| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
B via PKC-dependent tyrosine and serine phosphorylation of IB-
1Departments of Physiology and Medicine, Cardiovascular Research Laboratories, University of California, Los Angeles, California 90095; and 2Experimental Research Laboratory, Division of Cardiology, University of Louisville, Louisville, Kentucky 40202
Submitted 8 May 2003 ; accepted in final form 5 June 2003
 |
ABSTRACT |
|---|
 TOP ABSTRACT METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
|---|
B. However, the molecular mechanisms underlying activation of NF-B during ischemic PC remain unknown. In the hearts of conscious rabbits, it was found that ischemic PC (6 cycles of 4-min coronary occlusion and 4-min reperfusion) significantly induced both tyrosine (+226.9 ± 42%) and serine (+137.0 ± 36%) phosphorylation of the NF-B inhibitory protein IB-
, concomitant with increased activation of the IB- kinases IKK (+255.0 ± 46%) and IKK
(+173.1 ± 35%). Furthermore, both tyrosine and serine phosphorylation of IB- were blocked by pretreatment with either the nonreceptor tyrosine kinase inhibitor lavendustin-A (LD-A) or the PKC inhibitor chelerythrine (Che) (both given at doses previously shown to block ischemic PC). Interestingly, Che completely abolished PC-induced activation of IKK/, whereas LD-A had no effect. In addition, IB- protein level did not change during ischemic PC. Together, these data indicate that ischemic PC-induced activation of NF-B occurs through both tyrosine and serine phosphorylation of IB- and is regulated by nonreceptor tyrosine kinases and PKC.
ischemic preconditioning; Src tyrosine kinase; protein kinase C; signaling module; posttranslational modification
B, participate in the genesis of ischemic preconditioning (PC). Specific inhibition of PKC (2, 9, 22, 32), tyrosine kinases (1, 24, 28), or NF-B (31) can abolish the cardioprotective effects afforded by ischemic PC. Despite this information, the detailed signal transduction mechanisms involved in the interaction between these three molecules are not fully understood.
The transcription factor NF-B is maintained in an inactive form in the cytoplasm by interaction with its inhibitory protein, IB-. In response to various extracellular stimuli, NF-B is activated by one of two distinct mechanisms (4, 10, 11, 15, 33), both of which require biochemical modification of IB- at the protein level. The first is via serine phosphorylation of IB-. This posttranslational modification triggers ubiquitination of IB-, followed by proteolytic degradation, thus releasing NF-B to the nucleus, where it binds target gene promoters. In mammalian cells, several homologs of IB- have been identified, which include IB-, IB-, and IB-
. Phosphorylation of the specific serine 32/36 sites of IB- or serine 19/23 of IB- is critical for the degradation of IBs and hence activation of NF-B (11, 26). Serine phosphorylation of IB- is generally carried out by a multiprotein kinase complex, which includes IKK and IKK (11, 26).
The second major pathway for NF-B activation is via tyrosine phosphorylation of IB-. In contrast to the serine pathway, tyrosine phosphorylation of IB- does not lead to the degradation of this inhibitory protein but rather is, in itself, sufficient to release NF-B, which then translocates to the nucleus (11, 26). Despite these proposed paradigms in noncardiac cells, it remains unknown whether PC induces posttranslational modifications of NF-B molecular machinery, and if so, what specific molecular changes are involved. Furthermore, the signaling events that underlie activation of NF-B in cardiac myocytes during cardioprotection are also poorly defined.
The present study examined activation of NF-Bina conscious rabbit model of ischemic PC. The data demonstrate that ischemic PC induces both tyrosine and serine phosphorylation of IB- via a mechanism that appears to involve PKC and nonreceptor tyrosine kinases.
|
METHODS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
|---|
Experimental protocol for conscious rabbits. The conscious rabbit model of ischemic PC was previously described in detail (22, 31). Rabbits were preconditioned with ischemia (6 cycles of 4-min coronary occlusion followed by 4 min of reperfusion) and received the nonreceptor tyrosine kinase inhibitor lavendustin-A (LD-A; 1 mg/kg iv 10 min before first occlusion), the PKC inhibitor chelerythrine (Che; 5 mg/kg iv 10 min before first occlusion), or vehicle. Sham-operated rabbits did not undergo the ischemic PC protocol and were used as controls. These doses of LD-A and Che have been independently shown to block cardioprotection in this model (6, 22). Thirty minutes after the ischemic PC protocol, rabbit hearts were excised and the anterior wall of the left ventricle was dissected, rapidly frozen in liquid nitrogen, and stored at –80°C until biochemical assays were conducted. Five animals were used in each group.
