Article |
2 Mount Sinai Bone Program, Department of Medicine, Mount Sinai School of Medicine, and Bronx VA Geriatric Research Education and Medical Center, New York, NY 10029
Address correspondence to Narayan G. Avadhani, Dept. of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104. Tel.: (215) 898-8819. Fax: (215) 573-6651. E-mail: narayan{at}vet.upenn.edu
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: calcineurin; IBß; mitochondrial stress signaling; dephosphorylation; NFKB/Rel activation
* Abbreviations used in this paper:
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Major differences between IB
and I
Bß factors have been reported in terms of tissue/cell distribution, rates of synthesis/degradation, response to different stimuli both at transcription and posttranslation levels, mechanisms by which they sequester Rel factors in the cytoplasm, and finally, their nuclear entry and exit (Chu et al., 1996; Johnson et al., 1999). Some studies show that I
Bß plays a more important role in the constitutive phase of NF
B/Rel function, whereas I
B
regulates the stress-induced activation of the factors (Tam and Sen, 2001). IKK-dependent phosphorylation at the NH2-terminal sites (Ser32 and -36 in the case of I
B
and S19 and -23 in the case of I
Bß) is an important regulatory step for ubiquitin-dependent degradation of these inhibitory factors and activation of NF
B/Rel (Ghosh and Karin, 2002). Recently, Fenwick et al. (2000) also showed that members of
B-Ras G proteins bind to I
Bß and regulate the rate of its degradation. Phosphorylation at the COOH-terminal acidic PEST domain has also been shown to be important for the function of I
Bß (Chu et al., 1996; McKinsey et al., 1997; Tran et al., 1997), though unphosphorylated inhibitory protein seems to bind to Rel proteins with equal efficiency (Thompson et al., 1995; Chu et al., 1996; Weil et al., 1997). However, the precise mechanisms of inactivation of I
Bß and the release of NF
B/Rel dimeric proteins remain unclear.
The possible regulation of the NFB/Rel pathway by Ca2+ signaling was indirectly implied in studies showing that transcription activation of IL-2, an NF
B target gene, was adversely affected by known inhibitors of calcineurin (Frantz et al., 1994; Chu et al., 1996). However, the details of this pathway remain unclear. The Ca2+ and calmodulin-dependent phosphatase calcineurin (Cn)* plays important roles in various physiological and pathological processes including T cell activation, Ca2+-induced apoptosis, endocytosis of synaptic vesicles, muscle development, and skeletal and cardiac muscle hypertrophy (Clipstone and Crabtree, 1992; Molkentin et al., 1998; Crabtree, 1999; Lai et al., 1999; Wang et al., 1999; Rusnak and Mertz, 2000). Cn elicits these varied physiological functions by dephosphorylation of key phosphoproteins of the pathways (Crabtree, 1999; Li et al., 2000). A classic example of Cn-mediated activation is dephosphorylation of transcription factor NFATc, facilitating the nuclear localization of the active factor (O'Keefe et al., 1992; Beals et al., 1997).
Recently, we described a novel mitochondria to nucleus stress signaling in C2C12 rhabdomyocytes and human pulmonary A549 cells that involves altered Ca2+ fluxes (Biswas et al., 1999; Amuthan et al., 2002) and altered expression of several nuclear gene targets (Amuthan et al., 2001). We showed that depletion of mitochondrial DNA (mtDNA) or treatment with mitochondrial-specific inhibitors, leading to the disruption of mitochondrial membrane potential (m), results in elevated steady-state Ca2+ ([Ca2+]c). These changes were also accompanied by a three- to fivefold increase in cytoplasmic Cn, increased nuclear NFATc level, increased cytoplasmic I
B
, and reduced RelA (p65) in the nucleus (Biswas et al., 1999). In the present study, we show that nuclear p50 and cRel are markedly increased in cells subjected to mitochondrial stress, suggesting the activation of an alternate pathway. We demonstrate that an increase in Cn activity and attendant inactivation of I
Bß are distinctive features of the mitochondrial stressinduced activation of NF
B/Rel proteins. These results for the first time provide a critical link between mitochondrial stress, Ca2+ signaling, and activation of NF
B/Rel factors. Results also demonstrate the novel features of the mitochondrial stress signaling that are distinctly different from the known cytokines and TNF
-induced signaling.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Immunoblot in Fig. 1 A shows that the cytoplasmic IB
increased two- to threefold in mtDNA-depleted C2C12 cells, whereas I
Bß reduced markedly and returned to near control cell levels in reverted cells. Additionally, the nuclear cRel and p50 levels were increased markedly in mtDNA-depleted cells and returned back to near control cell level in reverted cells. In keeping with its chaperone-like function (Tam and Sen, 2001), a significant amount of I
B
was detected in the nuclear extract from control cells that was reduced by 5060% in the mtDNA-depleted cells (Fig. 1 A). However, there was no detectable I
Bß in the nuclear fractions of all three cell types. The levels of Na+/K+ ATPase and nuclear transcription factor YY1 used as loading controls for postnuclear and nuclear fractions, respectively, did not vary by mtDNA depletion and reversal. Finally, there was no detectable YY1 in the postnuclear fraction and Na+/K+ ATPase in the nuclear fraction, indicating the purity of the subcellular fractions used.
