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Association of Deficient Type II Protein
Kinase A Activity with Aberrant Nuclear
Translocation of the RIIβ Subunit in Systemic
Lupus Erythematosus T Lymphocytes
Nilamadhab Mishra, Islam U. Khan, George C. Tsokos and
Gary M. Kammer
J Immunol 2000; 165:2830-2840; ;
doi: 10.4049/jimmunol.165.5.2830
http://www.jimmunol.org/content/165/5/2830
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Copyright © 2000 by The American Association of
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References
Association of Deficient Type II Protein Kinase A Activity with
Aberrant Nuclear Translocation of the RII␤ Subunit in
Systemic Lupus Erythematosus T Lymphocytes1
Nilamadhab Mishra,* Islam U. Khan,* George C. Tsokos,†‡ and Gary M. Kammer2*
S
ystemic lupus erythematosus (SLE)3 is an autoimmune
disorder of indeterminate etiology characterized by impaired cellular immunity (1) that often results in anergy to
recall Ags (2, 3). Within the T cell compartment, both the CD3,
CD4 helper and CD3, CD8 cytotoxic/suppressor subsets are dysfunctional, resulting in an imbalance of exaggerated helper and
diminished cytotoxic/suppressor activities (4). A marker of altered
CD4 helper function is skewing of the Th1 and Th2 responses (5)
to antigenic challenge. There is diminished Th1 and enhanced Th2
cytokine production, yielding reduced generation of IL-2 and
*Section on Rheumatology and Clinical Immunology, Department of Internal Medicine, Wake Forest University School of Medicine, Winston-Salem, NC 27157; †Department of Cellular Injury, Walter Reed Army Institute of Research, Silver Spring,
MD 20910; and ‡Department of Medicine, Uniformed Services University of the
Health Sciences, Bethesda, MD 20814
Received for publication April 6, 2000. Accepted for publication June 12, 2000.
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 grants from the National Institutes of Health (RO1
AR39501 to G.M.K. and RO1 AI42269 to G.C.T. and G.M.K.), the Lupus Foundation
of America (to I.U.K. and G.M.K.), the General Clinical Research Center of the Wake
Forest University School of Medicine (MO1 RR07122), the National Cancer Institute
(5P30 CA12197), and the North Carolina Biotechnology Center (9510-1DG-1006).
N.M. is a postdoctoral research fellow of the Arthritis Foundation.
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, protein kinase A; PKA-I or -II, type I or II isozyme of PKA; RII␣/␤,
␣ or ␤ isoform of regulatory subunit of PKA-II; RII␣/␤2C2 holoenzyme, homodimer
of RII␣ or RII␤ isoform with C subunit; SLEDAI, SLE disease activity index; AC,
adenylyl cyclase; PI, propidium iodide; ECL, enhanced chemiluminescence; ADU,
arbitrary densitometric units; 8-Cl-cAMP, 8-chloro-cAMP; AKAP, A kinase anchor
protein; CREB, cAMP response element binding protein; NES, nuclear export signal.
Copyright © 2000 by The American Association of Immunologists
IFN-␥ by Th1 cells and overproduction of IL-6 and IL-10 by Th2
cells (6 –9). However, the mechanisms contributing to T cell immune dysfunctions in SLE are still incompletely understood (10).
One mechanism that may contribute to T cell dysfunction in
SLE is altered signal transduction. We have identified the presence
of several, discrete signaling defects in SLE T cells (10, 11). Deficient type I protein kinase A (PKA-I) activity is a signaling disorder that results in marked underphosphorylation of substrates
(12, 13) and occurs with a prevalence of 80% in SLE T cells (14).
Kinetic analyses of this isozyme deficiency revealed a significant
reduction in both the maximal enzyme velocity (Vmax) and cAMPbinding capacity of the type I regulatory (RI) subunit (Bmax), but
a significant increase in the cAMP half-maximal activation (Ka) of
the PKA-I holoenzyme compared with controls (15). This altered
isozyme kinetics is the result of a significant reduction of both RI␣
and RI␤ isoforms in SLE T cells (16). The association of diminished RI protein content with reduced amounts of steady state RI
transcripts raises the possibility that the PKA-I isozyme deficiency
could reflect a pretranslational disorder in SLE T cells. By hindering efficient phosphorylation of multiple substrates, deficient
PKA-I activity would be expected to significantly impede signaling downstream. Because PKA-catalyzed phosphorylation is a
principal posttranslational process that regulates widely divergent
cellular functions, including chaperonin activities (17), binding of
agonists to intracellular receptors (18, 19), catalysis (20, 21), and
activation of transcription factors (22–24), deficient PKA-I activity
may contribute to altered helper activity and cytotoxicity in SLE T
cells.
PKA is a serine/threonine kinase that is composed of two
isozymes, type I PKA (PKA-I) and type II PKA (PKA-II) (25).
These isozymes differ in their subcellular localization; in the human T cell, PKA-I localizes predominantly with the plasma membrane fraction whereas PKA-II is present chiefly in the cytosol
0022-1767/00/$02.00
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Systemic lupus erythematosus (SLE) is an autoimmune disorder of indeterminate etiology characterized by abnormal T cell signal
transduction and altered T cell effector functions. We have previously observed a profound deficiency of total protein kinase A
(PKA) phosphotransferase activity in SLE T cells. Here we examined whether reduced total PKA activity in SLE T cells is in part
the result of deficient type II PKA (PKA-II) isozyme activity. The mean PKA-II activity in SLE T cells was 61% of normal control
T cells. The prevalence of deficient PKA-II activity in 35 SLE subjects was 37%. Deficient isozyme activity was persistent over time
and was unrelated to SLE disease activity. Reduced PKA-II activity was associated with spontaneous dissociation of the cytosolic
RII␤2C2 holoenzyme and translocation of the regulatory (RII␤) subunit from the cytosol to the nucleus. Confocal immunofluorescence microscopy revealed that the RII␤ subunit was present in ⬃60% of SLE T cell nuclei compared with only 2–3% of
normal and disease controls. Quantification of nuclear RII␤ subunit protein content by immunoprecipitation and immunoblotting
demonstrated a 54% increase over normal T cell nuclei. Moreover, the RII␤ subunit was retained in SLE T cell nuclei, failed to
relocate to the cytosol, and was associated with a persistent deficiency of PKA-II activity. In conclusion, we describe a novel
mechanism of deficient PKA-II isozyme activity due to aberrant nuclear translocation of the RII␤ subunit and its retention in the
nucleus in SLE T cells. Deficient PKA-II activity may contribute to impaired signaling in SLE T cells. The Journal of Immunology, 2000, 165: 2830 –2840.