Tissue sample preparation. Tissue samples were prepared as previously described (22–24). Briefly, frozen myocardial tissue samples were powdered and then homogenized in sample buffer (22–24) with a glass-glass technique. The cytosolic and particulate fractions were separated by 30-min centrifugation at 45,000 g. Protein concentration was determined with the Bradford method (Bio-Rad).
Immunoprecipitation. Immunoprecipitation was performed as previously described (23). For kinase activity assays, 200 µg of protein from either cytosolic or particulate fraction was incubated with 2 µg of IKK or IKK antibodies (Santa Cruz) and 20 µl of protein A/G agarose beads (Santa Cruz) in 400 µl of RIPA buffer (150 mmol/l NaCl, 50 mmol/l Tris, pH 7.4, 1 mmol/l EDTA, 1 mmol/l EGTA, 1 mmol/l sodium orthovanadate, 1 mmol/l PMSF, 16 µg/ml benzamidine HCl, 10 µg/ml phenanthroline, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, and 1% Nonidet P-40). IB- was immunoprecipitated from 200 µg of protein overnight at 4°C (IB- antibody, Santa Cruz). Immunocomplexes were then washed and subjected to either kinase activity assays or immunoblotting.
IKK activity assay. The phosphorylation activities of IKK and IKK were determined by immunoprecipitation followed by phosphorylation activity assay as described by Mercurio et al. (19) with minor modifications. Briefly, the IKK/ immunoprecipitant was subjected to a phosphorylation assay with glutathione S-transferase (GST)-IB- (2 µg/assay; Santa Cruz) as the substrate. The kinase reaction was performed at 30°C for 30 min in 30 µl of kinase reaction buffer (20 mmol/l HEPES, pH 7.7, 10 mmol/l -glycerophosphate, 2 mmol/l MgCl2, 2 mmol/l MnCl2, 300 µmol/l Na3VO4, 1 mmol/l dithiothreitol, 1 mmol/l benzamidine, 2 mmol/l PMSF, 10 µg/ml aprotinin, 1 µg/ml leupeptin, and 10 µmol/l ATP) with 10 µCi of [-32P]ATP. The reaction was terminated by addition of 5x Laemmli buffer, and phosphorylation activity was determined by autoradiography after SDS-PAGE.
Immunoblotting. Standard electrophoresis and immunoblotting techniques were used (22, 24). Antibodies were anti-phosphotyrosine (Upstate Biotechnology), anti-phosphoserine-32 IB- (Calbiochem), and anti-phosphoserine (Zymed). Western blot signal was detected with the ECL chemiluminescence system (Amersham Biosciences).
Statistical analysis. Data are reported as means ± SE. Differences among the four experimental groups were analyzed with one-way ANOVA. If the ANOVA showed an overall significance, post hoc contrasts were performed with Student t-tests for unpaired data (22, 24, 30).
|
RESULTS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
|---|
B-. To examine whether ischemic PC could induce phosphorylation of the inhibitory protein IB-, we used an ischemic PC protocol (6 cycles of 4-min coronary occlusion and 4-min reperfusion) that has been shown to induce activation of NF-B and late PC against myocardial infarction and stunning in conscious rabbits (3, 22, 31). Tissue samples were obtained 30 min after the ischemic PC protocol. IB- was immunoprecipitated from total tissue lysates and immunoblotted with phosphotyrosine or phosphoserine antibodies. Phospho-specific serine-32 IB- serum was also used to detect the serine phosphorylation of IB-. Ischemic PC was found to significantly induce not only tyrosine, but also serine, phosphorylation of IB-. Compared with the control group, ischemic PC increased the tyrosine phosphorylation of IB- by 226.9 ± 42% (P < 0.05; Fig. 1) and the serine-32 phosphorylation of IB- by 137 ± 36% (P < 0.05; Fig. 2). These data indicate dual tyrosine and serine phosphorylation of the IB- protein during cardioprotection that may contribute to the activation of NF-B in this model.
|
|
Ischemic PC-induced tyrosine and serine phosphorylation of IB- are tyrosine kinase- and PKC-dependent. To further investigate the signaling mechanisms whereby tyrosine kinases and/or PKC participate in the activation of NF-B, i.e., phosphorylation of IB-, rabbits were pretreated with either the nonreceptor tyrosine kinase inhibitor LD-A or the PKC inhibitor Che before being subjected to the PC protocol. LD-A and Che were given at doses previously shown to block ischemic PC (22, 24). As illustrated in Figs. 1 and 2, ischemic PC-induced tyrosine and serine phosphorylation of IB- were completely abolished by administration of either LD-A or Che. These data demonstrate that both tyrosine and serine phosphorylation of IB- during ischemic PC occur via nonreceptor tyrosine kinase- and PKC-dependent signaling pathways. That is, tyrosine and serine phosphorylation of IB- are distal to tyrosine kinase and PKC activation by ischemic PC.
