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Protein Kinase A RIβ Subunit Deficiency in
Lupus T Lymphocytes: Bypassing a Block in
RIβ Translation Reconstitutes Protein Kinase
A Activity and Augments IL-2 Production
Islam U. Khan, Dama Laxminarayana and Gary M. Kammer
J Immunol 2001; 166:7600-7605; ;
doi: 10.4049/jimmunol.166.12.7600
http://www.jimmunol.org/content/166/12/7600
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Copyright © 2001 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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References
Protein Kinase A RI␤ Subunit Deficiency in Lupus T
Lymphocytes: Bypassing a Block in RI␤ Translation
Reconstitutes Protein Kinase A Activity and Augments IL-2
Production1
Islam U. Khan, Dama Laxminarayana, and Gary M. Kammer2
S
ystemic lupus erythematosus (SLE)3 is an idiopathic autoimmune disease characterized by defective cellular immunity. An imbalance of CD4 Th function relative to CD8
T cytotoxic effector activity promotes dysregulated production of
natural Abs and pathogenic autoantibodies by B cell clones, polyclonal hypergammaglobulinemia, and, ultimately, immune complex-mediated organ parenchymal inflammation (1). The morbidity and mortality in SLE is an outcome of chronic inflammation,
which can eventuate in end-stage renal disease and infections (2).
Aberrant signal transduction is one mechanism that may contribute to diverse T cell dysfunctions in SLE (3). T cells from 80%
of subjects with SLE exhibit impaired cAMP-dependent protein
phosphorylation due to a profound deficiency of the type I isozyme
of protein kinase A (PKA-I or the holoenzyme homodimer of regulatory isoforms of PKA-I (RI␣/RI␤) with catalytic subunit (C
subunit) (RI␣/␤2C2)) (4 –7). RI␣/␤2C2 holoenzyme is comprised
of two C subunits joined to either two ␣ or two ␤ regulatory (RI)
isoforms, resulting in RI␣2C2 and RI␤2C2 holoenzymes that
broaden the functional diversity of the PKA-I isozyme (8). Deficient RI␣/␤2C2 phosphotransferase activity in SLE T cells is a
product of significantly reduced amounts of RI subunit proteins,
Section on Rheumatology and Clinical Immunology, Department of Internal Medicine, Wake Forest University School of Medicine, Winston-Salem, NC 27157
particularly the ␤ isoform of RI (RI␤) (9). Low RI␤2C2 holoenzyme may significantly impair cAMP-inducible PKA-I activity because the concentration for half-maximal activation of this holoenzyme by cAMP is 2- to 7-fold lower than that of RI␣2C2 (10, 11).
This would have the effect of raising the threshold for the concentration of the cyclic nucleotide required to activate the PKA-I
isozyme.
The PKA-I isozyme is rapidly activated following an antigenic
stimulus to the T cell (12), and functions to inhibit T cell activation
(13). PKA-catalyzed substrate phosphorylation, including enzymes (14, 15) and transcription factors (16), is integral for physiologic effector functions. Deficient activity of the isozyme hinders
substrate phosphorylation (5), which may contribute to altered T
cell activation by hindering feedback inhibition and, ultimately,
may lead to the imbalance in effector activities that promote polyclonal hypergammaglobulinemia (1, 3).
To explore the mechanism underlying deficient RI␤ isoform expression, we determined the capacity of SLE T cells to translate
RI␤. Here, we demonstrate that deficient RI␤ protein is the result
of an apparent block in its translation. Transient transfection of
cDNAs from SLE subjects that span the RI␤ coding region into
autologous SLE T cells bypassed the block, resulting in RI␤ protein synthesis and a significant increase in both PKA-I phosphotransferase activity and IL-2 production.
Received for publication February 9, 2001. Accepted for publication April 2, 2001.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health Grants RO1 AR39501 and
RO1 AI42269, the Lupus Foundation of America, and the General Clinical Research
Center of the Wake Forest University School of Medicine (MO1 RR07122).