The Journal of Immunology
Materials and Methods
Patient and control populations
Thirty-five consecutive, unselected SLE subjects with a mean age (⫾SD)
of 35.5 ⫾ 11 years (range, 12– 66 years) were prospectively studied. All
subjects fulfilled four or more of the criteria for the classification of SLE
(28). Of these SLE subjects, 29 were female, 26 were white, and 9 were
black. Utilizing the SLE disease activity index (SLEDAI), a standardized
scale, to gauge the extent of disease manifestations (29), the mean (⫾SD)
SLEDAI was 11.9 ⫾ 7.4 (range, 2–32). A SLEDAI of 1–10 denotes mild
activity, 11–20 moderate activity, and ⱖ21 severe disease activity (13–16).
Thirty-five healthy controls with a mean age of 35.1 ⫾ 9.5 years (range,
24 – 65 years) were studied. Of these controls, 23 were female, 24 were
white, and 11 were black. Eleven subjects with primary Sjögren’s syndrome (SS) (30) with a mean age of 44.2 ⫾ 5.0 years (range, 28 –56 years)
served as disease controls. Of these, all subjects were white and female.
Subjects were studied according to our previous protocols (12, 13, 15,
16). Individuals experiencing a flare of SLE activity were studied before
initiation of corticosteroid and/or immunosuppressive therapy; none had
been treated with immunosuppressive agents for at least 3 mo. Only SLE
subjects treated with low dose corticosteroids (ⱕ10 mg/day prednisone)
were entered into this protocol; these individuals were studied 24 h after
their last oral dose. Nonsteroidal antiinflammatory agents and hydroxychloroquine were withheld for 72 h and 7 days, respectively, before study
when clinically feasible. Informed consent to participate in this study and
to obtain specimens by venipuncture or by leukapheresis were obtained
from subjects and controls. The research protocols and consent forms were
approved by the institutional review board of the Wake Forest University/
Baptist Medical Center.
T lymphocyte isolation and phenotypic characterization
SLE and control T lymphocytes were isolated and enriched from PBMC or
cells obtained by leukapheresis by the high gradient magnetic cell separation system, Midi MACS (Miltenyi Biotec, Auburn, CA) (16). Cytofluorographic analysis revealed that enriched, viable T cells expressed a mean
(⫾SEM) of 96 ⫾ 1.2% CD3. The proportions of CD3 T cells expressing
other cell surface markers have been previously detailed (14).
T cell lines
Freshly isolated T cells were stained with propidium iodide (PI), and the
proportions of cells in G0 /G1, S, and G2-M phases of the cell cycle were
quantified by flow cytometry (16). T cell lines were propagated in vitro as
previously described (16).
PKA assay
T cell PKA-I and PKA-II isozymes were fractionated and isolated by tandem DE52-cellulose and carboxymethyl-Sephadex chromatography as previously described (13). PKA phosphotransferase activity was quantified as
previously detailed (27). The physiologic range of T cell PKA-II activity is
given in Table I.
Nuclear extracts
Nuclear extracts were prepared from the T cells of normal and SS disease
controls and SLE subjects. T cells (80 ⫻ 106) were washed twice in PBS
and resuspended in 1 ml ice-cold lysis buffer (Dignam buffer A: 10 mM
Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 0.1 mM EGTA, 0.5 mM
DTT, 1 mM PMSF, and 2 ␮g/ml each leupeptin and aprotinin). After 10
min on ice, 50 ␮l 10% Nonidet P-40 were added, and the cells were centrifuged at 9000 rpm for 30 s at 4°C. Pelleted nuclei were washed twice in
Dignam buffer A and lysed in 200 ␮l Dignam high salt buffer C (20 mM
HEPES (pH 7.9), 420 mM NaCl, 1.5 mM MgC12, 0.1 M EDTA, 25%
glycerol, 0.5 mM DTT, 0.5 mM PMSF, 2 ␮g/ml each leupeptin and aprotinin) for 15 min at 4°C. After lysis, nuclear extracts were centrifuged at
12,000 rpm for 10 min at 4°C, and the resulting supernatants were diluted
1:1 (v/v) with Dignam buffer D (20 mM HEPES (pH 7.9), 100 mM KCl,
0.1 mM EDTA, 20% glycerol, 0.5 mM DTT, 0.5 mM PMSF, 2 ␮g/ml each
leupeptin and aprotinin). Protein concentration was determined by the
Bradford method (31) (Bio-Rad Laboratories, Hercules, CA).
Immunoprecipitation and immunoblotting
Immunoprecipitation and immunoblotting were performed as previously
described (16, 27). Nuclear extracts (20 ␮g) were incubated overnight with
1:250 anti-RII␤ mAb (Transduction Laboratories, Lexington, KY). Immune complexes were isolated by using affinity-purified goat anti-mouse
IgG conjugated to protein A-Sepharose. RII␤ was then eluted by boiling
the immune complexes in 25 ␮l buffer A (50 mM Tris-HCl (pH 6.8), 30%
(v/v) glycerol, 0.025% bromphenol blue, 2% SDS, and 10% 2-ME) for
3 min at 95°C. Samples were resolved by 10% one-dimensional SDSPAGE. Immunoblots were prepared, probed with 1:1,000 anti-RII␤ mAb,
and developed with enhanced chemiluminescence (ECL) (16).
Nuclei-free T cell homogenate was also prepared as previously described (16). Following separation of T cell homogenate (200 ␮g) by onedimensional SDS-PAGE and transfer to Immobilon-P (Millipore, Bedford,
MA), membranes were immunoblotted with 1:250 anti-RII␣ mAb (Transduction Laboratories) or 1:1000 anti-RII␤ mAb, washed with buffer C (100
mM Tris-HCl (pH 7.5), 500 mM NaCl, and 0.1% Tween 20), and probed
with 1:4000 HRP-labeled sheep anti-mouse IgG in Blotto. After four washings with buffer C, the blots were developed by ECL. Primary and secondary Abs were then extracted from the membrane using buffer D (62.5
mM Tris-HCl (pH 6.7), 100 mM 2-ME, and 2% SDS) (16) and were reprobed with 1:100 polyclonal rabbit anti-human actin, 1:4000 HRP-labeled
sheep anti-rabbit IgG, and ECL. Quantification of RII␣ and RII␤ proteins
in total T cell protein or isolated nuclei was performed by laser densitometry, the amounts calculated from reference standard curves, and expressed
as arbitrary densitometric units (ADU) (16).