|
Immunoblot in Fig. 2 A shows a fivefold reduced cytoplasmic IBß and three- to sixfold increased nuclear p50 and cRel levels in CCCP-treated C2C12 cells and in mtDNA-depleted cells. In addition, it shows that all of these changes are sensitive to FK506, a Cn inhibitor (O'Keefe et al., 1992) and also Ca2+ chelator EGTA/AM. Therefore, we investigated the role of Cn in I
Bß inactivation and associated nuclear translocation of NF
B/Rel factors by overexpressing CnA
, the catalytic subunit of the enzyme in control C2C12 myocytes. FK506 treatment was used as a control to ascertain the role of Cn in I
Bß inactivation. Immunoblot in Fig. 2 B shows that overexpression of wild-type CnA
(CnA) caused a 40% reduction in cytoplasmic I
Bß levels but no reduction of I
B
. Furthermore, addition of 10 nM FK506 resulted in increased I
Bß in the cytoplasm of both control cells and cells transfected with CnA cDNA constructs. The reduced I
Bß in cells overexpressing CnA is likely due to increased degradation, since we did not detect any increase in the nuclear I
Bß (unpublished data). The results also suggest that the basal Ca2+ (
75 nM), prevalent in control C2C12 cells (Biswas et al., 1999), is sufficient to activate ectopically expressed CnA.
|
Immunoblot in Fig. 3 A shows that transfection with CnA cDNA caused about a threefold increase in cytoplasmic CnA level and about a twofold increase in nuclear p65, p50, cRel, and NFATc levels. FK506 caused a 3040% reduction in nuclear p65 and a more pronounced reduction (7080%) of nuclear cRel, p50, and NFATc. Overexpression of IBß resulted in a 7080% reduction in nuclear cRel and p50 and a 2530% reduction of RelA (p65) (unpublished data). YY1 and Na+/K+ ATPase used as controls for nuclear and postnuclear proteins, respectively, were not detected significantly in reciprocal cell fractions, suggesting their purity. These results suggest that Cn plays a role in modulating the activity of NF
B/Rel family factors.
|
Physical interaction and dephosphorylation of IBß in vitro by calcineurin
Dephosphorylation of IBß by Cn was tested in vitro using purified Cn and 32P-labeled I
Bß. Fig. 4 A shows that Cn alone had a negligible effect when the reaction was performed at 4°C for 10 min. Under these conditions, a combination of Cn and calmodulin had a marginal effect (Fig. 4 A, lane 4), which was enhanced by adding Ca2+ and Mn2+ (Fig. 4 A, lane 5). However, parallel reactions run at 30°C for 5 min yielded a 1250-fold more pronounced dephosphorylation of I
Bß (Fig. 4 A, lanes 6 and 7), which was Cn dependent (Fig. 4 A, lanes 7 and 8). Immunoblot analysis of parallel reactions (Fig. 4, A and B, bottom panels) showed comparable levels of I
Bß, suggesting no protein degradation during in vitro incubation. Furthermore, dephosphorylation of I
Bß by Cn was inhibited by RII peptide in a dose-dependent manner (Fig. 4 B). These results show for the first time that Cn plays a direct role in the dephosphorylation of I
Bß.
|
|
The role of COOH-terminal PEST domain in binding to Cn was further tested by the antibody pull-down assay. Cytoplasmic protein from C2C12 cells transfected with various hIBß cDNA constructs was immunoprecipitated with flag antibody, and the immunoprecipitates were analyzed for the level of Cn and I
Bß. Immunoblot in Fig. 5 H shows that flag antibody immunoprecipitated comparable levels of I
Bß from cells expressing WT and mutant (
PEST, S313/315A and S315A) constructs. The level of Cn coimmunoprecipitated with different I
Bß proteins varied, however. The WT I
Bß pulled down the highest level of CnA, whereas its level was significantly reduced by 60% with the S315A mutant I
Bß. However, in the case of
PEST and S313,315A mutant proteins, no significant CnA was pulled down. Although not shown, immunoprecipitation of in vitro translation products yielded a similar pattern of coimmunoprecipitation. These results further support the possibility that I
Bß binds to CnA through its COOH-terminal PEST domain, and S313 and 315 residues are critical for this binding.