2
Address correspondence and reprint requests to Dr. Gary M. Kammer, Section on
Rheumatology and Clinical Immunology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157. E-mail address:
[email protected]
3
Abbreviations used in this paper: SLE, systemic lupus erythematosus; SS, Sjögren’s
syndrome; PKA-I or PKA-II, type I or type II isozyme of protein kinase A; RI␣/␤,
␣/␤ regulatory isoforms of PKA-I; C subunit, catalytic subunit; RI␣/␤2C2, holoenzyme homodimer of RI␣ or RI␤ isoform with C subunit; ADU, arbitrary densitometric unit; ALLM, N-acetyl-leucyl-leucyl-methional; FPLC, fast protein liquid chromatography; pI, isoelectric point; 5⬘ UTR, 5⬘ untranslated region.
Copyright © 2001 by The American Association of Immunologists
Materials and Methods
Patient and control groups
To study the mechanism of altered RI␤-subunit protein expression in SLE
T cells, subjects with SLE were selected from a previously studied cohort
(7, 9) based on the presence of deficient PKA-I activity (i.e., PKA-I specific activities ⱕ2 SD below the mean) (7). Normal and disease control
groups included age-, sex-, and racially matched normal individuals and
subjects with Sjögren’s syndrome (SS) (9). These studies were reviewed
and approved by the Institutional Review Board of the Wake Forest University/Baptist Medical Center.
T cell separation
SLE and control T cells were isolated and enriched from PBMCs or leukopheresis packs by the high-gradient magnetic cell separation system
0022-1767/01/$02.00
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A profound deficiency of type I protein kinase A (PKA-I or RI␣/␤2C2) phosphotransferase activity occurs in the T lymphocytes
of 80% of subjects with systemic lupus erythematosus (SLE), an autoimmune disorder of unknown etiology. This isozyme deficiency is predominantly the product of reduced or absent ␤ isoform of the type I regulatory subunit (RI␤). Transient transfection
of RI␤ cDNAs from SLE subjects into autologous T cells that do not synthesize the RI␤ subunit bypassed the block, resulting in
RI␤ subunit synthesis and restoration of the PKA-I␤ (RI␤2C2) holoenzyme. Transfected T cells activated via the T cell surface
receptor complex revealed a significant increase of cAMP-activatable PKA activity that was associated with a significant increase
in IL-2 production. These data demonstrate that a disorder of RI␤ translation exists, and that correction of the PKA-I deficiency
may enhance T lymphocyte effector functions in SLE. The Journal of Immunology, 2001, 166: 7600 –7605.
The Journal of Immunology
Midi MACS (Miltenyi Biotech, Auburn, CA) as described (17). Cytofluorographic analysis of T cells demonstrated that ⱖ96% expressed CD3,
which defines mature T cells.
T cell lines
was then immediately collected, and T cells were isolated as described
above. Thereafter, T cells isolated at each time point were washed three
times in cold PBS, and resuspended in iso-osmolar lysis buffer (5 mM
Tris-HCl (pH 7.2), 0.05% Triton X-100, 250 mM sucrose, 1 mM PMSF,
0.1 mM DTT, and protease inhibitor mixture). After eliminating nuclei by
centrifugation, RI- and C␣-subunits were immunoprecipitated with anti-RI
and anti-C␣ subunit mAbs (1/250 dilution), and the immunoprecipitates
were separated by 10% one-dimensional SDS-PAGE. Gels were then
treated with En3Hance according to the manufacturer’s protocol (NEN,
Boston, MA), dried, and subjected to autoradiography. Quantification of
35
S incorporated into proteins was determined by computerized scanning
laser densitometry.
In vitro transcription and translation
cDNAs that span the RI␤ coding region (hereafter referred to as RI␤
cDNA) (19) were subcloned into the pBluescript SK⫹ vector under the
control of a T7 promoter. Plasmid DNA was purified using a Qiagen DNA
purification kit (Qiagen, Valencia, CA). Before in vitro transcription, the
construct was linearized downstream of the cDNA insert to achieve ordered termination of transcription. Plasmid DNA (⬃100 ␮g) was used as
a template for the large-scale production of RNA using a RiboMax kit
(Promega, Madison, WI). In vitro translation of synthesized mRNA (1–2
␮g) was performed in a rabbit reticulocyte system according to the manufacturer’s protocol. The final volume was 50 ␮l in the presence or absence
of [35S]methionine (specific activity ⬎1000 Ci/mmol; Amersham Pharmacia Biotech, Piscataway, NJ). Reaction mixtures were boiled for 7 min in
Laemmli buffer, and 25 ␮l of the denatured reaction mix was analyzed on
10% SDS-PAGE. The proteins were then transferred to polyvinylidene
difluoride membrane, and autoradiography was performed.