Confocal microscopy
Confocal immunofluorescence microscopy was performed as previously
detailed (17). Cells were centrifuged at 600 ⫻ g, transferred to Eppendorf
tubes, and centrifuged at 4000 rpm for 4 min at 4°C. The pellet was resuspended in 100 ␮l fixation buffer (4.0% paraformaldehyde, 120 mM
sucrose in PBS) per tube, and fixation was allowed to proceed at 4°C for
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(26). The R subunits, RI and RII, are comprised of highly homologous ␣ and ␤ isoforms (i.e., RI␣/␤ and RII␣/␤) and the catalytic
(C) subunits of ␣, ␤, and ␥ isoforms. In their holoenzyme forms,
both isozymes exist primarily as homodimers, RI␣/␤2C2 and RII␣/
␤2C2. In the T cell, PKA isozymes can be activated via two mechanisms. Occupancy by an agonist of Gs-bound stimulatory receptors (Rs) activates adenylyl cyclase (AC), hydrolyzing ATP to
cAMP. Alternatively, binding of antigenic peptide to the TCR initiates a signal from the CD3 complex that bifurcates at the level of
protein kinase C, phosphorylates AC, and stimulates AC catalysis
and cAMP turnover (27). Binding of cAMP to the A- and B-binding domains of the R subunits activates the holoenzymes, as shown
by the equation: R2C2 ⫹ 4cAMP 7 R2cAMP4 ⫹ 2C (25).
The present study was undertaken to explore the idea that the
profound deficiency of total PKA activity in SLE T cells is in part
the result of deficient PKA-II phosphotransferase isozyme activity.
During the course of our work, we have observed that markedly
diminished total PKA activity in SLE T cells was associated with
a concomitant reduction of PKA-II activity in the cells of some
subjects with deficient PKA-I isozyme activity (13). To document
PKA-II isozyme deficiency, we performed a prospective analysis
of 35 unselected, consecutive SLE subjects and controls. This
analysis revealed that: 1) SLE T cells can harbor a significant,
co-existent reduction of PKA-II activity; 2) the prevalence of deficient PKA-II activity in this cohort is 37%; 3) like deficient
PKA-I activity, there is no apparent relationship of deficient
PKA-II activity and SLE disease activity; and 4) the mechanism of
this isozyme deficiency is aberrant translocation of the RII␤ isoform to the nucleus from the cytosol and its retention in the nucleus. This association of nuclear translocation of a protein kinase
regulatory subunit with a persistent deficiency of protein kinase
activity is a novel mechanism in primary T cells. Moreover, this
mechanism is distinct from that of deficient PKA-I isozyme activity (16). Together, deficient PKA-I and PKA-II activities yield a
profound reduction of total cAMP-activatable PKA activity, which
may significantly impede TCR-initiated signaling (27) and contribute to compromised T cell effector functions in SLE (10).
2831
2832
DEFICIENT T CELL PKA-II ACTIVITY IN LUPUS
30 min (32). PBS, 1 ml, with 1 mg/ml BSA (PBS/BSA) was added to each
tube and centrifuged, and the pellet was resuspended in 100 ␮l quench
solution (50 mM NH4Cl in PBS) to stop the fixation. After the pellet was
washed twice in PBS/BSA, fixed cells were resuspended in 100 ␮l permeabilization buffer (0.2% Triton X-100, 1 mg/ml BSA in PBS) containing
1:50 anti-RII␣, anti-RII␤, or anti-C␣ subunit mAbs, and the cells were
incubated for 60 min at room temperature. After permeabilization of the
cells, the resulting pellet was resuspended in permeabilization buffer containing 1:50 FITC-F(ab)2 goat anti-mouse IgG. This suspension was incubated for 45 min at room temperature, the labeled cells were washed twice
with permeabilization buffer, and the cells were then incubated in 5 ␮M PI
to label nuclei. After the cells were washed, the pellet was resuspended in
20 ␮l of the DABCO/Mowiol solution (33, 34) and examined with a Zeiss
LSM 510 confocal microscope.
Table I. T cell PKA-II isozyme activities in SLE and normal control
subjects
Statistical analysis
lence of deficient PKA-II isozyme activity in this SLE cohort is
37% (13 of 35 subjects).
Results
Deficiency of PKA-II isozyme activity in SLE T lymphocytes
To quantify PKA-II isozyme phosphotransferase activity, we isolated PKA-II holoenzyme in T cell homogenates by tandem DE52cellulose and carboxymethyl-Sephadex column chromatography
using a salt gradient. Compared with the PKA-I holoenzyme that
elutes between 85 and 110 mM NaCl, the PKA-II holoenzyme
characteristically elutes from DE52-cellulose columns between
180 and 220 mM NaCl (26). The mean (⫾SD) PKA-II phosphotransferase sp. act. in normal T cells from 35 subjects was 481.2 ⫾
144.1 pmol/min/mg protein. By contrast, T cells from SLE subjects exhibited a mean (⫾SD) PKA-II activity of 293.8 ⫾ 192
pmol/min/mg (Fig. 1A and Table I). Although there was some
overlap between SLE and controls, the mean PKA-II activity in
SLE T cells was 61% of controls ( p ⱕ 0.001 by paired Student’s
t test). If deficient PKA-II isozyme is defined as phosphotransferase activity ⱕ 193 pmol/min/mg (i.e., ⫽ 2 SD), then the preva-
Mean ⫾ SD
Range
25th–75th percentiles
Normal range
Controls
(n ⫽ 35)
SLE
(n ⫽ 35)
481.2 ⫾ 144.1
223–847
394–555
193–769
293.8 ⫾ 192
0–992
148–413
Relationship of PKA-II isozyme activity to disease activity or
therapy
The relationship between PKA-II isozyme activity and SLE disease activity was analyzed to establish whether or not deficient
isozyme activity is related to SLE disease activity. There were no
significant differences between mean PKA-II activities in subjects
with severe, moderate, or mild disease activity. Moreover, the 13
subjects with deficient PKA-II activities were distributed among
these groups. Their mean PKA-II activity was 103.8 ⫾ 56.8 pmol/
min/mg (25–75%, 72–140 pmol/min/mg; Fig. 1B, p ⱕ 0.001). Unexpectedly, the mean SLEDAI score of SLE subjects with physiologic PKA-II activities was 12.4 compared with 10.6 in subjects
with deficient PKA-II activities (Fig. 1B). Although this difference
between the SLEDAI scores was not statistically significant, SLE
subjects with physiologic PKA-II activities actually exhibited
greater clinical disease activity than those with lower isozyme activities. These data suggest that deficient PKA-II isozyme activity
may not necessarily be associated with SLE disease activity.
