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,
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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
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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60% of SLE T cell nuclei compared with only 2–3% of
normal and disease controls. Quantification of nuclear RIIß subunit
protein content by immunoprecipitation and immunoblotting demonstrated
a 54% increase over normal T cell nuclei. Moreover, the RIIß 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 RIIß subunit
and its retention in the nucleus in SLE T cells. Deficient PKA-II
activity may contribute to impaired signaling in SLE T
cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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by Th1 cells and overproduction of IL-6 and IL-10 by Th2
cells (6, 7, 8, 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 cAMP-binding
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 RIß 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, 23, 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 (26). The R
subunits, RI and RII, are comprised of highly homologous and ß
isoforms (i.e., RI/ß and RII/ß) and the catalytic (C)
subunits of , ß, and isoforms. In their holoenzyme forms, both
isozymes exist primarily as homodimers,
RI/ß2C2 and
RII/ß2C2. 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 
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 RIIß 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).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 
21 severe disease
activity (13, 14, 15, 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 (
10 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
.
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Nuclear extracts were prepared from the T cells of normal and SS disease controls and SLE subjects. T cells (80 x 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 µg/ml each leupeptin and aprotinin). After 10 min on ice, 50 µl 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 µl 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 µg/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 µg/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 µg) were incubated overnight with 1:250 anti-RIIß mAb (Transduction Laboratories, Lexington, KY). Immune complexes were isolated by using affinity-purified goat anti-mouse IgG conjugated to protein A-Sepharose. RIIß was then eluted by boiling the immune complexes in 25 µl 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 SDS-PAGE. Immunoblots were prepared, probed with 1:1,000 anti-RIIß 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 µg)
by one-dimensional 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-RIIß 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 RIIß 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 x g, transferred to Eppendorf tubes, and
centrifuged at 4000 rpm for 4 min at 4°C. The pellet was resuspended
in 100 µl fixation buffer (4.0% paraformaldehyde, 120 mM sucrose in
PBS) per tube, and fixation was allowed to proceed at 4°C for 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 µl
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 µl permeabilization buffer (0.2% Triton
X-100, 1 mg/ml BSA in PBS) containing 1:50 anti-RII,
anti-RIIß, 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 µM PI to label nuclei. After the cells
were washed, the pellet was resuspended in 20 µl of the DABCO/Mowiol
solution (33, 34) and examined with a Zeiss LSM 510
confocal microscope.
Statistical analysis
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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To quantify PKA-II isozyme phosphotransferase activity, we
isolated PKA-II holoenzyme in T cell homogenates by tandem
DE52-cellulose 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 0.001 by paired
Student’s t test). If deficient PKA-II isozyme is defined
as phosphotransferase activity 193 pmol/min/mg (i.e., = 2 SD),
then the prevalence of deficient PKA-II isozyme activity in this SLE
cohort is 37% (13 of 35 subjects).
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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 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 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
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.
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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 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 FCS-containing 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 RIIß and/or 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-RIIß mAbs. Fig. 3 demonstrates that freshly isolated
control T cells possessed both cytosolic RIIß and RII isoforms.
After 10 passages, cycling T cell progeny from controls expressed
increased amounts of cytosolic RII protein, but no detectable
cytosolic RIIß isoform. After resting cells for 72 h, at which
time 95% of cells were in G0/G1 phase,
control T cells reexpressed cytosolic RIIß protein, and the amount of
RII returned toward baseline. Thus, RIIß was depleted from the
cytosol whereas RII accumulated in the cytosol of cycling normal T
cells. This depletion of RIIß protein, and therefore
RIIß2C2 holoenzyme, may
account for the reduction of PKA-II activity during mitogenesis.
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shows that
cytosolic RIIß, but not RII, was absent in freshly isolated SLE T
cells. Like cycling normal T cells, cycling SLE T cells revealed no
cytosolic RIIß; instead, there was accumulation of RII (Fig. 3).