Role of Cn and IBß in mitochondria to nucleus stress signaling
The physiological significance of IBß's interaction with Cn was investigated using the muscle tissue from CnA
knockout mouse (Zhang et al., 1996) based on the rationale that reduced Cn should result in increased cytoplasmic I
Bß and reduced nuclear NF
B/Rel. RNase protection analysis showed that the skeletal muscle from adult CnA
2/- mice contained significantly reduced CnAß, and CnA
mRNAs (unpublished data). Muscle extracts from CnA
2/- mouse used in this study had very low Cn activity. Immunoblot in Fig. 6 A shows detectable CnA in skeletal muscle from wild-type but not CnA
2/- mice. As expected, the nuclear extract from skeletal muscle of CnA
2/- mouse showed no detectable NFATc. The level of I
B
remains the same in muscle tissue from wild-type and CnA
2/- mice, whereas the level of antibody reactive I
Bß increased by approximately two- to threefold. Additionally, the nuclear p65 level was reduced by
60%, whereas the p50 and cRel levels were reduced by >7080% in the CnA
2/- mouse tissue. These results support the view that Cn plays an important role in the regulation of the NF
B pathway in vivo.
|
The distinctive nature of mitochondrial stress signaling and the involvement of IBß in the signaling cascade were investigated using fibrosarcoma cell line HT1080I, which carries a superrepressor mutant of I
B
with vastly diminished phosphorylation and proteasome-mediated degradation (Wang et al., 1996). The superrepressor protein binds to NF
B/Rel proteins with higher affinity, inhibiting their nuclear translocation and DNA binding (Wang et al., 1998). The mutant cell line HT1080I is sensitive to TNF
-mediated apoptosis because the I
B
pathway is refractory to stress response. The HT1080V cells carrying wild-type I
B
exhibit normal response to TNF
(Wang et al., 1998).
Immunoblot in Fig. 7 A shows that, as reported before for C2C12 cells, induction of mitochondrial stress by CCCP treatment caused a reduction in postnuclear IBß in both HT1080V and HT1080I cell lines. On the other hand, treatment with TNF
did not alter the level of cytoplasmic I
Bß. The cytoplasmic I
B
level was increased twofold by CCCP treatment only in the wild-type cells but had no effect on the mutant HT1080I cells. Similarly, TNF
treatment drastically reduced the cytoplasmic I
B
level in wild-type cells but had no effect on mutant cells. The effect on cytoplasmic I
B
became apparent in less than 1 h of TNF
treatment and remained unchanged up to 4 h of treatment (unpublished data). The cytoplasmic CnA was induced in both cell types by CCCP but not by TNF
. CCCP treatment caused a reduction in cytoplasmic cRel and p50 levels with concomitant increase of these proteins in the nuclear compartment in both cell lines. The nuclear cRel and p50 were increased in response to TNF
in wild-type cells but did not increase in mutant cell line. Finally, the nuclear fraction from both cell lines contained no detectable I
Bß. The levels of Na+/K+ ATPase used as a loading control for postnuclear fraction, and YY1 for nuclear fraction, did not vary under these conditions. These results show that both mutant and wild-type cells respond similarly to CCCP-induced mitochondrial stress, and Cn and NF
B/Rel were activated in both cases. Furthermore, results show that TNF
signaling does not involve a change in Cn level and Cn-mediated inactivation of I
Bß.
|
Distinctive nature of TNF and mitochondrial stressinduced NF
B targets
Previous studies from our laboratory showed that mitochondrial stress induced the expression of several marker genes (Biswas et al., 1999; Amuthan et al., 2001, 2002). In the present study, using RyR1 and cathepsin L genes as markers, we investigated whether TNF-mediated and mitochondrial stressinduced signaling pathways affect the same or different gene targets. Immunoblot in Fig. 8 A shows that expression of both RyR1 Ca2+ channel and cathepsin L proteins were induced by CCCP treatment in both HT1080V and HT1080I cells, but the level of IP3 channel protein remained the same (Fig. 8 A). TNF
-mediated stress signaling in both HT1080V and HT1080I cells failed to induce the expression of RyR1 and cathepsin L proteins, further demonstrating the distinctive nature of the two signaling pathways. Although not shown, the induction was at the transcription level as indicated by increased mRNA levels. Furthermore, overexpression of
PEST domain or S313A mutants of I
Bß in cells treated with CCCP resulted in complete reversal of both RyR1 and cathepsin L gene expression (Fig. 8 B). However, overexpression of S315A I
Bß resulted in partial reversal (Fig. 8 B). These results provide direct evidence that Cn-mediated inactivation of I
Bß is critical for mitochondrial stressinduced activation of marker genes.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Nuclear entry and exit of IB
and I
Bß are important parts of signal-mediated activation of NF
B/Rel. The nuclear I
B
plays an important role in the exit of Rel factors from the nucleus at the end of cytokine or other signaling events. Furthermore, LMB-sensitive CRM1 serves as an essential vehicle for the exit of nuclear I
B
to the cytoplasm (Turpin et al., 1999; Huang et al., 2000; Tam et al., 2000). I
Bß, on the other hand, lacks the CRM1 binding domain (Turpin et al., 1999; Tam et al., 2000), which raises questions about its nuclear entry and exit. In support of recent studies (Malek et al. 2001; Tam and Sen, 2001), we also failed to detect I
Bß in the nuclear compartments of C2C12 and HT1080 cells (Fig. 1, A and B, and Fig. 7 A). Therefore, I
Bß appears to be a strictly cytoplasmic protein, indicating yet another distinctive feature different from I
B
.