PKA-I and PKA-II fractionation
CD3, CD4 or CD3, CD8 subpopulations were enriched from PBMC (7, 9).
Nuclei-free T cell lysates were prepared in a buffer containing 10 mM
K2PO4 (pH 7.2), 1 mM EDTA, 0.1 mM DTT, and protease inhibitor mixture (Complete Mini EDTA-free Protease Inhibitor; Roche Diagnostics,
Indianapolis, IN) (7, 9). The PKA isozymes were partially purified by fast
protein liquid chromatography (FPLC; GradiFrac; Amersham Pharmacia
Biotech, Piscataway, NJ) using an anion exchange MonoBead column
(Amersham Pharmacia Biotech). Briefly, 750 ␮g of lysate was applied to
a 1-ml HiTrap column; the column was rinsed with 20 mM Tris-HCl (pH
7.2) buffer; and the column was eluted with this buffer containing either
200 mM NaCl (fraction I contains PKA-I holoenzymes) or 400 mM NaCl
(fraction II contains PKA-II holoenzymes). Eluates were desalted and concentrated 5-fold through a Centricon-30 filter (Amicon, Beverly, MA), lyophilized, diluted in water to a concentration of 1 ␮g/␮l, then 20 ␮g of
protein per lane was loaded onto a 10% one-dimensional SDS-PAGE.
SDS-PAGE
One- and two-dimensional SDS-PAGEs were performed as described (18).
The isoelectric points (pIs) and Mr values of proteins in two-dimensional
SDS-PAGE were determined by using a mixture of marker proteins (BioRad, Hercules, CA). Protein (150 ␮g) was loaded onto each two-dimensional gel. Proteins were focused in a pI range of 3.5–10 using isoelectric
focusing tube gels; the second dimension and immunoblotting were performed as described.
Immunoprecipitation and immunoblotting
Immune complexes were isolated by using affinity-purified goat anti-mouse
IgG conjugated to protein A-Sepharose as described (18). Immunoblots
were probed with 1:1000 anti-RI mAb or anti-C␣-subunit mAb (Transduction Laboratories, Lexington, KY).
RI␤ cDNA overexpression
RI␤ cDNAs from the T cells of five SLE subjects were amplified by RTPCR and cloned into the mammalian expression vector, pCR3.1 (containing a CMV promoter) using a TA cloning kit (Invitrogen, Carlsbad, CA).
The resulting pCR3.1/RI␤ construct was used for transfection into autologous SLE T cells.
PBMCs were cultured in RPMI 1640 supplemented with 10% FCS, 2
mM L-glutamine, 10 mM HEPES, antibiotics, and 1 ␮g/ml PHA for 22 h
at 37°C in 5% CO2 (20). After isolation of T cells, 35 ␮g DNA and 1 ⫻
107 cells were resuspended in 0.4 ml RPMI 1640 in prechilled 0.4-cm gap
width cuvettes. The reporter gene or pCR3.1 (mock or empty vector) or
pCR3.1/RI␤ construct was electroporated at 950 ␮F and 270 V at room
temperature using a Bio-Rad Gene Pulser w/Cap extender. Subsequently,
transfected T cells were cultured in a 3:1 ratio of RPMI 1640 and HL-1
supplemented with 5% FCS, 25 mM HEPES, 2 mM L-glutamine, antibiotics, and 1 ␮g/ml PHA. Cells were recovered after 24 and 48 h.
The conditions for electroporation and optimum time for PHA stimulation to make peripheral T cells competent for transient transfection were
determined by using a ␤-galactosidase reporter gene, pHook-2lacZ (Invitrogen). PHA-stimulated T cells were transiently transfected with either
pHook-2lacZ or pHook-2 (Invitrogen) reporter genes. A negative control
and a positive control along with lysate from transiently transfected T cells
were assayed for ␤-galactosidase activity using a commercially available
kit (Promega). An increase of ⬃10-fold in ␤-galactosidase activity in T
cells transiently transfected with pHook-2lacZ was found at 22 h post PHA
stimulation.
