binding and activation, was
required for both TCR-dependent and TCR-independent
diacylglycerol synthesis. The TCR-dependent diacylglycerol
synthesis is most likely mediated by phospholipase C-, which has 24 conserved CK2 phosphorylation sites. Similarly, several other molecules
that are involved in TCR/BCR-CD5 proximal events of CD5 signaling
contain sites for CK2-dependent phosphorylation, and
studies are under way to address whether they are inducibly
phosphorylated by CK2 are in progress.
In summary, this study is the first to demonstrate the activation of CK2 by direct association with a cell surface receptor. The ability to separate inactive CK2 from active CK2 will facilitate studies to define the properties that regulate its kinase activity. The findings presented here have the potential to expand the role of CK2 as regulator of membrane proximal signals in addition to previously described intermediate and distal events.
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FOOTNOTES |
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* This work was supported by an Arthritis Investigator award from the Arthritis Foundation (to C. R.) and by National Institutes of Health Grant P60-38520-08.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Tel.: 205-934-2472; Fax: 205-934-1564; E-mail: craman{at}uab.edu.
¶ Present address: Box 47, Rockefeller University, 1230 York Ave., New York, NY 10021.
1 The abbreviations used are: TCR, T cell receptor; BCR, B cell receptor; CK2, casein kinase 2; mAb, monoclonal antibody; BD, binding domain; AD, activation domain; UT, untranslated.
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REFERENCES |
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