We present multiple lines of evidence for the physical and functional interaction of IBß with the catalytic subunit of Cn. Both endogenous and ectopically expressed I
Bß form a ternary complex of
250 kD with Cn and NF
B/Rel proteins, as tested by reversible cross-linking with dithiobis(succinimidylpropionate) (DSP). Direct proof of the functional association between I
Bß and Cn comes from in vitro experiments showing that 32P-labeled I
Bß is dephopshorylated by purified Cn in the presence of calmodulin and other necessary cofactors. In vivo and in vitro association of I
Bß with Cn and also its Cn-dependent dephosphorylation are inhibited by FK506 and RII peptide, well-known inhibitors of catalytic function of Cn (O'Keefe et al., 1992; Enz et al., 1994). Notably, the effect of FK506 is targeted to complexes containing Cn but not the 150-kD binary complexes of I
Bß and NF
B/Rel proteins. This is probably the first demonstration of a functional interaction between I
Bß and Cn.
Cn binds to diverse protein substrates including type II cAMP-dependent protein kinase, protein phosphatase inhibitor-1, myosin light chains, -subunit of phosphorylase kinase, G substrate, DARPP-32, dynamin, tau factor, NMDA receptor, NO synthase, and NFATc (for reviews see Rusnak and Mertz, 2000; Shibasaki et al., 2002) through its phosphatase active site leading to protein dephosphorylation. A sequence domain spanning residues 8199 of type II cAMP-dependent protein kinase, designated as RII domain, is involved in interaction with the active site of Cn. The RII peptide has been extensively used as a site-specific inhibitor of Cn phosphatase. A common feature among the protein substrates of Cn is the conservation of phosphoprotein sequence similar to the RII domain. The COOH-terminal PEST domain from the mouse and human I
Bß (sequence 306322) shows
80% structural homology (ß-sheet structure) of RII peptide (Blumenthal et al., 1986) and contains acidic phosphorylatin sites. Indeed, RII peptide inhibited binding of I
Bß to Cn (Fig. 4 B). Results of chemical cross-linking and antibody pull-down assay (Fig. 5, D and H) show that I
Bß, lacking the COOH-terminal PEST domain, or substituted S313 and S315 CKII target sites from this domain, failed to interact with Cn. Although not shown, even S to E substituted protein failed to bind to Cn, suggesting that phosphorylated Ser residues at 313 and 315 positions are critical for binding. Therefore, binding of I
Bß with Cn appears to vary from that of NFAT family members in two ways. First, the 13-aa segment (Cn binding peptide A) from the NH2-terminal domain of NFAT binds to Cn even in the absence of phosphorylation (Aramburu et al., 1998). Second, I
Bß binding to Cn does not appear to involve a second COOH-terminal site similar to that shown for some members of NFAT proteins (Park et al., 2000). Furthermore, mutations targeted to PEST domain phosphorylation sites of I
Bß also acted as a persistent inhibitor of Cn-mediated activation of NF
B/Rel (Fig. 8).
Previous studies show that phosphorylation at the COOH-terminal PEST domain sites (Thompson et al., 1995; Chu et al., 1996; Schwarz et al., 1996; McKinsey et al., 1997; Tran et al., 1997; Weil et al., 1997) are important for the function of IBß. It was shown that dephosphorylation of purified I
Bß or discrete PEST domain mutations affected its ability to bind NF
B/Rel dimmers (Chu et al., 1996; McKinsey et al., 1997). In extension of these studies, we demonstrate that mutations targeted to Ser 313 and 315 of the PEST domain abolished Cn binding but had minimal effect for binding to NF
B/Rel factors. Our results suggest that Cn-mediated dephosphorylation is critical for the inactivation of I
Bß and release of NF
B/Rel.
TNF, interleukin 1, and other receptor-mediated signaling pathways induce the activation of NF
B/Rel factors mostly through signal-mediated phosphorylation and inactivation of I
B
, without significantly affecting the steady-state levels of I
Bß. Using the HT1080I mutant cell line, which does not respond to TNF
and interleukin-mediated signaling (Wang et al., 1996), we demonstrate that mitochondrial stressinduced activation of NF
B/Rel occurs through a mechanism distinctly different from the TNF
signaling. First, the activation of Cn and reduction in the steady-state level of I
Bß in response to CCCP treatment occur in both wild-type HT1080V cells and mutant HT1080I cell lines. However, TNF
-mediated reduction in I
B
occurs only in HT1080V cells (Fig. 8). Furthermore, TNF
treatment in both cell lines has no effect on the level of I
Bß. Second, mitochondrial stress response genes RyR1 and cathepsin L are induced in both cell types in response to CCCP, a specific inducer of mitochondrial stress but not by TNF
. Results presented in this study therefore suggest that the mitochondrial stress signaling follows a pathway distinctly different from TNF
and interleukin 1 signaling. A direct proof for the involvement I
Bß in the mitochondrial stressinduced signaling comes from experiments showing that ectopic expression of PEST domaindeleted or S313,315Asubstituted I
Bß caused a dramatic reduction in the expression of RyR1 and cathepsin L genes in CCCP-treated HT1080V cells and mtDNA-depleted C2C12 cells (Fig. 8). A similar dominant-negative effect of the S313,315A mutant of I
Bß on the mitochondrial stress signaling, reversal of invasive property, and also cell morphology of mtDNA-depleted A549 and C2C12 cells was shown (Amuthan et al., 2002; unpublished data).