To determine transfection efficiency, peripheral T cells were cotransfected with pCR3.1/RI␤ and pEGFP-C1 (Clontech, Palo Alto, CA) constructs. The proportion of cells exhibiting green fluorescence at 24 and 48 h
posttransfection in viable cell populations were determined by cytofluorography. Based on this analysis, we routinely achieved 12–15% GFP⫹ cells.
35
S biosynthetic labeling of RI and C subunits
Three SLE subjects with significantly reduced RI␤ mRNA transcripts
(0.071 ⫾ 0.010 amol/␮g of total RNA) compared with healthy control RI␤
mRNA transcripts (0.209 ⫾ 0.037 amol/␮g of total RNA) ( p ⫽ 0.01), as
determined by competitive PCR (9), were selected to analyze the turnover
of RI- and C␣-subunit proteins. PBMCs (4 ⫻ 108) were incubated in methionine-free medium for 30 min at 37°C. Cells were then metabolically
labeled with 250 ␮Ci/ml trans-[35S]methionine (⬎1000 Ci/mmol; ICN,
Irvine, CA) for 12 h at 37°C in the presence of 2.5 mM dibutyryl cAMP
and 200 ␮M isobutylmethylxanthine. Cells were thoroughly washed and
resuspended in RPMI 1640 containing unlabeled methionine supplemented
with 10% FCS, 2 mM L-glutamine, 25 mM HEPES (pH 7.4), and antibiotics. Cells were then cultured in duplicate for 3, 6, 12, 24, or 48 h in 5%
CO2 at 37°C. Cell death at each time point was ⬍10%. The 0-h time point
PKA assay
PKA-specific phosphotransferase activity was quantified by measuring the
transfer of phosphate-32 from [␥-32P]ATP to synthetic heptapeptide, leuarg-arg-ala-ser-leu-gly. The specific phosphotransferase activity is expressed as pmol/min/mg protein (21).
T cell activation
T cells (6 ⫻ 106) were activated via the TCR/CD3 complex with 4 ␮g/ml
anti-CD3 (Beckman Coulter, Miami, FL) plus 100 ng/ml anti-CD28 (BD
Biosciences, San Jose, CA) ⫹ 100 U/ml recombinant human IL-1␣ (R&D
Systems) for 12, 24, and 48 h. Supernatants were then collected, and IL-2
release was measured by ELISA (R&D Systems).
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To propagate T cell lines in vitro, PBMCs were cultured in 3:1 RPMI 1640
and HL-1 (BioWhittaker, Walkersville, MD) supplemented with 5% heatinactivated FCS (HyClone, Logan, UT), 25 mM HEPES, 2 mM L-glutamine, 10 ␮g/ml streptomycin, 10 IU/ml penicillin, and 1 ␮g/ml PHA, as
described (9). After 2 days, T lymphoblasts were passaged and cultured in
the above medium supplemented with 20 U/ml recombinant human IL-2
and 40 U/ml of recombinant human IL-4 (R&D Systems, Minneapolis,
MN). After propagating through 10 passages, T cells were harvested and
incubated in 50 ␮g/ml propidium iodide overnight at 4°C, and the cell
cycle was quantified by cytofluorography. To force cells to re-enter G0/G1,
T cells were transferred to RPMI 1640 supplemented with only 2% FCS,
antibiotics, and L-glutamine. At 72 h, rested T cells were harvested and
stained with propidium iodide, and the proportion of cells in each phase of
the cell cycle was quantified. Fewer than 2% of control and SLE T cells
underwent apoptosis during this time due to withdrawal of cytokines, as
determined by the absence of hypodiploid cells.
The specific protease and ubiquitin inhibitors, ALLM (N-acetyl-leucylmethional) and lactacystin (Calbiochem, San Diego, CA), were dissolved
in DMSO, and were used at a final concentration of 100 and 20 ␮M,
respectively. T cell lines were propagated with or without protease inhibitors for 10 passages before T cell lysates were prepared. To force cells to
reenter G0/G1, T cells were cultured as described above for 72 h in the
presence or absence of inhibitors. At 72 h, rested T cells were harvested,
and T cell lysates were prepared.
