| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Subunit Deficiency in Lupus T Lymphocytes: Bypassing a Block in RI Translation Reconstitutes Protein Kinase A Activity and Augments IL-2 Production1
Section on Rheumatology and Clinical Immunology, Department of Internal Medicine, Wake Forest University School of Medicine, Winston-Salem, NC 27157
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
/
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
(RI2C2) 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. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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, 5, 6, 7).
RI/2C2 holoenzyme is
comprised of two C subunits joined to either two or two
regulatory (RI) isoforms, resulting in
RI2C2 and
RI2C2 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, particularly the
isoform of RI (RI) (9). Low
RI2C2 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
RI2C2 (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.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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 
2 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 Midi MACS (Miltenyi Biotech, Auburn, CA) as described
(17). Cytofluorographic analysis of T cells demonstrated
that 
96% expressed CD3, which defines mature T cells.
T cell lines
To propagate T cell lines in vitro, PBMCs were cultured in 3:1 RPMI 1640 and HL-1 (BioWhittaker, Walkersville, MD) supplemented with 5% heat-inactivated FCS (HyClone, Logan, UT), 25 mM HEPES, 2 mM L-glutamine, 10 µg/ml streptomycin, 10 IU/ml penicillin, and 1 µg/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 µg/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-leucyl-methional) and lactacystin (Calbiochem, San Diego, CA), were dissolved in DMSO, and were used at a final concentration of 100 and 20 µM, 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.
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 µg 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 µg/µl, then 20 µg 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 (Bio-Rad, Hercules, CA). Protein (150 µg) 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).
35S biosynthetic labeling of RI and C subunits
Three SLE subjects with significantly reduced RI mRNA
transcripts (0.071 ± 0.010 amol/µg of total RNA) compared with
healthy control RI mRNA transcripts (0.209 ± 0.037 amol/µg
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 x
108) were incubated in methionine-free medium for
30 min at 37°C. Cells were then metabolically labeled with 250
µCi/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 µM 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 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 35S
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 µg) 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 µg) was
performed in a rabbit reticulocyte system according to the
manufacturer’s protocol. The final volume was 50 µl 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 µl
of the denatured reaction mix was analyzed on 10% SDS-PAGE. The
proteins were then transferred to polyvinylidene difluoride membrane,
and autoradiography was performed.
RI cDNA overexpression
RI cDNAs from the T cells of five SLE subjects were amplified
by RT-PCR 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 µg/ml PHA
for 22 h at 37°C in 5% CO2
(20). After isolation of T cells, 35 µg DNA and 1
x 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 µF 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 µg/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.
PKA assay
PKA-specific phosphotransferase activity was quantified by
measuring the transfer of phosphate-32 from
[
-32P]ATP to synthetic heptapeptide,
leu-arg-arg-ala-ser-leu-gly. The specific
phosphotransferase activity is expressed as pmol/min/mg protein
(21).
T cell activation
T cells (6 x 106) were activated via
the TCR/CD3 complex with 4 µg/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).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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 nuclei-free 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. 1
A). 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.
|
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. 1
B 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).
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).
|
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 were
established (9) in the presence (treated) or absence
(untreated) of 10 µM lactacystin, a specific 26S proteasome inhibitor
(23, 24), and 100 µM 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 derived
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.
|
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
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
RI2C2 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.
|
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
RI2C2 holoenzyme may
convey a signal involved in the regulation of IL-2 synthesis. |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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 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 |
|---|
|
Footnotes |
|---|
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: gmkammer{at}wfubmc.edu
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.
Received for publication February 9, 2001. Accepted for publication April 2, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and RI transcripts and proteins in systemic lupus erythematosus T lymphocytes. J. Immunol. 162:5639. of cAMP-dependent protein kinases: expression, holoenzyme formation and microinjection into living cells. Exp. Cell Res. 214:595.[Medline]
by cAMP-dependent protein kinase. J. Biol. Chem. 264:20167. of cAMP-dependent protein kinases. Biochem. Biophys. Res. Comm. 176:166.[Medline]
B1 precursor protein and the activation of NF-B. Cell 78:773.[Medline]
This article has been cited by other articles:
|
|
 |
|
  K. Tenbrock, Y.-T. Juang, V. C. Kyttaris, and G. C. Tsokos Altered signal transduction in SLE T cells Rheumatology, October 1, 2007; 46(10): 1525 - 1530. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. J. Loza, S. Foster, S. P. Peters, and R. B. Penn Beta-agonists modulate T-cell functions via direct actions on type 1 and type 2 cells Blood, March 1, 2006; 107(5): 2052 - 2060. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. V. Schillace, S. F. Andrews, S. G. Galligan, K. A. Burton, H. J. Starks, H. G. A. Bouwer, G. S. McKnight, M. P. Davey, and D. W. Carr The Role of Protein Kinase A Anchoring via the RII{alpha} Regulatory Subunit in the Murine Immune System J. Immunol., June 1, 2005; 174(11): 6847 - 6853. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. R. Elliott, R. A. Shanks, I. U. Khan, J. W. Brooks, P. J. Burkett, B. J. Nelson, V. Kyttaris, Y.-T. Juang, G. C. Tsokos, and G. M. Kammer Down-Regulation of IL-2 Production in T Lymphocytes by Phosphorylated Protein Kinase A-RII{beta} J. Immunol., June 15, 2004; 172(12): 7804 - 7812. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. R. Elliott, M. Tolnay, G. C. Tsokos, and G. M. Kammer Protein Kinase A Regulatory Subunit Type II{beta} Directly Interacts with and Suppresses CREB Transcriptional Activity in Activated T Cells J. Immunol., October 1, 2003; 171(7): 3636 - 3644. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Tenbrock, Y.-T. Juang, M. F. Gourley, M. P. Nambiar, and G. C. Tsokos Antisense Cyclic Adenosine 5'-Monophosphate Response Element Modulator Up-Regulates IL-2 in T Cells from Patients with Systemic Lupus Erythematosus J. Immunol., October 15, 2002; 169(8): 4147 - 4152. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) in lanes 1–3. The blots were
stripped and reprobed with anti-C
; SLE subjects, 