To determine whether PKA-II activity was associated with therapy, the medical regimen of each subject at enrollment and at three
subsequent intervals during 4 years was analyzed. There was no
statistical relationship between any medical therapy and PKA-II
isozyme activities in SLE subjects with mild, moderate, or severe
FIGURE 1. Relationship of PKA-II isozyme activity to disease activity in SLE. A, T cell PKA-II activities in SLE and control populations (n ⫽ 35
subjects, respectively). B, SLE subjects were divided into two groups based on their PKA-II activities: group I, physiologic PKA-II activities (n ⫽ 22
subjects); and group II, deficient PKA-II activities (n ⫽ 13 subjects). Comparison of SLEDAI scores between groups I and II reveals that the scores were
higher in group I with physiologic PKA-II activities than in group II with deficient PKA-II activities. u, PKA-II activity in normal controls; f, PKA-II
activities for SLE; o, SLEDAI scores for SLE subjects.
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The prevalence of PKA-II isozyme deficiency is defined as the probability
of currently having that isozyme deficiency regardless of the duration of
time one has had the disorder. It is calculated by dividing the number of
subjects with the isozyme deficiency by the number of subjects in the study
population. Statistical significance ( p ⫽ 0.05) was calculated by the paired
Student t test, Mann-Whitney U rank-sum test, or ANOVA (SigmaStat,
Jandel Scientific, Corte Madera, CA). Except where indicated, means
(⫾SEM) are used throughout the text.
PKA-II sp. act. (pmol/min/mg protein)
The Journal of Immunology
SLE activity. In five instances, comparison of PKA-II activities
before and after corticosteroid therapy demonstrated no significant
differences between PKA-II activities. Moreover, five subjects
whose disease became clinically inactive and were able to discontinue therapy revealed no significant change in their PKA-II activities over time (data not shown). Together, these results suggest
that therapy is unlikely to modify T cell PKA-II activity and, therefore, is also unlikely to be implicated in this T cell isozyme deficiency in SLE.
Persistence of PKA-II isozyme deficiency over time
In vitro culture does not reverse deficient SLE T cell PKA-II
isozyme activity
To determine whether or not deficient SLE T cell PKA-II isozyme
activity is reversible, we established T cell lines and studied cells
that had been propagated over 10 passages. The advantage of this
approach is that it is possible to study the progeny of SLE T cells
that were originally isolated from PBMC but have not been exposed to the disease process. Thus, any identified defects cannot be
attributed to extracellular stimuli, such as cytokines or immune
complexes, that may be present in the lupus microenvironment
in vivo.
T cell lines from three untreated SLE patients with markedly
reduced PKA-II activities and three healthy controls were established concomitantly. After 10 passages, cycling T cells were harvested, the proportions of cells in each phase of the cell cycle were
determined, and PKA-II isozyme activities were quantified. Both
SLE and control T cell lines had equivalent proportions of cells in
FIGURE 2. Comparison of PKA-II isozyme activities and SLEDAI
scores in 15 SLE subjects during a 4-year interval. PKA-II activities remained stable despite significant improvement in SLEDAI scores during
this interval. Left three-bar group, PKA-II activity; right three-bar group,
SLEDAI scores.
each phase of the cell cycle; 35% of the cells were in S phase.
Compared with a mean PKA-II activity of 164.5 ⫾ 53 pmol/
min/mg in freshly isolated SLE T cells in G0 /G1, cells in S phase
had a 29.2% reduction of the mean PKA-II isozyme activity to
116.5 ⫾ 10.6 pmol/min/mg ( p ⫽ NS). By contrast, the mean
PKA-II isozyme activity in control T cell lines in S phase was
reduced by a mean 58.6% (224.7 ⫾ 146 pmol/min/mg) compared
with freshly isolated T cells in G0 /G1 (542.6 ⫾ 187 pmol/min/mg)
( p ⫽ 0.017). These results reveal that a proportion of the total
PKA-II holoenzyme is activated in cycling T cells, resulting in a
reduction in the amount of residual PKA-II holoenzyme. A similar
effect of T cell proliferation on PKA-I activity has been previously
observed (35). That the magnitude of PKA-II activation and utilization in SLE T cells was diminished by one-half (29.2% vs
58.6%) may reflect the low PKA-II activity in G0 /G1 cells.
SLE and control T cells were then rested for 72 h in low FCScontaining media in the absence of cytokines and mitogen to force
cells to reenter G0 /G1 phase of the cell cycle, and PKA-II activity
was quantified in these cells. Although ⬎95% of cells had returned
to G0 /G1 phase, PKA-II activity remained depressed in SLE cells
but had increased 30% (298.3 ⫾ 90 pmol/min/mg) toward baseline
levels in control cells. Quantification of PKA-II activity in T cells
cultured for ⬎72 h in media containing very low concentrations of
FCS and no cytokines or mitogens was unreliable due to increasing
cell death. Together, these data suggest that the progeny of SLE T
cells have a persistent deficiency of PKA-II activity that is independent of cell activation and mitogenesis as well as the lupus
microenvironment.
Analysis of RII isoform content in normal and SLE T cell lines
That normal cycling T cells undergo a reduction of PKA-II holoenzyme activity that is partially repleted during the resting phase
of the cell cycle raised the possibility that the content of a RII
isoform comprising the holoenzyme could be altered. Because the
PKA-II isozyme is predominantly localized within the cytosol in
human T cells (26), a reduced amount of cytosolic RII␤ and/or
FIGURE 3. Expression of RII␣ and RII␤ protein in SLE and control T
cells. Freshly isolated CD3 T cells were ⬎98.5% G0 /G1 phase of the cell
cycle. T cell lines were established as detailed in Materials and Methods.
