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Department of Veterinary Pathobiology, University of Missouri, Columbia, MO 65211
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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) and SAF-1. Immunofluorescence
and combined immunoprecipitation-Western blot analyses revealed
colocalization and interaction between SAF-1 and PKA-C. In vivo
evidence of SAF-1 and PKA-C interaction was further revealed by
coimmunoprecipitation of these two proteins in cAMP-activated liver
cells. We further show that SAF-1 is phosphorylated in vitro by
PKA-C and that addition of cAMP markedly induces in vivo
phosphorylation of SAF-1 and transcription of SAF-regulated reporter
genes. These results showed that SAF1-PKA-C interaction is involved
in functional activation of SAF-1. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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either alone or in combination and inflammatory mediators like PMA can
increase transcription of SAA. Regulation of SAA
gene expression has been studied and characterized by different groups,
including ours (3, 4, 5, 6, 7, 8, 9). Multiple cis-acting
elements have been defined to be important for transcriptional
induction of SAA genes, which include CCAAT enhancer-binding
protein (C/EBP), NF-
B, YY-1, Sp1, and SAA-activating factor
(SAF) transcription factor DNA-binding elements. Nonhepatic
expression of rabbit SAA2 is primarily regulated by the SAF DNA-binding
element given that mutation of this region abolishes 60–80% of the
SAA2 promoter activity in many nonhepatic cells (9).
Because nonhepatic expression of SAA seems to play a critical role in
the pathogenesis of SAA-linked inflammatory diseases, SAF might have a
significant role in the disease outcome.
SAF is a family of zinc finger transcription factors containing
multiple Cys2-His2-type zinc finger motifs at their C terminus
(10). DNA-binding activity of SAF is induced in response
to LPS (11) or IL-1 and IL-6 treatment of cells
(12). Structural analysis showed that one member of this
family, SAF-1, is a homologue of human myc-associated zinc
finger protein (MAZ) (13) and murine Pur-1
(14). To date, SAF-1/MAZ/Pur-1 is identified as a
regulator of c-myc, insulin, serotonin 1A receptor, CD4,
-fibrinogen, phenylethanolamine N-methyltransferase, and
CLC-K1 genes (13, 14, 15, 16, 17, 18, 19). These findings suggest that the
SAF-1/MAZ/Pur-1 family of proteins have a wide regulatory role in
governing many cellular processes.
Protein-protein interaction between transcription factors and other
accessory proteins is a unique mechanism through which strict cellular
control of gene expression is maintained. We hypothesized that
association of other proteins is necessary for SAF-1/MAZ/Pur-1 to
regulate a variety of genes in a cell-type or stimulus-specific manner.
The yeast two-hybrid system is a powerful tool for identifying
protein-protein interactions that occur in vivo in the course of
regulating many physiological activities including signal transduction
process. By two-hybrid assay, we detected interaction between SAF-1
protein and the catalytic subunit of cAMP-dependent protein
kinase (PKA-C). The interaction between these two proteins was
subsequently demonstrated by both in vitro and in vivo analyses.
Further studies showed that activation of endogenous PKA by
intercellular elevation of cAMP leads to increased expression of
SAF-1-regulated promoters.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A truncated form of SAF-1 (aa 187–477), hereafter designated as
SAF-1187–477, was cloned into the pAS2-1
vector (CLONTECH Laboratories, Palo Alto, CA), and the
resultant pAS2-1-SAF1187–477 plasmid was used as
a bait to screen a mouse brain MATCHMAKER cDNA library
(CLONTECH Laboratories) following the manufacturer’s
protocol. CG1945 yeast cells were transformed with
pAS2-1-SAF1187–477 plasmid DNA. A 200-µg
sample of the cDNA library cloned in pACT2 plasmid was then transformed
into CG1945 cells containing pAS2-1-SAF1187–477.
Transformed cells were plated onto medium lacking tryptophan, leucine
and histidine but containing 15 mM 3-amino-1,2,4-triazole. Growth on
His- plates indicated an interaction between
SAF-1187–477 and the protein coded by an unknown
cDNA. Selected colonies were further assayed for 
-galactosidase
activity. After 3 rounds of selection, 20 colonies were chosen for
further analysis by DNA sequencing.