7601
7602
RI␤ SUBUNIT DEFICIENCY AND ITS RECONSTITUTION IN LUPUS T CELLS
Results
Analysis of semipurified RI␤ protein in primary T cells
To explore the mechanism of diminished/absent RI␤ protein in
SLE T cells, the PKA-I isozyme was partially purified from nucleifree T cell homogenates of SLE and normal subjects by FPLC, as
detailed in Materials and Methods. Using an anti-RI mAb that
recognizes both RI␣ and RI␤ isoforms, an immunoblot of the fractions containing PKA-I isozymes (i.e., fraction I) from six healthy
subjects revealed both RI␣ and RI␤ isoforms in a ratio of 4.4:1
(Fig. 1A). In contrast to controls, fractions containing PKA-I
isozymes from six SLE subjects had reduced amounts of RI␣ protein and no detectable RI␤ protein (Fig. 1A). Absence of detectable
RI␤ protein was not the result of its elution with RII proteins
associated with the PKA-II isozyme (i.e., fraction II) (data not
shown). Because the column is eluted with 200 mM NaCl, the
absence of RI␤ protein could be the result of a molecular charge
shift due to variation of pI values for RI␤ protein in SLE T cells.
Determination of pI of RI proteins
FIGURE 1. Immunoblots of T lymphocyte PKA RI- and C subunit proteins. A, Nuclei-free T cell lysates from
six SLE and normal control subjects,
respectively, were prepared; RI proteins were partially purified by FPLC;
RI␣ and RI␤ isoforms were identified
by immunoblotting with anti-RI mAb;
and the amounts of each isoform were
quantified by laser densitometry. B, Immunoblots of PKA RI␣, RI␤, and C␣
subunits from six healthy controls, SS
disease controls, and SLE subjects, respectively, separated by two-dimensional SDS-PAGE. Protein (150 ␮g)
was loaded onto each gel. Diminution
or absence of the RI␤ isoform in SLE
samples is denoted by the arrowhead
() in lanes 1–3. The blots were
stripped and reprobed with anti-C␣
mAb to identify the C␣ subunit. Representative gels from healthy and SS
controls and a SLE subject are shown in
lane 4.
In vivo synthesis of RI and C subunit proteins in SLE T cells
We have previously demonstrated by competitive PCR that the
amount of RI␤ transcript in SLE T cells is significantly reduced by
about one-half compared with control T cells (9). To determine
whether SLE T cells can translate RI␤ protein from existing RI␤
mRNA, we performed pulse-chase [35S]methionine metabolic labeling experiments. In these experiments, we used T cells from
SLE subjects that expressed significantly reduced amounts of RI␤
transcript (see Materials and Methods). Fig. 2A shows the kinetics
of RI␣-, RI␤-, and C␣-subunit expression. Compared with normal
and SS disease control T cells, there is a striking absence of detectable RI␤ protein synthesis by SLE T cells over 48 h (Fig. 2, A
and C). By contrast, over the same time normal and SS T cells
produced a mean 196 arbitrary densitometric units (ADU) and 116
ADU of RI␤ protein, respectively (SLE vs normal or SS, p ⫽
0.002, respectively). Moreover, SLE T cells also synthesized only
301 ADU of RI␣ protein compared with the production of 525 and
438 ADU of RI␣ protein by normal and SS T cells (Fig. 2B),
respectively (SLE vs normal, p ⫽ 0.004; SLE vs SS, p ⫽ 0.025).
However, there were no significant differences in the amounts of
C␣-subunit proteins between SLE and control T cells. This absence of RI␤ protein synthesis demonstrates that SLE T cells apparently have a selective block in the translation of the RI␤ isoform. This resultant alteration of RI␣ and RI␤ protein expression
may account for the markedly skewed ratio of RI␣ and RI␤ proteins previously identified in SLE T cells (9).