The CEM-SS T cell leukemia and rat pituitary cell lines were used as
controls for RII␣ and RII␤ protein expression, respectively. Normal control T cells (N) expressed RII␤ and RII␣ proteins in a ratio of ⬃4:1. By
contrast, SLE T cells (L) had increased RII␣ protein, but no detectable
RII␤ protein. Cycling normal and SLE T cells were 35% S phase, respectively. During cycling, neither normal nor SLE T cells expressed RII␤
protein. After resting cells for 72 h in low FCS-containing medium, ⬎95%
normal and SLE T cells were in G0 /G1 and ⬎98% were viable. Although
rested control T cells in G0 /G1 reexpressed RII␤, rested SLE T cells had no
detectable RII␤ protein. The amount of RII␣ was heightened in SLE compared with normal control T cells. Values are representative of three independent experiments using normal control and SLE T cell lines.
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To determine whether deficient PKA-II activity persists over time
and remains independent of disease activity, a group of 15 SLE
patients with an initial mean (⫾SD) SLEDAI score of 12.1 ⫾ 7.2
(25–75%, 6.5–15.5) was followed up to 4 years and restudied on
at least three occasions. Of these, six initially had mild SLE activity, eight had moderate activity, and one had severe activity.
Fig. 2 demonstrates that there were essentially no differences in
PKA-II activities during the follow-up interval. In contrast, subjects being treated for SLE experienced a significant reduction in
their SLEDAI scores between the first and second follow-up studies ( p ⫽ 0.02), but no significant change between the second and
third follow-up analyses. Thus, low PKA-II activities persist over
time and are independent of disease activity.
2833
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DEFICIENT T CELL PKA-II ACTIVITY IN LUPUS
FIGURE 4. RII␣ and RII␤ protein expression in SLE and control T cells. Freshly isolated T cells from 6 normal controls (A), SS controls (B), and SLE
subjects (C) were lysed; the lysates (200 ␮g protein/lane) comprising plasma membrane and cytosolic proteins were separated by 10% SDS-PAGE, and
the proteins were immunoblotted with anti-RII␣, anti-RII␤, and anti-actin mAbs. Equivalent amounts of actin protein demonstrated that equal amounts of
lysate were loaded into each lane. To quantify the amounts of RII␣ and RII␤ proteins by laser densitometry, we used reference standard curves to obviate
any potential differences in ECL expression on autoradiographs exposed for 1 min. The amounts of each isoform protein are expressed as ADU.
Quantification of T cell RII␣ and RII␤ protein content in SLE
and controls
To determine whether cytosolic RII␤ protein is reduced or absent
in SLE T cells, we examined freshly isolated T cells from 21 SLE
subjects, 21 healthy controls, and 11 SS disease controls. The immunoblots shown in Fig. 4 demonstrated 1) the presence of cytosolic RII␤ and RII␣ proteins in normal controls in a ratio of 3.95:1,
2) decreased cytosolic RII␣ and increased cytosolic RII␤ in SS
subjects yielding an increased ratio of 5.35:1, and 3) absence of
cytosolic RII␤ and increased cytosolic RII␣ protein in SLE subjects. The T cells of all SLE subjects shown in Fig. 4 had deficient
PKA-II activity. Table II shows that, on average, there was a 60%
reduction of cytosolic RII␤ in SLE T cells, yielding a significantly
reduced RII␤:RII␣ ratio of 1.28:1 compared with normal and SS
controls. That both normal and SS control T cells have significantly greater cytosolic RII␤ content than SLE T cells suggests
disease specificity. In sum, these results demonstrate that deficient
PKA-II activity in SLE T cells is associated with reduced cytosolic
RII␤ protein content. In 29% (6 of 21) of subjects, there was no
detectable cytosolic RII␤ protein.
Aberrant nuclear translocation of RII␤ from the cytosol in SLE
T cells
Utilizing confocal immunofluorescence microscopy, we have recently demonstrated that activation of the PKA-II isozyme in normal T cells by either 8-chloro-cAMP (8-Cl-cAMP) or anti-CD3 ⫹
anti-CD28 ⫹ rIL-1␣ induced nuclear translocation of RII␤ within
30 min that peaked by 1 h (36). Here, we observed that SLE T cells
exhibited spontaneous translocation of RII␤ from the cytosol to the
nucleus in the absence of in vitro cell activation (Fig. 5B). On
average, 60% of SLE T cells had detectable nuclear RII␤, whereas
only 3% of normal T cells exhibited nuclear translocation of RII␤
by confocal immunofluorescence microscopy (Fig. 6). None of the
T cells from normal or SS controls shown in Fig. 5B had detectable
nuclear RII␤. By contrast, the RII␣ isoform remained localized to
the cytosol in SLE T cells (Fig. 5A). Because it has been well
demonstrated that the C subunit can diffuse between the cytoplasm
and nucleus and can, therefore, be present in both compartments
Table II. Quantification of T cell RII␣ and RII␤ protein content in SLE and controlsa
Controls
Disease
RII
Isoforms
Normal
SS
SLE
p
RII␣
RII␤
RII␤:RII␣
1.00 ⫾ 0.13
3.95 ⫾ 0.42
3.95:1
1.58 ⫾ 0.36
8.46 ⫾ 1.01
5.35:1
1.24 ⫾ 0.18
1.59 ⫾ 0.49
1.28:1
0.371,b 0.46,c 0.377d
⬍0.001,b ⬍0.001,c ⬍0.001d
ADU ⫾ SEM.
Normal vs SS.
Normal vs SLE.
d
SS vs SLE.
a
b
c
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RII␣ protein is one mechanism that could yield diminished PKA-II
phosphotransferase activity. To test this idea, nuclei-free T cell
homogenates were prepared from 1) freshly isolated T cells, 2)
cycling cells (after 10 passages), and 3) rested T cells (at 72 h).