Cell culture and transfection
BNL CL2 liver cells, a normal embryonic liver cell line derived
from a BALB/c mouse, were obtained from American Type Culture
Collection (Manassas, VA) and cultured in DMEM containing high glucose
(4.5 g/L) supplemented with 7% FCS. These cells retained many of the
features of normal liver cells. Transient transfections were conducted
by the calcium phosphate method (20). Reporter plasmids
and expression vectors containing SAF-1 cDNA were transfected in
duplicate, along with 1 µg pSV--gal (Promega, Madison, WI). The
pSV--gal plasmid DNA was used as an internal control for measuring
transfection efficiency. The amount of DNA in each transfection assay
was kept same by using carrier DNA. Some cells were treated with
8-bromo (8-Br)-cAMP (0.4 mM), myristoylated cell-permeable PKA
inhibitor 14–22 amide, (PKI) (5 µM), and H89 (5 µM). Cells were
harvested 48 h posttransfection, and cell extracts containing
equivalent amounts of -galactosidase activity were used for
chloramphenicol acetyltransferase (CAT) assay as described previously
(9, 10, 11). -Galactosidase activity was assayed with the
substrate
O-nitrophenyl--D-galactopyranoside
as described (20). All transfection experiments were
performed at least three times.
Plasmid constructs
The CAT reporter plasmids wtSAF-CAT and mtSAF-CAT were constructed by ligating three copies of wild-type or mutant SAF DNA-binding element, -254 to -226 bp, of the SAA promoter (9) into pBLCAT2 plasmid. The sequences of the wild-type and mutant SAF DNA-binding elements are 5'-CCCTTCCTCTCCACCCACAGCCCCCATGG-3' and 5'-CCATTACTGTCGACTGACAGCTACCATGG-3', respectively. Underlined bases represent altered sequences. The pCMV-SAF1 expression plasmid was prepared by inserting a full length SAF-1 cDNA (10) under the control of CMV promoter in pCDNA3 vector (Invitrogen, San Diego, CA). Bacterially expressed SAF-1 protein was obtained by using FLAG-SAF1 plasmid constructed as described earlier (21) and affinity purified from anti-FLAG-agarose gel as described (21).
Immunoprecipitation, coimmunoprecipitation, and Western blotting
Bacterially expressed FLAG-SAF1 protein (0.5 µg) was incubated with 1.0 U (1.3 µg) of a purified preparation of catalytic subunit of PKA (Calbiochem-Novabiochem, La Jolla, CA) in 20 µl 10 mM HEPES (pH 7.9), 10 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, 0.1 mM ZnCl2, 0.5 mM sodium orthovanadate, 10 mM NaF, 1 mM PMSF, 0.5 mg/ml benzamidine buffer at 25°C for 1 h. After incubation, 0.5 ml immunoprecipitation buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2.5 mM PMSF, 1% Nonidet P-40, 0.1% SDS, 0.5 mg/ml benzamidine was added to the mixture, and proteins were immunoprecipitated with anti-PKA Ab (Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C for 16 h. Next, 50 µl protein G-agarose slurry was added to the reaction mixture and incubated for 2 h at 4°C, and bound proteins were eluted and separated by SDS-PAGE. The proteins were transferred to nitrocellulose membrane and probed with anti-FLAG Ab (Sigma, St. Louis, MO). Chemiluminescence reaction was performed with ECL detection kit using the manufacturer’s protocol (Amersham, Arlington Heights, IL).
For coimmunoprecipitation analysis, BNL CL2 liver cells were grown in
duplicate in 100-mm dishes. One set of cells was not treated with any
agent, and the other set was treated with 8-Br-cAMP (0.4 mM, final
concentration) for 4 h. After incubation, cells were harvested,
washed in PBS, and resuspended in a lysis buffer containing 50 mM
Tris-HCl (pH 8.0), 100 mM NaCl, 0.5% Nonidet P-40, 2.5 mM PMSF, 0.5
mg/ml benzamidine, 5 µg/ml leupeptin, 10 µg/ml aprotinin, 0.2 mM
sodium orthovanadate, and 100 mM NaF. Clarified cell extracts were
further immunoprecipitated with either anti-SAF1 Ab or anti-PKA
Ab (Santa Cruz Biotechnology). Immunoprecipitated proteins were
fractionated by SDS-PAGE, transferred onto nitrocellulose membrane, and
further probed with anti-PKA or anti-SAF1 Ab, as indicated in
Figs. 2
and 3. The migration position of PKA or SAF-1 in the immunoblot
was verified by fractionating a sample of purified catalytic subunit of
PKA (Calbiochem-Novabiochem) or bacterially expressed pure SAF-1
protein.