Several protein kinases, including CKII, GSK3, PKC, PKC
, IKK
, and IKKß regulate the activity of NF
B, though the precise mechanisms in some cases is not clear (Ghosh and Karin, 2002). For example, CKII and GSK3 are thought to modulate the DNA-binding activity of the nuclear Rel proteins by subunit phosphorylation (Ghosh and Karin, 2002), though CKII may also activate NF
B by phosphorylation of I
Bß and I
B
at their COOH-terminal PEST domain sites (Chu et al., 1996; McElhinny et al., 1996). Some studies suggest that CKII may phosphorylate free I
B
and I
Bß, whereas IKKs may phosphorylate Rel proteinbound inhibitory proteins (Pando and Verma, 2000). Using a combination of gene knock out and an active site inhibitor of activator protein, NEMO, it was shown that IKK
and IKKß play critical roles in the activation of NF
B in response to lymphotoxin ß, interleukin, and TNF
-induced stress signaling (DiDonato et al., 1997; May et al., 2000; Pando and Verma, 2000; Ghosh and Karin, 2002). In further support of the distinctive nature of the mitochondrial stressinduced activation of NF
B different from TNF
-induced activation described above, CCCP, a potent inducer of mitochondrial stress, induced NF
B activation in IKK
2/- and IKKß2/- cells. These results further suggest the possibility that the mitochondrial stressinduced activation occurs through a pathway other than that involving the IKK
- and IKKß-mediated phosphorylation of I
Bß. Alternatively, IKK
- and IKKß-mediated phosphorylation of COOH-terminal sites of I
Bß may be a downstream event that follows the Cn-dependent dephosphorylation of PEST domain sites.
In summary, our results provide evidence for the distinctive nature of the mitochondria to nucleus stress signaling different from various cytokines and receptor-mediated stress. Results also provide mechanistic insights into a novel-signaling pathway by which NFB/Rel factors respond to Ca2+- and calmodulin-dependent process as summarized in Fig. 9. Our working model proposes that activated Cn binds to the cytoplasmic I
BßRel complex and catalyzes the dephosphorylation of I
Bß at its COOH-terminal PEST domain. The Cn-mediated dephosphorylation in turn causes inactivation of I
Bß, resulting in the release of active p50/cRel heterodimers for translocation into the nuclear compartment.
|
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Subcellular fractionation and immunoblot analysis
Cultured cells and skeletal muscle tissue were homogenized in homogenization buffer (0.3 M sucrose, 10 mM Tris-HCl, pH 8.0, 10 mM NaCl, 3 mM MgCl2, 0.2 mM EDTA) containing phosphatase inhibitors (1 mM NaVO4, 100 µM molybdic acid, 10 mM NaF) and protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 50 µg/ml each leupeptin, pepstatin, aprotinin, chymostatin, and antipain). Subcellular fractions were isolated by differential centrifugation as described previously (Biswas et al., 1999). Proteins were resolved by electrophoresis on 10 or 12% SDSpolyacrylamide gels (Laemmli, 1970) and subjected to immunoblot analysis as described by Towbin et al. (1979). Blots were developed using Super Signal West Femto maximum sensitivity substrate from Pierce Chemical Co.
Gel mobility shift assays
DNAprotein binding was assayed by gel mobility shift as described before (Amuthan et al., 2001). Binding was performed with 32P endlabeled NFB consensus DNA (5'-AGTTGAGGGGACTTTCCAGGC-3') of ZBP-89 DNA (5'-GGGTGGGGGGG-3'). Binding reactions (20 µl) contained
0.10.2 ng of labeled DNA (10,00015,000 cpm), 15 µg of nuclear extract, and 2 µg of poly (dI-dC) under conditions described previously (Amuthan et al., 2001). DNAprotein complexes were resolved on 4% nondenaturing polyacrylamide gels in 0.5% x Tris-Glycine (25 mM Tris, 100 mM glycine, 1 mM EDTA, pH 8.3). Competition with 20 molar excess unlabeled DNA and antibody supershift were performed as described (Amuthan et al., 2001).