However, in contrast to normal rested T cells, cytosolic RIIß failed
to be reexpressed in rested SLE T cells (Fig. 3) in which 4% of
cells were in S phase. Moreover, the amount of cytosolic RII
remained increased. Taken together, these results suggested that a
disorder of RIIß regulation might be associated with deficient PKA-II
activity in SLE T cells.
Quantification of T cell RII and RIIß protein content in SLE
and controls
To determine whether cytosolic RIIß 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 RIIß and RII proteins in normal
controls in a ratio of 3.95:1, 2) decreased cytosolic RII and
increased cytosolic RIIß in SS subjects yielding an increased ratio
of 5.35:1, and 3) absence of cytosolic RIIß 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 RIIß in SLE T cells, yielding a
significantly reduced RIIß:RII ratio of 1.28:1 compared with
normal and SS controls. That both normal and SS control T cells have
significantly greater cytosolic RIIß 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 RIIß protein content. In 29% (6 of 21) of subjects, there
was no detectable cytosolic RIIß protein.
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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 RIIß within 30 min that
peaked by 1 h (36). Here, we observed that SLE T
cells exhibited spontaneous translocation of RIIß 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 RIIß, whereas only 3% of normal T cells
exhibited nuclear translocation of RIIß by confocal
immunofluorescence microscopy (Fig. 6).
None of the T cells from normal or SS controls shown in Fig. 5B had detectable nuclear RIIß. 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 (37), we
anticipated the presence of the C subunit in both compartments of T
cells (Fig. 5C).
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D). It is likely that RIIß was
identified by immunoblotting, but not by confocal immunofluorescence
microscopy, because the amount of constitutive nuclear RIIß 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 RIIß protein in SLE T cells compared with
normal and SS controls, respectively (n = 6, SLE vs
normal controls, p 0.001). Thus, exaggerated nuclear
translocation of RIIß appears to be the mechanism responsible for
deficient PKA-II activity in SLE T cells. Persistence of nuclear RIIß after in vitro passage of SLE T cells
Absence of cytosolic RIIß protein in freshly isolated SLE T cells and its persistent absence in rested T cell progeny raised the question, "What is the disposition of RIIß?" To determine whether RIIß 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-RIIß mAb. Based on quantification by densitometry, there was no appreciable change in the amount of nuclear RIIß 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 RIIß in SLE T cells and suggested that, after its enhanced nuclear translocation, RIIß is retained in the nucleus of SLE T cells and that this retention is independent of the cell cycle.
Dexamethasone does not alter nuclear RIIß 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-RIIß, 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-Cl-cAMP-induced nuclear
RIIß 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 µM 8-Cl-cAMP;
the nuclei were separated; and, RIIß was isolated by
immunoprecipitation and quantified after immunoblotting with
anti-RIIß 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
RIIß (Fig. 7). These results suggest
that dexamethasone is unlikely to modify T cell RII subunit protein
content or its subcellular localization.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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,
RII2C2 and
RIIß2C2
(26). However, the amount of RIIß protein is 4-fold
higher than that of RII. Interestingly, T cells from SS disease
controls actually express significantly increased amounts of cytosolic
RIIß compared with normal controls, yielding a markedly skewed
RIIß: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 RIIß isoform is
constitutively present in the nucleus of normal primary T cells. The
presence of constitutive nuclear RIIß 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 RIIß subunits from the C subunit and the rapid
translocation of RIIß, but not RII, to the nucleus from the
cytosol (36). Because this translocation enhances the
amount of nuclear RIIß, this process can be monitored by confocal
immunofluorescence microscopy. RIIß 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 RIIß subunit or impede its translocation to the
nucleus after activation of the
RIIß2C2 holoenzyme by
8-Cl-cAMP. Because both RII isoforms possess the nuclear localization
sequence, KKRK, in their carboxyl-terminal regions (38),
it is uncertain why RIIß, but not RII, translocates to the nucleus
after activation of PKA-II. At present, the role of RIIß in the
nucleus is being studied.