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BNL CL2 cells were transfected in duplicate, with pCMV-FLAG-SAF-1 plasmid DNA. After transfection, one set of cells was stimulated with 8-Br-cAMP (0.4 mM). The cells were fixed with acetone-chloroform (1:1), incubated at -20°C for 10 min, and permeabilized in PBS-0.2% Triton X-100 for 5 min at room temperature. After a washing in PBS, cells were blocked for 1 h in a blocking buffer (PBS-1% BSA-1% goat serum) and further incubated for another hour with anti-FLAG mouse IgG plus anti-PKA rabbit IgG in the blocking buffer. After three washings in PBS, cells were incubated with a mixture of goat anti-mouse IgG Alexa Fluor 546, goat anti-rabbit IgG Alexa Fluor 488, and DAPI (all three reagents were obtained from Molecular Probes, Eugene, OR) in the blocking buffer for 30 min in the dark and then washed three times in PBS. A few drops of Fluoromount-G (Fisher Scientific, Pittsburgh, PA) were added, the coverglass was placed, and the edges of the coverglass were sealed with clear nail polish. Fluorescent-labeled cells were visualized in an epifluorescence microscope (Axiophot; Zeiss, Oberkochen, Germany).
In vivo phosphorylation of SAF-1
BNL CL2 liver cells were transfected with pCMVSAF-1 expression plasmid. The cells were metabolically labeled with [32P]orthophosphate (32Pi; 0.5 mCi/ml) in phosphate free DMEM for 6 h in the absence or presence of 8-Br-cAMP (0.4 mM). Before the labeling, cells were grown in DMEM plus 5% FCS for 24 h. The 32Pi-labeled cells were harvested, washed quickly in phosphate-free DMEM, and lysed by adding hot lysis buffer (50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 0.5% Nonidet P-40, 2.5 mM PMSF, 0.5 mg/ml benzamidine, 5 µg/ml leupeptin,10 µg/ml aprotinin, 0.2 mM sodium orthovanadate, 100 mM NaF plus 1% SDS). After lysis, SDS concentration was reduced to 0.1% by adding lysis buffer without SDS. The 32P-labeled SAF-1 protein was isolated by immunoprecipitation with anti-SAF1 Ab, fractionated in an SDS-11% polyacrylamide gel, and detected by autoradiography.
In vitro phosphorylation and immunoprecipitation of SAF-1
The phosphorylation reaction was performed in 50 mM Tris-HCl (pH
7.5), 10 mM MgCl2, 100 µM unlabeled ATP, 10
µCi [-32P]ATP (6000 Ci/mmol), 0.5 µg
purified FLAG-SAF1 protein, 1.0 U (1.3 µg) of purified catalytic
subunit of PKA (Calbiochem-Novabiochem) at 30°C for 30 min in a total
volume of 50 µl. In some reactions, PKI inhibitor peptide (5 µM)
was added (Calbiochem-Novabiochem). After incubation, one-half of in
vitro phosphorylated reaction was immunoprecipitated with anti-FLAG
Ab (Sigma).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To identify proteins that might in vivo interact with SAF-1 and
therefore be involved in regulating its activity, we used the yeast
two-hybrid system. We generated a bait that produced a fusion protein
containing the GAL4 DNA-binding domain (GAL4-DBD) and a truncated form
of SAF-1187–477 protein using pAS2-1 vector.
This bait construct did not autoactivate the HIS3 and
lacZ reporter genes on transformation in CG1945 yeast cells.
The GAL4DBD-SAF1187–477 bait (i.e.,
pAS2-1-SAF1187–477 plasmid) was used for
screening a mouse brain MATCHMAKER cDNA library constructed using pACT2
vector that produces a fusion protein containing the GAL4 activation
domain (GAL4-AD) and proteins coded by the cDNA molecules in the
library. Of the 1 x 106 colonies screened,
20 clones displayed growth on His- medium and
-galactosidase activity indicating interaction between
SAF1187–477 and the selective cloned proteins.