Immunoprecipitation
Immunoprecipitation was performed by the protein ASepharose pull-down method as described previously (Anandatheerthavarada et al., 1999) using 1 mg equivalent of cytosolic proteins from control or transfected cells. The immunoprecipitates were extracted with 2x Laemmli buffer devoid of ß-mercaptoethanol at 95°C for 5 min. Immunoprecipitation of in vitrotranslated proteins was performed essentially as described previously (Bhat and Avadhani, 1985).
Chemical cross-linking of NFB with Cn
Cross-linking was performed in vitro using 1 mg of cytosolic proteins diluted to 500 µl volume with 50 mM Hepes (pH 7.5). The cross-linking reaction was performed with 500 µM reversible cross-linker, DSP (Pierce Chemical Co.) at 25°C for 30 min. Reaction was stopped by adding 100 mM Tris buffer (pH 7.2). Cross-linked products were subjected to immunoprecipitation using monoclonal flag antibody and subjected to immunoblot analysis using different antibodies to Rel factors, IBß, I
B
, and calcineurin.
Protein phosphatase assay
IBß synthesized in vitro with unlabeled amino acids was phosphorylated by adding [
-32P]ATP and 10 U CKII (New England Biolabs Inc.). Phosphatase assays were performed in 50 µl vol using 32P-labeled proteins (80,000 cpm) as the substrate in 50 mM Hepes (pH 7.5) containing 1 mM CaCl2, 1 mM MnCl2, 3 µM calmodulin (CaM), and 1.5 µg of purified bovine brain Cn (Calbiochem). The reactions were run at 4 or 30°C for 5 min and subjected to immunoprecipitation as described above using I
Bß antibody. Immunoprecipitates were subjected to SDSpolyacrylamide gel, and the gels were imaged using GS525 Molecular Imager (Bio-Rad Laboratories). A parallel set was subjected to immunoblot analysis using I
Bß antibody for protein level.
Metabolic labeling of IBß in intact cells
Control C2C12 cells were transfected with CnAWT, flag-tagged IBßWT, or S313,315A cDNAs, and at 60 h posttransfection, cells were labeled for 4 h with 32Pi (1 mCi/ml) in phosphate-depleted DME with or without added FK506 (10 nM) as described by Chu et al. (1996). Cytosolic proteins (0.5 mg each, 100,000 g supernatant fraction) were immunoprecipitated with either I
Bß or flag antibody. Immunoprecipitates were resolved by 12% SDS-PAGE, and the labeled I
Bß was visualized by autoradiography. Companion gels were subjected to immunoblot analysis using I
Bß and Cn antibodies.
Immunocytochemistry
C2C12 cells were grown on coverslips were transfected with IB
or I
Bß cDNAs (5 µg) for 60 h. In some cases LMB (5 nM) was added during the last 4 h of culturing. Fixing and staining of cells with primary and secondary antibodies were performed as described previously (Biswas et al., 1999). Confocal microscopy was performed with a TCS laser confocal microscope (Leica).
![]() |
Acknowledgments |
---|
This work was supported in part by National Institutes of Health grant CA-22762 to N.G. Avadhani.
Submitted: 22 November 2002
Revised: 17 March 2003
Accepted: 17 March 2003
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Amuthan, G., G. Biswas, S.Y. Zhang, A. Klein-Szanto, C. Vijayasarathy, and N.G. Avadhani. 2001. Mitochondria-to-nucleus stress signaling induces phenotypic changes, tumor progression and cell invasion. EMBO J. 20:19101920.
Amuthan, G., G. Biswas, H.K. Ananadatheerthavarada, C. Vijayasarathy, H.M. Shephard, and N.G. Avadhani. 2002. Mitochondrial stress-induced calcium signaling, phenotypic changes and invasive behavior in human lung carcinoma A549 cells. Oncogene. 21:78397849.[CrossRef][Medline]
Anandatheerthavarada, H.K., G. Biswas, J. Mullick, N.B. Sepuri, L. Otvos, D. Pain, and N.G. Avadhani. 1999. Dual targeting of cytochrome P4502B1 to endoplasmic reticulum and mitochondria involves a novel signal activation by cyclic AMP-dependent phosphorylation at Ser128. EMBO J. 18:54945504.
Aramburu, J., F. Garcia-Cozar, A. Raghavan, H. Okamura, A. Rao, and P.G. Hogan. 1998. Selective inhibition of NFAT activation by a peptide spanning the calcineurin targeting site of NFAT. Mol. Cell. 1:627637.[Medline]
Beals, C.R., N.A. Clipstone, S.N. Ho, and G.R. Crabtree. 1997. Nuclear localization of NF-ATc by a calcineurin-dependent, cyclosporin-sensitive intramolecular interaction. Genes Dev. 11:824834.[Abstract]
Beg, A.A., and A.S. Baldwin, Jr. 1993. The IB proteins: multifunctional regulators of Rel/NF-
B transcription factors. Genes Dev. 7:20642207.[CrossRef][Medline]
Bhat, N.K., and N.G. Avadhani. 1985. Transport of proteins into hepatic and nonhepatic mitochondria: specificity of uptake and processing of precursor forms of carbamoyl-phosphate synthetase I. Biochemistry. 24:81078113.[Medline]
Biswas, G., O.A. Adebanjo, B.D. Freedman, H.K. Anandatheerthavarada, C. Vijayasarathy, M. Zaidi, M. Kotlikoff, and N.G. Avadhani. 1999. Retrograde Ca2+ signaling in C2C12 skeletal myocytes in response to mitochondrial genetic and metabolic stress: a novel mode of inter-organelle crosstalk. EMBO J. 18:522533.