Because reduced or undetectable RI␤ could also reflect accelerated proteolysis and ubiquitination of RI␤ protein (22), we determined whether the ubiquitin-proteasome proteolytic pathway
might augment the loss of RI␤ created by this putative block in its
translation in SLE T cells. T cell lines from three SLE subjects
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To determine whether a charge shift of RI␤ exists, T cell homogenates from SLE subjects with putatively absent RI␤ protein and
normal and SS disease controls were separated by two-dimensional SDS-PAGE (18) and immunoblotted with anti-RI mAb, and
the pI of RI proteins was quantified. Fig. 1B demonstrates that
normal and SS T cell lysates express both RI␣ and RI␤ proteins
with the expected Mr values of 49 and 53.5 kDa (9), respectively,
and a pI range of 5.8 – 6.4. Thus, under physiologic conditions,
RI␣ and RI␤ subunits consist of proteins with a spectrum of pI
values. Of note is that there was no charge shift of RI␤ proteins in
SLE T cells. Instead, the more basic isoforms were uniformly absent, and the acidic isoforms were markedly diminished or absent
(Fig. 1B). This observation suggests that SLE T cells make none of
the basic RI␤ isoforms and only small amounts or none of the
acidic isoforms of RI␤ protein. When the same blots were probed
for the presence of PKA C␣ subunit by anti-C␣ subunit mAb, there
were no differences in the amounts of this subunit between SLE
and healthy or SS controls (Fig. 1B). That RI␤ protein was diminished or undetectable in SLE T cell homogenates on two-dimensional immunoblots is in accord with our previous findings by
one-dimensional immunoblots (9).
The Journal of Immunology
7603
were established (9) in the presence (treated) or absence (untreated) of 10 ␮M lactacystin, a specific 26S proteasome inhibitor (23,
24), and 100 ␮M ALLM (25), the cysteine protease calpain inhibitor. In a representative experiment shown in Fig. 2D, freshly isolated SLE T cells exhibited only the RI␣ isoform. After propagating the cells through 10 passages in vitro, ⬃35% of cells were in
S phase of the cell cycle and RI␤ isoform remained undetectable,
independent of treatment with inhibitors (Fig. 2D). This persistent
absence of RI␤ protein during S phase is consistent with our previous inability to detect this isoform in cycling SLE T cell progeny
(9). Thus, the absence of RI␤ in cycling SLE T cells would not
appear to be the result of proteolysis. By contrast, there was a
modest, but statistically insignificant increase in RI␣ protein content in cells treated with lactacystin and ALLM (Fig. 2D). After
resting for 72 h in vitro, ⬎96% of SLE T cells returned to G0/G1
phase of the cell cycle. Notwithstanding, RI␤ protein remained
undetectable in SLE T cell progeny in the presence or absence of
both the proteasome and calpain inhibitors. Again, these results are
consistent with our previous inability to detect RI␤ isoform in
nondividing SLE T cell progeny (9). Taken together, our findings
suggest that the absence of RI␤ protein in SLE T cells is not the
consequence of either enhanced proteolysis or proteasome
degradation.
In vitro synthesis of RI␤ protein by SLE T cell cDNA
Our identification of a putative translational block of RI␤ in SLE
T cells prompted us to determine whether RI␤ cDNA from these
cells could be transcribed and translated in vitro. RI␤ cDNA de-
rived from SLE or normal T cells was subcloned into the pBluescript SK⫹ vector under the control of the T7 promoter (26). Fig.
3A demonstrates that translation of in vitro transcribed [35S]methionine-labeled RI␤ mRNAs in a cell-free system yielded comparable amounts of the 53.5-kDa RI␤ protein in SLE and normal
controls. These findings demonstrate that RI␤ cDNA from SLE T
cells driven by an exogenous promoter can be efficiently translated
to RI␤ protein.
Reconstitution of RI␤ protein expression in SLE T cells
To determine whether the defect could be bypassed, we transiently
transfected a pCR3.1/RI␤ construct under the control of a CMV
promoter into primary SLE T cells. The constructs were made
from RI␤ cDNAs of five RI␤-deficient SLE subjects, and were
transfected into autologous T cells from each of these persons.
Compared with freshly isolated or mock-transfected cells, there
was a statistically significant 8- and 10-fold increase in RI␤ protein
expression at 24 and 48 h after transfection, respectively (Fig. 3, B
and C) (24 h, p ⫽ 0.031; 48 h, p ⫽ 0.004). This resulted in a mean
ratio of RI␣/RI␤ protein of 4.1:1 and 3.8:1 at 24 and 48 h, respectively, values very similar to the mean ratio of RI␣:RI␤ protein of
3.2:1 in normal T cells (9). Thus, by transfecting SLE RI␤ cDNAs
coupled to an exogenous promoter, we were able to bypass the
putative block in RI␤ translation.