In SLE T cells, we observed an association between deficient PKA-II
activity and enhanced translocation of RIIß to the nucleus from the
cytosol. On average, 60% of freshly isolated SLE T cells had
identifiable nuclear RIIß 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 RIIß was quantified in isolated nuclear and cytosolic
extracts, the content of nuclear RIIß was increased by 54%, and that
of cytosolic RIIß was reduced by 60% compared with normal T cells.
This shift produced a significant reduction in the ratio of cytosolic
RIIß:RII to 1.28:1 from 3.95:1 in normal T cells. The 6%
difference in the amount of RIIß 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
RIIß protein. That both normal and SS control T cells have
significantly higher cytosolic RIIß protein content than do SLE T
cells underscores the disease specificity of nuclear RIIß
translocation.
The association of a skewed RIIß:RII ratio with diminished PKA-II
activity in freshly isolated SLE T cells raised the possibility that
nuclear RIIß 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 environment. Once
these T cell progeny from established T cell lines reentered
G0/G1 phase of the cell cycle, there was
still no detectable cytosolic RIIß; by contrast, control T cell
progeny again expressed cytosolic RIIß. 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 RIIß with which to form the holoenzyme,
RIIß2C2, and 2) RIIß 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, 45, 46, 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
RII2C2 and
RIIß2C2 holoenzymes are
compartmentalized to cytoskeletal elements and cytosolic organelles by
attachment to anchoring structures termed A kinase anchor proteins
(AKAPs) (48). RIIß 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,
RIIß2C2 holoenzyme
dissociates to free RIIß and C subunits and RIIß 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 RIIß is then exported from the nucleus to the
cytosol where it can then bind AKAP75 and reform holoenzyme. However,
the mechanism by which RIIß is exported remains to be established. On
the basis of our current data, we propose that in SLE T cells
spontaneous activation of the
RIIß2C2 holoenzyme
promotes aberrant nuclear RIIß translocation and sequestration,
resulting in deficient PKA-II activity (Fig. 8B).
|
Second, why does the RIIß subunit accumulate in the nucleus of SLE T cells? The RIIß subunit has a nuclear localization signal (NLS), KKRK, in its carboxyl terminus. This sequence accounts for the capacity of RIIß 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 shuttling observed in normal T cells as they reenter the G0/G1 phase of the cell cycle. Because RIIß protein does not possess an apparent consensus nuclear retention signal, which could override the nuclear export signal resulting in retention of the protein in the nucleus (57), this mechanism cannot be invoked to account for the overexpression of nuclear RIIß in SLE T cells. Currently, the mechanism of nuclear retention of RIIß in SLE T cells remains to be established.
In summary, our results reveal that SLE T cells may harbor a deficiency
of PKA-II isozyme activity that persists over time and 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 these isozyme deficiencies
are different. There is a significant reduction of RIß > RI
protein that is associated with a marked reduction of RIß >
RI transcripts in SLE T cells (16). Indeed, our current
data suggest that there may be a pretranslational block of RIß
protein synthesis in the T cells of some SLE subjects (I. U. Khan and
G. M. Kammer, unpublished data). By contrast, deficient
RIIß2C2 activity is a
consequence of spontaneous activation of this holoenzyme, release of
RIIß, and its translocation to and retention in the nucleus.
Long-term overexpression of nuclear RIIß may alter transcriptional
activation of genes, such as c-fos (Fig. 8B).
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Footnotes |
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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.
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/ß, or ß isoform of regulatory subunit of PKA-II; RII/ß2C2 holoenzyme, homodimer of RII or RIIß 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.
Received for publication April 6, 2000. Accepted for publication June 12, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, PKA-II activity in normal controls;
, PKA-II activities for SLE; 
, SLEDAI scores for SLE subjects.