Blumenthal, D.K., K. Takio, R.S. Hansen, and E.G. Krebs. 1986. Dephosphorylation of camp-dependent protein kinase regulatory subunit (type II) by calmodulin-dependent protein phosphatase. Determinants of substrate specificity. J. Biol. Chem. 261:81408145.
Chu, Z.L., T.A. McKinsey, L. Liu, X. Qi, and D.W. Ballard. 1996. Basal phosphorylation of the PEST domain in IBß regulates its functional interaction with the c-rel proto-oncogene product. Mol. Cell. Biol. 16:59745984.[Abstract]
Clipstone, N.A., and G.R. Crabtree. 1992. Identification of calcineurin as a key enzyme in T-lymphocyte activation. Nature. 357:695697.[CrossRef][Medline]
Crabtree, G.R. 1999. Generic signals and specific outcomes: signaling through Ca2+, calcineurin and NF-AT. Cell. 96:611614.[Medline]
DiDonato, J.A., M. Hayakawa, D.M. Rothwarf, E. Zandi, and M. Karin. 1997. A cytokine-responsive IkappaB kinase that activates the transcription factor NF-kappaB. Nature. 388:548554.[CrossRef][Medline]
Enz, A., C. Shapiro, and A. Dattler. 1994. Nonradioactive assay for protein phosphatase 2B (calcineurin) activity using a partial sequence of the subunit of cAMP-dependent protein kinase as substrate. Anal. Biochem. 216:147153.[CrossRef][Medline]
Frantz, B., E.C. Nordby, G. Bren, N. Steffan, C.V. Paya, R.L. Kincaid, M.J. Tocci, S.J. O'Keefe, and E.A. O'Neill. 1994. Calcineurin acts in synergy with PMA to inactivate I kappa B/MAD3, an inhibitor of NF-kappa B. EMBO J. 15:861870.
Fenwick, C., S.Y. Na, R.E. Voll, H. Zhong, S.Y. Im, J.W. Lee, and S. Ghosh. 2000. A subclass of Ras proteins that regulate the degradation of IkappaB. Science. 287:869873.
Feo, S., V. Antona, G. Barbieri, R. Passantino, L. Cali, and A. Giallongo. 1995. Transcription of the human beta enolase gene (ENO-3) is regulated by an intronic muscle-specific enhancer that binds myocyte-specific enhancer factor 2 proteins and ubiquitous G-rich-box binding factors. Mol. Cell. Biol. 15:59916002.[Abstract]
Grilli, M., J.S. Chiu, and M.J. Lenardo. 1993. NF-B and Rel-participants in a multiform transcriptional regulatory system. Int. Rev. Cytol. 143:162.[Medline]
Ghosh, S., and M. Karin. 2002. Missing pieces in the NF-kappaB puzzle. Cell. 109:S81S96.[Medline]
Huang, T.T., N. Kudo, M. Yoshida, and S. Miyamoto. 2000. A nuclear export signal in the N-terminal regulatory domain of IkappaBalpha controls cytoplasmic localization of inactive NF-kappaB/IkappaBalpha complexes. Proc. Natl. Acad. Sci. USA. 97:10141019.
Israel, A. 1995. A role for phosphorylation and degradation in the control of NF-B activity. Trends Genet. 11:203205.[CrossRef][Medline]
Johnson, C., D. Van Antwerp, and T.J. Hope. 1999. An N-terminal nuclear export signal is required for the nucleocytoplasmic shuttling of IkapppaBalpha. EMBO J. 18:66826693.
Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227:680685.[Medline]
Lai, M.M., J.J. Hong, A.M. Ruggiero, P.E. Burnett, V.I. Slepnev, P. De Camilli, and S.H. Snyder. 1999. The calcineurin-dynamin 1 complex as a calcium sensor for synaptic vesicles endocytosis. J. Biol. Chem. 274:2596325966.
Li, L., D. Guerini, and E. Carafoli. 2000. Calcineurin controls the transcription of Na+/Ca2+ exchanger isoforms in developing cerebellar neurons. J. Biol. Chem. 275:2090320910.
Malek, S., Y. Chen, T. Huxford, and G. Ghosh. 2001. IBß but not I
B
functions as a classical cytoplasmic inhibitor of NF-
B dimers by masking both NF-
B nuclear localization sequences in resting cells J. Biol. Chem. 276:4522545235.[CrossRef]
May, M.J., F. D'Acquisto, L.A. Madge, J. Glockner, J.S. Pober, and S. Ghosh. 2000. Selective inhibition of NF-kappaB activation by a peptide that blocks the interaction of NEMO with the IkappaB kinase complex. Science. 289:15501554.