For further confirmation, DNA was isolated from these yeast colonies,
transformed into bacteria, and reintroduced into freshly grown yeast
with pAS2-1-SAF1187–477 and tested for growth on
His- medium. Ten clones passed this final round of
selection and were used for DNA sequence analysis. Four independent
cDNAs were identified as coding for the catalytic subunit of PKA-C
(22). Two clones contained the full length sequence, and
the other clones contained partial sequences of PKA-C, 21–350 and
33–350 aa, respectively. As shown in Fig. 1, yeast containing both
GAL4DBD:SAF1187–477 and pACT2- PKA-C grew on
plate lacking histidine. As a negative control,
pAS2-1-SAF1187–477 was cotransformed with pACT2
empty vector, which did not grow on histidine-lacking plates. This data
suggested that PKA-C has affinity for SAF-1 that results in a
protein-protein interaction between these two proteins.
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To test whether SAF-1 and PKA proteins might interact in vitro as
well, we used a combined immunoprecipitation/Western blot analysis.
Bacterially expressed FLAG-SAF1 protein was incubated with purified
preparation of the catalytic subunit of PKA (Calbiochem-Novabiochem).
After incubation, proteins were immunoprecipitated with anti-PKA Ab
and Western blotted with anti-FLAG Ab. As seen in Fig. 2
, lane 2, FLAG-SAF1 was
immunoprecipitated with anti-PKA and further detected by
anti-FLAG Ab. In the absence of PKA (Fig. 2, lane 3),
SAF-1 could not be immunoprecipitated and thus remained absent in
Western blot analysis. We show that anti-FLAG Ab is specific
because it detects only FLAG-SAF1 protein (lane 4)
but not PKA (lane 5). These results showed that SAF-1
interacts with PKA under in vitro conditions.
PKA interacts with SAF in vivo
To test whether PKA-C and SAF interact in vivo with each other,
we used coimmunoprecipitation assay. Cellular proteins of BNL CL2 liver
cells were treated with anti-SAF-1 Ab to selectively
immunoprecipitate intracellular SAF-1 (Fig. 3). When anti-SAF-1
immunoprecipitated proteins were probed with anti-PKA Ab, a band
(Fig. 3, lane 2) was preferentially detected in cAMP-treated
cells that comigrated with the catalytic subunit of PKA (Fig. 3, lane 3). This result suggested that in vivo SAF and PKA
interact with each other. Similar to our finding, a recent report
showed interaction between PKA-C and SOX-9, a transcription factor
involved in chondrocyte differentiation (23).
Surprisingly, the level of PKA coimmunoprecipitated with SAF-1 protein
was much less in untreated BNL cells (Fig. 3, lane 1)
indicating that cAMP treatment of the cells is necessary either for
optimal release of the catalytic subunit of PKA from its native form or
for optimal interaction of SAF-1 with PKA. In a reciprocal experiment,
immunoprecipitation of cellular proteins with anti-PKA-C Ab
pulled down SAF-1 protein at a favorable level when cAMP was added to
the medium (Fig. 3, compare lanes 4 and 5). The
role of cAMP in SAF-PKA interaction in vivo is interesting and
suggested that cAMP-mediated actions facilitate interaction between
these two proteins.
Colocalization of SAF-1 and PKA in the cells after cAMP stimulation
For an interaction between two proteins to have a functional and
physiological consequence, they must be expressed in the same cell. To
address this, BNL CL2 cells were transfected with pCMV-FLAG-SAF-1
plasmid and grown on coverslips. Next, one set of cells was stimulated
with 8-Br-cAMP and processed for immunofluorescence assay using both
anti-FLAG and anti-PKA Abs. As seen in Fig. 4A, SAF protein was seen in
the cytoplasm but also at a lower level in the nucleus of unstimulated
cells. In contrast, PKA was seen predominantly in the cytoplasm of
unstimulated cells. However, in response to cAMP stimulation (Fig. 4B), both PKA and SAF proteins were present at a higher
level in the nuclei. These results demonstrated that SAF and PKA
colocalize under some in vivo conditions.