We next determined whether reconstitution of RI␤ protein in
SLE T cells was associated with restoration of PKA phosphotransferase activity (21). This is a pathophysiologically relevant issue,
for 80% of SLE subjects harbor a profound T cell deficiency of
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FIGURE 2. Synthesis of PKA RI␣-, RI␤-, and C␣-subunits by SLE and control T cells. A, T cells were biosynthetically labeled in the presence of 2.5
mM dibutyryl cAMP and 200 ␮M isobutylmethylxanthine. RI- and C␣-subunits associated with the plasma membrane and cytosolic compartments were
immunoprecipitated with anti-RI and anti-C␣ subunit mAbs. B and C, Kinetics of RI␣ and RI␤ isoform synthesis over 48 h (normal controls, F; SS disease
controls, f; SLE subjects, ). D, Determination of RI␤ isoform proteolysis by protease and proteasome inhibitors. Freshly isolated, cycling, and rested
T cells were cultured in the absence or presence of 10 ␮M lactacystin and 100 ␮M ALLM. Cycling T cells were harvested at the completion of the 10th
passage or washed, resuspended in medium lacking mitogen and IL-2, and rested for 72 h. Nuclei-free T cell lysates were prepared; 150 ␮g of protein/lane
was loaded, then proteins were separated by 10% one-dimensional SDS-PAGE, immunoblotted with anti-RI and anti-C␣ subunit mAbs, and developed with
ECL. This immunoblot is representative of three independent experiments from three SLE subjects.
RI␤ SUBUNIT DEFICIENCY AND ITS RECONSTITUTION IN LUPUS T CELLS
7604
cells (27). To determine whether restoration of PKA-I activity
could correct IL-2 production in SLE T cells, we activated T cells
from four SLE subjects in vitro via CD3, CD28, and IL-1␣ cell
surface receptors (12). The results, shown in Table I, reveal a
significant increase in secreted IL-2 over 48 h. Although the
amount of cytokine produced by transfected SLE T cells is 17-fold
lower than that of activated normal T cells, the capacity of these T
cells to significantly enhance their production of IL-2 suggests that
the RI␤2C2 holoenzyme may convey a signal involved in the regulation of IL-2 synthesis.
Discussion
PKA-I activity characterized by only 20 –25% of physiologic activity (6, 7). As shown in Table I, there was a mean 73% increase
in PKA activity in SLE T cells transiently transfected with the RI␤
construct (n ⫽ 5, p ⫽ 0.03). The presence of physiologic amounts
of C␣-subunit combined with RI␤ isoform to form the RI␤2C2
holoenzyme, thereby raising cAMP-activatable PKA-I enzymatic
activity to physiologic levels (7). Thus, transient transfection of
RI␤ cDNAs from SLE subjects into autologous T cells reconstituted RI␤ protein levels and restored physiologic PKA activity.
Altered cytokine production is a byproduct of T cell dysfunction
in SLE. Following in vitro activation via cell surface receptors,
SLE T cells produce significantly less IL-2 than normal control T
Table I. Association of increased PKA-specific activity with enhanced
IL-2 production by SLE T lymphocytes transiently transfected with the
pCR3.1/RI␤ construct
T Cells
NTa
Ta
a
PKA-Specific Activity
(pmol/min/mg protein)
IL-2
(pg/ml)
513.2 ⫾ 176b
887.5 ⫾ 196c
59.3 ⫾ 21
252 ⫾ 23d
NT and T, Nontransfected and transfected T cells.
Mean ⫾ SEM.
NT vs T, p ⫽ 0.03.
d
NT vs T, p ⫽ 0.005.
b
c
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FIGURE 3. Translation of RI␤ protein in SLE T cells. A, In vitro transcription and translation of RI␤ in the rabbit reticulocyte system. Lanes 1
and 2 used RI␤ cDNAs from two normal subjects; lanes 3–5 used RI␤
cDNAs from three SLE subjects. B, Fold increase in RI␤ protein expression over 24 and 48 h in T cells of five SLE subjects following transient
transfection of the pCR3.1/RI␤ construct or empty vector (mock). C, Representative immunoblot of RI␣ and RI␤ proteins following transient transfection of the pCR3.1/RI␤ construct or empty vector (mock). Lane 1, Nuclei-free T cell lysate from a normal control was prepared; 150 ␮g of
protein/lane was loaded, and proteins were separated on a 10% one-dimensional SDS-PAGE, immunoblotted with anti-RI mAb, and developed with
ECL (9). Lane 2, Freshly isolated SLE T cells; lanes 3 and 4, transiently
transfected SLE T cells with pCR3.1 vector only (mock) and harvested at
24 and 48 h; lanes 5 and 6, transiently transfected SLE T cells with
pCR3.1/RI␤ construct and harvested at 24 and 48 h.