McElhinny, J.A., S.A. Trushin, G.D. Bren, N. Chester, and C.V. Paya. 1996. Casein kinase II phosphorylates I kappa B alpha at S-283, S-289, S-293, and T-291 and is required for its degradation. Mol. Cell. Biol. 16:899906.[Abstract]
McKinsey, T.A., Z.-L. Chu, and D.W. Ballard. 1997. Phosphorylation of the PEST domain of IBß regulates the function of NF-
B/I
Bß complexes. J. Biol. Chem. 272:2237722380.
Molkentin, J.D., J.R. Lu, C.L. Antos, B. Markham, J. Richardson, J. Robbins, S.R. Grant, and E.N. Olson. 1998. A calcineurin-dependent transcriptional pathway for cardiac hypertrophy. Cell. 93:215228.[Medline]
O'Keefe, S.J., J. Tamura, R.L. Kincaid, M.J. Tocci, and E.A. O'Neill. 1992. FK-506- and CsA-sensitive activation of the Interleukin-2 promoter by calcineurin. Nature. 357:692694.[CrossRef][Medline]
Pahl, L.H. 1999. Activators and target genes of Rel/NF-B transcription factors. Oncogene. 18:68536866.[CrossRef][Medline]
Pando, M.P., and I.M. Verma. 2000. Signal-dependent and -independent degradation of free and NF-kappa B-bound IkappaBalpha. J. Biol. Chem. 275:2127821286.
Park, S., M. Uesugi, and G.L. Verdine. 2000. A second binding site on the NFAT regulatory domain. Proc. Natl. Acad. Sci. USA. 97:71307135.
Rusnak, F., and P. Mertz. 2000. Calcineurin: form and function. Physiol. Rev. 80:14831521.
Schwarz, E.M., D. Van Antwerp, and I.M. Verma. 1996. Constitutive phosphorylation of IkappaBalpha by casein kinase II occurs preferentially at serine 293: requirement for degradation of free IkappaBalpha. Mol. Cell. Biol. 16:35543559.[Abstract]
Shibasaki, F., U. Hallin, and H. Uchino. 2002. Calcineurin as a multifunctional regulator. J. Biochem. 131:115.[Abstract]
Tam, W.F., and R. Sen. 2001. IB family members function by different mechanisms. J. Biol. Chem. 276:77017704.
Tam, W.F., L.H. Lee, L. Davis, and R. Sen. 2000. Cytoplasmic sequestration of Rel proteins by IB
requires CRM1-dependent nuclear export. Mol. Cell. Biol. 20:22692284.
Thompson, J.E., R.J. Phillips, H. Erdjument-Bromage, P. Tempst, and S. Ghosh. 1995. IkB-b regulates the persistent response in a biphasic activation of NF-kB. Cell. 80:573582.[Medline]
Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA. 76:43504354.[Abstract]
Tran, K., M. Merika, and D. Thanos. 1997. Distinct functional properties of IB
and I
Bß. Mol. Cell. Biol. 17:53865399.[Abstract]
Turpin, P., R.T. Hay, and C. Dargemont. 1999. Characterization of IB
nuclear import pathway. J. Biol. Chem. 274:68046812.
Wang, C.Y., M.W. Mayo, and A.S. Baldwin, Jr. 1996. TNF- and cancer therapy-induced apoptosis: potentiation by inhibition of NF-kappaB. Science. 274:784787.
Wang, C.-Y., M.W. Mayo, R.G. Korneluk, D.V. Goeddel, and A.S. Baldwin Jr. 1998. NF-B antiapoptosis: Induction of TRAF1 and TRAF2 and c-IAP1 and c- IAP2 to suppress caspase-8 activation. Science. 281:16801683.
Wang, H.G., N. Pathan, I.M. Ethell, S. Krajewski, Y. Yamaguchi, F. Shibasaki, F. McKeon, T. Bobo, T.F. Franke, and J.C. Reed. 1999. Ca2+-induced apoptosis through calcineurin dephosphorylation of BAD. Science. 284:339343.
Weil, R., C. Laurent-Winter, and A. Israel. 1997. Regulation of IBß degradation. Similarities to and differences from I
B
. J. Biol. Chem. 272:99429949.
Zandi, E., Y. Chen, and M. Karin. 1998. Direct phosphorylation of IkappaB by IKKalpha and IKKbeta: discrimination between free and NF-kappaB-bound substrate. Science. 281:13601363.
Zhang, B.W., G. Zimmer, J. Chen, D. Ladd, E. Li, F.W. Alt, G. Wiederrecht, J. Cryan, E.A. O'Neill, C.E. Seidman, et al. 1996. T cell responses in calcineurin A alpha-deficient mice. J. Exp. Med. 183:413420.[Abstract]