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Interaction of SAF-1 with PKA provided a clue for the involvement
of the cAMP/PKA signaling pathway in modulating SAF function. To that
end, we sought to determine whether intracellular elevation of cAMP, an
inducer of PKA activity in cells, has any effect on SAF activity. A
CAT gene containing three tandem copies of SAF-binding
elements present in the SAA gene was chosen as a reporter gene system
to monitor the function of activated SAF. BNL CL2 liver cells were
transfected in duplicate with SAF-CAT reporter alone or together with
pCMV-SAF-1 expression plasmid DNA. After transfection, cells were
incubated for 24 h with or without 8-Br-cAMP. As shown in Fig. 5, addition of 8-Br-cAMP markedly induced
expression of SAF-CAT reporter while H89 and
PKI14–22 amide (Calbiochem-Novabiochem), two
specific inhibitors of endogenous PKA activity inhibited such
induction. The effect of cAMP and the PKA inhibitors was significantly
higher when exogenous SAF was provided to the cells by cotransfection
with pCMV-SAF-1. In contrast, the reporter gene expression from
mtSAF-CAT reporter remained virtually undetectable and unaffected by
any of these agents (data not shown). The inhibitory effect of PKI and
H89 and the stimulatory effect of 8-Br-cAMP were not due to a change in
the expression of SAF1 protein because the level of SAF-1 protein
remained same during these different treatments, as determined by
Western immunoblot analysis (data not shown). This suggested that
although SAF-1 is present in the cells, its activity is regulated by
PKA on activation by cAMP.
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To determine whether phosphorylation of SAF-1 in vivo is increased
by cAMP, we labeled pCMVSAF-1-transfected BNL CL2 cells with
32Pi in the presence or
absence of 8-Br-cAMP. The 32P-labeled cellular
phosphoproteins were immunoprecipitated with anti-SAF-1 Ab and
separated by SDS-PAGE. As seen in Fig. 6A, cells transfected with
SAF-1 expression plasmid revealed a phosphorylated protein band, the
intensity of which increased by severalfold when cells were labeled in
the presence of cAMP (Fig. 6A, lane 2) and
reduced in the presence of H89 (Fig. 6A, lane 3).
This increase of phosphorylation was not due to any increase in the
expression of the transfected SAF-1 gene, because the level of SAF-1
protein was the same during cAMP treatment (Fig. 6B, lanes
1–3).
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-32P]ATP (Fig. 6C). After
incubation, a portion of in vitro phosphorylated samples were
immunoprecipitated with anti-FLAG Ab (Fig. 6C,
lanes 2 and 3). Two bands, one comigrating with
FLAG-SAF1 protein and another comigrating with the catalytic subunit of
PKA at 38 kDa position were detected (Fig. 6C, lane
2). No radioactivity was seen in the lane that contained PKI
inhibitor during in vitro phosphorylation (Fig. 6C,
lane 3). These results clearly indicated that SAF-1 is a
substrate of PKA, and immunoprecipitation of both SAF-1 and PKA with
anti-FLAG Ab further confirmed their association. Taken together,
these results indicated that cAMP/PKA mediate cellular event regulates
functional activity of SAF-1 protein. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and acts as a substrate for PKA.
Interaction of PKA-C with SAF-1 seems to induce its function because
activators of PKA signaling pathway markedly increased expression of
SAF-1-regulated reporter genes and in vivo phosphorylation of SAF-1
protein. Colocalization of PKA-C and SAF-1 further correlated with
the notion that interaction of these two proteins have biological
implications for effective gene regulation.
Activation of transcription factors by extracellular signals is an
important step in regulating gene expression and protein
phosphorylation is a primary mechanism for rapid
activation/deactivation of many transcription factors. Among various
protein kinases, cAMP-dependent PKA is a key enzyme involved in the
regulation of many biological processes (reviewed in Refs.
24 and 25). An increase in the intracellular
levels of cAMP releases catalytic subunits of PKA from the regulatory
subunits, resulting in the activation of PKA. Activated PKA mediates
the effects of cAMP by phosphorylating various substrate transcription
factors and thereby affects both the regulation of key metabolic
enzymes and the expression of numerous target genes. CAT activity
generated from SAF-regulated reporter was markedly higher in cells
treated with PKA-activating agents. Reciprocally, specific inhibitors
of PKA inhibited SAF-mediated gene expression. Further, we have shown
that SAF-1 and the PKA-C colocalize during cAMP stimulation of
cells. SAF-1 protein is seen in nuclei of unstimulated cells. In
contrast, PKA is predominantly present in the cytoplasm of unstimulated
cells but migrates to the nucleus after 8-Br-cAMP treatment. Therefore,
it seems that nuclear localization of SAF-1 is not dependent on its
phosphorylation by PKA. These results are in agreement with previous
observations, which detected SAF protein in nuclear extract of
unstimulated cells (10) and migration of the PKA-C in
the nucleus on intracellular elevation of cAMP (26).