The homogenates were fractionated by 10% one-dimensional
SDS-PAGE, electroblotted to Immobilon membrane, and immunoblotted with anti-RII␣ and anti-RII␤ mAbs. Fig. 3 demonstrates
that freshly isolated control T cells possessed both cytosolic RII␤
and RII␣ isoforms. After 10 passages, cycling T cell progeny from
controls expressed increased amounts of cytosolic RII␣ protein,
but no detectable cytosolic RII␤ isoform. After resting cells for
72 h, at which time ⱖ95% of cells were in G0 /G1 phase, control
T cells reexpressed cytosolic RII␤ protein, and the amount of RII␣
returned toward baseline. Thus, RII␤ was depleted from the cytosol whereas RII␣ accumulated in the cytosol of cycling normal
T cells. This depletion of RII␤ protein, and therefore RII␤2C2
holoenzyme, may account for the reduction of PKA-II activity
during mitogenesis.
Recognition that reduced PKA-II activity was associated with
depletion of RII␤ in normal T cells prompted us to determine
whether cytosolic RII␤ is present in freshly isolated SLE T cells.
Fig. 3 shows that cytosolic RII␤, but not RII␣, was absent in
freshly isolated SLE T cells. Like cycling normal T cells, cycling
SLE T cells revealed no cytosolic RII␤; instead, there was accumulation of RII␣ (Fig. 3). However, in contrast to normal rested T
cells, cytosolic RII␤ failed to be reexpressed in rested SLE T cells
(Fig. 3) in which ⱕ4% of cells were in S phase. Moreover, the
amount of cytosolic RII␣ remained increased. Taken together,
these results suggested that a disorder of RII␤ regulation might be
associated with deficient PKA-II activity in SLE T cells.
The Journal of Immunology
2835
FIGURE 6. Nuclear translocation of the RII␤ subunit in SLE T cells. A, Confocal immunofluorescence
microscopy reveals that ⱕ3% of normal control T cells
have detectable RII␤ nuclear translocation. PI stains the
nucleus red. Anti-RII␤ mAb/FITC-labeled F(ab)2 goat
anti-mouse IgG stains the cytosol green, indicating the
presence of RII␤ subunit in the cytosol. Computer-assisted superimposition of the images confirms the presence of RII␤ in the cytosol. B, Confocal immunofluorescence microscopy reveals that, on average, 60% of
SLE T cells have detectable RII␤ nuclear translocation.
Anti-RII␤ mAb/FITC-labeled F(ab)2 goat anti-mouse
IgG stains the nucleus green in cells exhibiting nuclear
RII␤. Computer-assisted superimposition of the PI and
FITC images shows yellow staining of the nucleus, confirming the colocalization of PI and FITC images and
indicating the presence of nuclear RII␤.
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
FIGURE 5. Subcellular localization of RII␣-, RII␤- and C subunits in SLE and control T cells. A, Confocal immunofluorescence microscopy of RII␣
subunit localization in the cytosol in T cells from SLE subjects and normal and SS controls. PI stains the nucleus red. Anti-RII␣ mAb/FITC-labeled F(ab)2
goat anti-mouse IgG stains the cytosol green. Computerized superimposition of the 2 images gives a green cytosol and red nucleus. B, Confocal immunofluorescence microscopy of RII␤ subunit localization in the cytosol of normal and SS T cells and in the nucleus of SLE T cells. Anti-RII␤ mAb/FITClabeled F(ab)2 goat anti-mouse IgG staining demonstrates the presence of RII␤ in the nucleus. This is verified by computer-assisted superimposition of the
PI and FITC images, which reveals a yellow color in the nucleus. Compared with normal and SS T cells, there is no detectable cytosolic RII␤. C, Confocal
immunofluorescence microscopy of the C␣ subunit reveals its presence in both the nucleus and cytosol of normal and SS control T cells; in this SLE T
cell sample, there was no detectable nuclear C subunit. D, Quantification of constitutive nuclear RII␤ subunit in SLE and control T cells by immunoprecipitation and immunoblotting. The RII␤ control in lane 1 is from the Raji B cell line.
2836
DEFICIENT T CELL PKA-II ACTIVITY IN LUPUS
(37), we anticipated the presence of the C subunit in both compartments of T cells (Fig. 5C).
To quantify the amounts of nuclear RII␤ protein in SLE and
controls, we immunoprecipitated RII␤ from nuclear extracts and
quantified RII␤ proteins on immunoblots by densitometry. Unexpectedly, RII␤ protein was constitutively present in the nuclei of
both normal and SS disease controls (Fig. 5D). It is likely that RII␤
was identified by immunoblotting, but not by confocal immunofluorescence microscopy, because the amount of constitutive nuclear RII␤ in control T cells was below the limits of detection by
confocal immunofluorescence microscopy. By immunoblotting,
there was a 54 and 39% increase in nuclear RII␤ protein in SLE T
cells compared with normal and SS controls, respectively (n ⫽ 6,
SLE vs normal controls, p ⱕ 0.001). Thus, exaggerated nuclear
translocation of RII␤ appears to be the mechanism responsible for
deficient PKA-II activity in SLE T cells.
Persistence of nuclear RII␤ after in vitro passage of SLE T cells
Dexamethasone does not alter nuclear RII␤ subunit content in
normal T cells
Although we observed no in vivo effect of corticosteroids on
PKA-II activity, we considered the possibility that corticosteroids
might modify T cell RII subunit protein content or its subcellular
localization. To test this possibility, freshly isolated T cells from
three healthy controls were cultured in the absence or presence of
10 nM dexamethasone for 18 h, and total T cell lysates were separated by SDS-PAGE, transferred to Immobilon membrane, and
immunoblotted with anti-RII␣, anti-RII␤, and anti-actin mAbs.
Compared with untreated cells, dexamethasone did not alter the
total T cell content of either RII isoform or actin over time (data
not shown). To test whether or not dexamethasone alters 8-ClcAMP-induced nuclear RII␤ translocation, normal T cells were
cultured in the absence or presence of 10 nM dexamethasone for
18 h; the cells were then incubated for 1 h in the absence or presence of 20 ␮M 8-Cl-cAMP; the nuclei were separated; and, RII␤
was isolated by immunoprecipitation and quantified after immunoblotting with anti-RII␤ mAb. Compared with untreated cells,
dexamethasone produced no significant change in cell viability.
Moreover, dexamethasone did not alter 8-Cl-cAMP-induced nuclear translocation of RII␤ (Fig. 7). These results suggest that
dexamethasone is unlikely to modify T cell RII subunit protein
content or its subcellular localization.