Our identification of impaired cAMP-dependent protein phosphorylation due to deficient PKA-I phosphotransferase activity was the
initial recognition of disordered signal transduction in SLE T cells
(4 – 6). That an intrinsic disorder of signaling exists raised the possibility that previously identified T cell effector dysfunctions (1, 3)
may in part be a consequence of this altered signaling. To establish
how aberrant signaling in SLE T cells relates to impaired effector
functions, we tested the hypothesis that deficient RI␤2C2 holoenzyme is a pan-T cell disorder. Indeed, both CD4 and CD8 SLE T
cells have significantly reduced amounts of both RI␣ and RI␤
isoforms in nuclei-free homogenates. In particular, our results in
this and previous analyses revealed that the amounts of RI␤ isoform are profoundly reduced or absent (9). By contrast, the
amounts of C subunit protein are comparable between SLE and
normal controls.
Herein, we have demonstrated that reduced or absent RI␤ isoform appears to be the result of a selective block in its translation
rather than global translational silencing of PKA RI␣, RI␤, and C␣
subunit translation. If there were global translational silencing of
these subunit genes, we would have expected to observe no in vivo
RI␣ and C␣ subunit protein by [35S]methionine biosynthetic labeling. Although the amount of RI␣ protein translated was significantly less than that of both normal and SS disease controls, translation of RI␣ protein was consistently observed in SLE. Moreover,
the amount of translated C␣ subunit protein was comparable with
controls.
We have previously found that the amounts of RI␤ mRNA are
significantly reduced in SLE (9). That RI␤ protein was not identified by biosynthetic labeling, but could be in vitro transcribed,
suggests that existing RI␤ transcripts are not being effectively processed through translation. To determine whether there is translational repression of RI␤ in SLE T cells, we transiently transfected
these cells with constructs made from cDNAs that span the RI␤
coding region. Full-length RI␤ cDNAs could not be used because
the 5⬘ untranslated region (5⬘ UTR) of the gene has not yet been
sequenced. Transient transfection of RI␤ cDNAs into autologous
SLE T cells was able to bypass the defect, resulting in correction
of the putative translational block, production of RI␤ isoform, and
a significant increase of PKA phosphotransferase activity. Importantly, restoration of PKA activity was associated with an enhanced T cell effector function, as reflected by a significant increase in IL-2 production by receptor-activated SLE T cells.
To date, our data demonstrate both reduced RI␤ mRNA and
translational repression of RI␤. Several mechanisms could be operative concurrently to account for these findings. One is the existence of an as yet unidentified polymorphism(s) or mutation(s) of
the 5⬘ UTR. Identification of such changes will await the sequencing of the RI␤ 5⬘ UTR. A second mechanism is alternative use of
exons due to splicing. Either mechanism could lead to reduced
amounts of RI␤ transcript (9). Indeed, our preliminary evidence
suggests that production of nascent RI␤ mRNA may be impaired.
A third mechanism is aberrant phosphorylation of an initiation
The Journal of Immunology
factor(s). Importantly, impaired translational responses in SLE T
cells have been recently linked to increased PKR-catalyzed phosphorylation of the initiation factor, eIF2␣ (28). It is conceivable
that this mechanism may coexist with a disorder of nascent transcript production and contribute to the impaired translation of
RI␤ ⬎ RI␣. However, it remains to be established whether the
translation of RI␤ and/or RI␣ genes is regulated by PKR and
eIF2␣.
This is the first identification of an apparent block in translation
of a signaling molecule whose genetic correction results in an enhanced T cell effector function in SLE. Understanding the precise
transcriptional abnormality(s) contributing to this putative block in
the translation of RI␤ will be integral to designing gene repair
strategies.
Acknowledgments
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We thank G. Sigmon for excellent technical assistance; members of the
Kammer laboratory for thoughtful discussions during the course of this
work and critical reading of the manuscript; and Drs. Doug Lyles and Steve
Mizel for their critical reading of this manuscript.
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