Several earlier studies speculated that function of SAF is modulated by serine/threonine phosphorylation (9, 10). Recently, we have shown that PKC plays a significant role in increasing the DNA-binding ability of SAF, which results in increasing SAF-regulated gene expression (21). Another study demonstrated increased level of DNA-binding activity of MAZ, a homologue of SAF-1, by casein kinase II (27). The results presented in this report strongly suggest a role of PKA in activating SAF and SAF-regulated metabolic processes. At present, although SAF is shown to be activated by PKC, casein kinase II, and PKA, it does not necessarily mean that this transcription factor is regulated only by these three protein kinases. In fact, because SAF is seen to be activated by different inflammatory signals, including LPS (11), cytokines (12), and minimally modified low density lipoprotein (28), we speculate that these diverse effects may be mediated by a combination of many signal transducing protein kinases. Further studies should reveal more about the regulatory mechanisms involving activation of SAF-1 protein.
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Acknowledgments |
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RI)59/60. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Alpana Ray, Department of Veterinary Pathobiology, University of Missouri, Columbia, MO 65211. E-mail address: rayal{at}missouri.edu
3 Abbreviations used in this paper: SAA, serum amyloid A; SAF, SAA-activating factor; PKA, protein kinase A; PKC, protein kinase C; CAT, chloramphenicol acetyltransferase; MAZ, myc-associated zinc finger protein; PKA-C, catalytic subunit of PKA; C/EBP, CCAAT enhancer-binding protein; 8-Br, 8-bromo; PKI, PKA inhibitory amide; 32Pi, [32P]orthophosphate.
Received for publication April 17, 2001. Accepted for publication June 13, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B and NF-IL6 transactivating factors in the synergistic activation of human serum amyloid A gene expression by interleukin-1 and interleukin-6. J. Biol. Chem. 268:25624.B like transcription factors. J. Biol. Chem. 266:15192. and C/EBP-
and their activation by protein phosphorylation. Mol. Cell. Biol. 14:4324.B and C/EBP heteromer in lipopolysaccharide induction of serum amyloid A gene expression in liver. J. Biol. Chem. 270:7365.-fibrinogen gene functions as an IL-6 response element. J. Immunol. 165:3411.This article has been cited by other articles:
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  D. Kumar, A. Ray, and B. K. Ray Transcriptional Synergy Mediated by SAF-1 and AP-1: CRITICAL ROLE OF N-TERMINAL POLYALANINE AND TWO ZINC FINGER DOMAINS OF SAF-1 J. Biol. Chem., January 16, 2009; 284(3): 1853 - 1862. [Abstract] [Full Text] [PDF]
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 M. Sastri, D. M. Barraclough, P. T. Carmichael, and S. S. Taylor A-kinase-interacting protein localizes protein kinase A in the nucleus PNAS, January 11, 2005; 102(2): 349 - 354. [Abstract] [Full Text] [PDF]
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A. Ray, D. Kumar, P. Ray, and B. K. Ray Transcriptional Activity of Serum Amyloid A-activating Factor-1 Is Regulated by Distinct Functional Modules J. Biol. Chem., December 24, 2004; 279(52): 54637 - 54646. [Abstract] [Full Text] [PDF]
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A. Ray, P. Ray, N. Guthrie, A. Shakya, D. Kumar, and B. K. Ray Protein Kinase A Signaling Pathway Regulates Transcriptional Activity of SAF-1 by Unmasking Its DNA-binding Domains J. Biol. Chem., June 13, 2003; 278(25): 22586 - 22595. [Abstract] [Full Text] [PDF]
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B. K. Ray, R. Murphy, P. Ray, and A. Ray SAF-2, a Splice Variant of SAF-1, Acts as a Negative Regulator of Transcription J. Biol. Chem., November 22, 2002; 277(48): 46822 - 46830. [Abstract] [Full Text] [PDF]
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A. Schmitt and A. R. Nebreda Inhibition of Xenopus oocyte meiotic maturation by catalytically inactive protein kinase A PNAS, April 2, 2002; 99(7): 4361 - 4366. [Abstract] [Full Text] [PDF]
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A. Schmitt and A. R. Nebreda Inhibition of Xenopus oocyte meiotic maturation by catalytically inactive protein kinase A PNAS, April 2, 2002; 99(7): 4361 - 4366. [Abstract] [Full Text] [PDF]
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