Discussion
The results of this work demonstrate that T lymphocytes from
subjects with SLE may harbor a deficiency of PKA-II isozyme
phosphotransferase activity. The prevalence of this isozyme deficiency in this study population was 37%. Our previous work has
FIGURE 7. Effect of dexamethasone on 8-Cl-cAMP-induced nuclear
translocation of the RII␤ subunit. Normal primary T cells were incubated
for intervals between 0 and 18 h in the absence or presence of 10 nM
dexamethasone. Cells were harvested and cultured in the absence or presence of 20 ␮M 8-Cl-cAMP for 1 h at 37°C; nuclei were isolated and a
lysate was prepared; the RII␤ subunit was isolated by immunoprecipitation
with anti-RII␤ mAb and identified by immunoblotting with anti-RII␤
mAb; and the amount of nuclear RII␤ subunit was quantified by laser
densitometry in ADU. Values are representative of three independent experiments performed.
also revealed the presence of deficient PKA-I activity in the T cells
of 80% of SLE subjects (13–16). In this cohort of SLE subjects,
one-third possessed a combined deficiency of both isozymes. On
average, the mean specific activity of PKA-II was ⬃60% that of
age-, gender-, and racially matched controls. However, three subjects had profoundly low isozyme activities, less than 10% of the
mean physiologic specific activity. By contrast, PKA-I isozyme
activity in SLE T cells ranges between 20 and 25% of controls
(16). Of particular interest is that there was no relationship between the extent of deficient PKA-II activity and SLE disease activity over a period of 4 years as quantified by a standardized
disease activity index. These results mirror our recent findings for
deficient PKA-I activity, in which diminished PKA-I activity was
found to be persistent over time and independent of disease activity (14). Considering these data, it is reasonable to conclude that
the profound decrement in total PKA activity reflects a deficiency
of PKA-I and/or PKA-II isozyme activities in SLE T cells. We
propose that a significant deficiency of PKA activity could markedly hinder effective signal transduction by impairing efficient substrate phosphorylation in SLE T cells.
In normal T cells, the PKA-II isozyme is predominantly found
in the cytosol in its holoenzyme forms, RII␣2C2 and RII␤2C2 (26).
However, the amount of RII␤ protein is ⬃4-fold higher than that
of RII␣. Interestingly, T cells from SS disease controls actually
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Absence of cytosolic RII␤ protein in freshly isolated SLE T cells
and its persistent absence in rested T cell progeny raised the question, “What is the disposition of RII␤?” To determine whether
RII␤ might be retained in the nucleus, nuclear extracts from
freshly isolated SLE T cells, cells cycled through 10 passages, and
cells rested over 72 h were immunoprecipitated, gel separated and
electrotransferred, and immunoblotted with anti-RII␤ mAb. Based
on quantification by densitometry, there was no appreciable
change in the amount of nuclear RII␤ protein in in vitro-rested T
cell progeny compared with freshly isolated or in vitro-propagated
T cells (data not shown). These results were consistent with the
continued absence of cytosolic RII␤ in SLE T cells and suggested
that, after its enhanced nuclear translocation, RII␤ is retained in
the nucleus of SLE T cells and that this retention is independent of
the cell cycle.
The Journal of Immunology
ronment. Once these T cell progeny from established T cell lines
reentered G0 /G1 phase of the cell cycle, there was still no detectable cytosolic RII␤; by contrast, control T cell progeny again expressed cytosolic RII␤. Moreover, PKA-II activities remained
markedly depressed and unchanged from that of freshly isolated
SLE T cells whereas that of control T cells increased toward baseline activities of quiescent cells. These results are consistent with
the idea that 1) PKA-II activity is diminished due to reduced/absent cytosolic RII␤ with which to form the holoenzyme, RII␤2C2,
and 2) RII␤ is retained in the nucleus.
Subcellular localization of PKA R and C subunits appears to be
a principal mechanism to juxtapose the kinase to cAMP and its
substrates. Longstanding evidence supports the concept that the
PKA isozymes are localized to discrete regions of cells and that
this is often cell-type specific (44 – 47). In the human T cell, PKA-I
associates with the plasma membrane fraction, whereas PKA-II is
localized to the cytosol (26). Within the cytosol, the RII␣2C2 and
RII␤2C2 holoenzymes are compartmentalized to cytoskeletal elements and cytosolic organelles by attachment to anchoring structures termed A kinase anchor proteins (AKAPs) (48). RII␤ binds
to AKAP75 in neuronal cells (49) and in T cells (Shook and G. M.
Kammer, unpublished data), where it is likely to be in its holoenzyme form. Here, on its activation by cAMP, RII␤2C2 holoenzyme
dissociates to free RII␤ and C subunits and RII␤ can shuttle to the
nucleus. This mechanism is shown in Fig. 8A. After its release
from the cAMP response element binding protein (CREB) heterodimer, our current evidence suggests that RII␤ is then exported
from the nucleus to the cytosol where it can then bind AKAP75
and reform holoenzyme. However, the mechanism by which RII␤
is exported remains to be established. On the basis of our current
data, we propose that in SLE T cells spontaneous activation of the
RII␤2C2 holoenzyme promotes aberrant nuclear RII␤ translocation
and sequestration, resulting in deficient PKA-II activity (Fig. 8B).
Two pertinent issues are currently under study. First, what is the
stimulus that initiates RII␤2C2 activation, resulting in RII␤ nuclear
translocation? One mechanism may be cytokine receptor-mediated
activation of the AC-cAMP-PKA pathway. To date, TGF-␤ is the
only known cytokine that directly activates the AC-cAMP-PKA
pathway in mesangial cells (50) and primary T cells (Choi and G.
M. Kammer, unpublished data). However, synthesis of activated
TGF-␤ by NK cells is markedly depressed in SLE (51), making it
unlikely that the AC/cAMP/PKA pathway is being spontaneously
activated via binding of TGF-␤ to its cell surface receptors. Moreover, PKA is unlikely to be activated by enhanced cAMP binding
to RII␤ subunits, for we know that basal intracellular cAMP concentrations are comparable in unstimulated SLE and control T
cells (52, 53). Additionally, if the PKA-II isozyme is like the
PKA-I isozyme (15), the apparent Ka for PKA-II may be increased
and would require higher concentrations of endogenous cAMP to
activate the isozyme.
Second, why does the RII␤ subunit accumulate in the nucleus of
SLE T cells? The RII␤ subunit has a nuclear localization signal
(NLS), KKRK, in its carboxyl terminus. This sequence accounts
for the capacity of RII␤ to enter the nucleus (38). However, the
protein does not possess the consensus nuclear export signal
(NES), XLXXXLXXLXLX (54). Instead, it has a partial sequence, VLDAMFEKLV (55), in which a hydrophobic phenylalanine (F) replaces the nonpolar leucine (L). This 10-aa stretch is
positioned in the cAMP A-binding region between residues 165
and 174. Such replacements of one hydrophobic amino acid for
another in NES sequences have been previously identified, as, for
instance, in cyclin B1 (56). At present, however, it remains uncertain whether this partial sequence is a functional NES. If it is a
functional NES, this may account for the nuclear-cytoplasmic
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express significantly increased amounts of cytosolic RII␤ compared with normal controls, yielding a markedly skewed RII␤:
RII␣ ratio of 5.35:1. At present, however, the mechanism underlying these alterations in RII isoform expression in SS T cells
remains uncertain. Although the RII isoforms are predominantly
localized to the cytosol, a small amount of RII␤ isoform is constitutively present in the nucleus of normal primary T cells. The
presence of constitutive nuclear RII␤ can be detected only by immunoblotting, for the amount is below the sensitivity of confocal
immunofluorescence microscopy. This was true for T cells from
both normal and SS disease controls. Activation of PKA-II by
anti-CD3 ⫹ anti-CD28 ⫹ rIL-1␣ or 8-Cl-cAMP results in the
separation of the RII␣ and RII␤ subunits from the C subunit and
the rapid translocation of RII␤, but not RII␣, to the nucleus from
the cytosol (36). Because this translocation enhances the amount of
nuclear RII␤, this process can be monitored by confocal immunofluorescence microscopy. RII␤ can first be detected in the nucleus
by 30 min and peaks at 1 h. Interestingly, treatment of normal T
cells with dexamethasone, a corticosteroid similar to that commonly used in the treatment of SLE, did not alter the subcellular
localization of the RII␤ subunit or impede its translocation to the
nucleus after activation of the RII␤2C2 holoenzyme by 8-ClcAMP. Because both RII isoforms possess the nuclear localization
sequence, KKRK, in their carboxyl-terminal regions (38), it is uncertain why RII␤, but not RII␣, translocates to the nucleus after
activation of PKA-II. At present, the role of RII␤ in the nucleus is
being studied.
In SLE T cells, we observed an association between deficient
PKA-II activity and enhanced translocation of RII␤ to the nucleus
from the cytosol. On average, 60% of freshly isolated SLE T cells
had identifiable nuclear RII␤ by confocal immunofluorescence microscopy. This compares with only 2–3% of normal and SS control
T cells or 4% of SLE T cells that did not have deficient PKA-II
activity. When the amount of RII␤ was quantified in isolated nuclear and cytosolic extracts, the content of nuclear RII␤ was increased by 54%, and that of cytosolic RII␤ was reduced by 60%
compared with normal T cells. This shift produced a significant
reduction in the ratio of cytosolic RII␤:RII␣ to 1.28:1 from 3.95:1
in normal T cells. The 6% difference in the amount of RII␤ between the cytosolic and nuclear compartments is probably insignificant, for it was within the error of the assay. However, it is
pertinent to point out that, of the 21 SLE subjects that we analyzed,
29% had no detectable T cell cytosolic RII␤ protein. That both
normal and SS control T cells have significantly higher cytosolic
RII␤ protein content than do SLE T cells underscores the disease
specificity of nuclear RII␤ translocation.
The association of a skewed RII␤:RII␣ ratio with diminished
PKA-II activity in freshly isolated SLE T cells raised the possibility that nuclear RII␤ translocation could be triggered by the
lupus microenvironment. SLE plasma often has increased levels of
cytokines (e.g., IFN-␣, IL-10) (39, 40), immune complexes (41),
and complement fragments (42), which may bind to and potentially alter T cell signaling. However, an effect of the lupus microenvironment seems unlikely for two reasons. First, our previous
experiments failed to demonstrate any effects of either IFN-␣ or
immune complexes on PKA-catalyzed protein phosphorylation in
normal T cells cultured in vitro. Contrariwise, culturing SLE T
cells in vitro did not reverse the observed defect in PKA-catalyzed
protein phosphorylation in SLE T cells (43). Second, in the present
experiments, SLE T cells were propagated through 10 passages,
and the progeny were analyzed. These T cell progeny had never
been exposed to the lupus microenvironment and would, therefore,
not be expected to exhibit any putative aberrant functions that
freshly isolated T cells might express from exposure to that envi-
2837
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DEFICIENT T CELL PKA-II ACTIVITY IN LUPUS
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FIGURE 8. Proposed models of the mechanism of RII␤ subunit nuclear translocation. A, Normal T cells. After initiation of a signal through the
TCR-CD3 complex, the signal is transduced via protein kinase C to activate the AC-cAMP-PKA pathway. Binding of cAMP to the R subunits of PKA-I
and PKA-II isozymes activates these isozymes. On activation of the PKA-II isozyme, the RII␤ subunit translocates from the cytosol to the nucleus. Current
evidence suggests that RII␤ forms a heterodimer with CREB, a nuclear transcription factor, and that the CREB-RII␤ heterodimer can bind to cAMP
response elements (CRE) of promoters on genes such as c-fos. The free C subunit can diffuse into the nucleus, where it can phosphorylate substrates. After
dissociation from CREB, RII␤ can translocate to the cytosol and reform a holoenzyme with C subunit. B, SLE T cells. Apparent spontaneous activation
of the RII␤2C2 holoenzyme by an unknown mechanism promotes aberrant nuclear translocation of RII␤ to the nucleus. RII␤ appears to be retained in the
nucleus. Together, these events lead to depletion of cytosolic RII␤, reduced formation of RII␤2C2 holoenzyme, and deficient PKA-II isozyme activity. SRE,
serum responsive element.
The Journal of Immunology
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is unassociated with disease activity. In about one-third of subjects, both PKA-I and PKA-II deficiencies can coexist. Of particular interest is the recognition that the mechanisms underlying
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