Ischemic PC increases activities of both IKK and IKK. In noncardiac cells, IKKs directly phosphorylate serine residues of IB-, leading to its degradation and the subsequent activation of NF-B. Accordingly, it was postulated that IKKs may be involved in ischemic PC-induced serine phosphorylation of IB-. To test this hypothesis, IKK and IKK activities were assessed in both cytosolic and particulate fractions from rabbit hearts preconditioned with ischemia. Cytosolic or particulate fractions were immunoprecipitated with IKK or IKK antibodies and then subjected to in vitro kinase phosphorylation assays using recombinant GST-IB- as substrate. As shown in Figs. 3 and 4, the activities of IKK (+255.0 ± 46% above control) and IKK (+173.1 ± 35% above control) were significantly elevated in the cytosolic fraction after ischemic PC. Pretreatment with LD-A slightly decreased the activation of IKK and IKK compared with the untreated ischemic PC group, but this change did not reach statistical significance, indicating that LD-A failed to block ischemic PC-induced activation of IKK and IKK. In contrast, pretreatment with Che completely abolished the increases in IKK and IKK activity (Figs. 3 and 4). No significant difference in particulate fraction IKK and IKK activation was observed between the different experimental groups (data not shown). These data suggest that IKK and IKK activities are upregulated in a manner dependent on PKC but independent of nonreceptor tyrosine kinases and that activation of IKK and IKK by itself was insufficient to afford protection in the presence of nonreceptor tyrosine kinase blockade by LD-A.
|
|
Ischemic PC does not alter IB- protein expression/degradation. Because two distinct and well-accepted regulatory mechanisms for NF-B activation have been described, and because the aforementioned results suggested that both of these mechanisms may play a role in ischemic PC, it was of interest to examine whether ischemic PC triggers IB- protein degradation. Cardiac cell lysates were analyzed by immunoblotting with anti-IB- antibodies. The data demonstrate that the total IB- protein expression was not changed during ischemic PC compared with shamoperated controls. LPS-treated rabbit tissue samples were used as a positive control for increased IB- protein expression (26). In contrast to ischemic PC, we found that LPS treatment dramatically decreased expression of IB- protein, as expected (Fig. 5). These data demonstrate that ischemic PC induces activation of IKK and IKK and increased DNA binding activity of NF-B (31) without alteration of IB- protein expression/degradation.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
|---|
B in the genesis of cardioprotection (3, 18, 31, 34). In addition, recent studies have indicated formation of PKC-tyrosine kinase signaling modules as a conserved cardioprotective signaling mechanism (21, 29) and NF-B as a downstream molecular target of these modules. Despite this, the manner in which PKC-tyrosine kinase modules initiate activation of NF-B during protection remains unknown. The present study provides direct evidence that ischemic PC mobilizes NF-B molecular machinery by posttranslational modification of IB- on both serine and tyrosine residues. Administration of LD-A, a nonreceptor tyrosine kinase inhibitor, blocked tyrosine and serine phosphorylation of IB- but not IKK activation. Moreover, ischemic PC-induced tyrosine and serine phosphorylation of IB-, as well as increased IKK and IKK activity, were abolished by pretreatment with the PKC inhibitor Che (given at a dose shown previously to block the infarct-sparing effects of ischemic PC), demonstrating that both tyrosine and serine phosphorylation of IB-, as well as increased IKK activity, are PKC dependent. Although other studies had collectively implicated PKC- and tyrosine kinase-dependent modulation of the NF-B pathway (12, 13, 16), the data herein are the first to indicate that PKC modules may be responsible for tyrosine and serine phosphorylation of IB- and for increased IKK and IKK activity during cardioprotection.
Role of NF-B pathway in cardioprotection. Many investigations have indicated that transcriptional regulation of protective genes is a crucial mechanism underlying the development of the delayed cardioprotection induced by ischemic PC or a host of pharmacological stimuli (3, 14, 18). It is now clear that late protection involves increased expression of specific proteins (3, 6, 17, 29) and that this increase is affected, in part, at the transcriptional level. This evidence extends to the PKC-
cardioprotective signaling system, which has been shown to contain a group of proteins that may be involved in transcriptional and translational regulation of proteins (7). Despite this, direct evidence detailing a mechanistic link between PKC and the regulation of protein expression was lacking.
Pharmacological evidence previously implicated the transcription factor NF-B as a necessary mediator of late cardioprotection (31), and subsequent studies implicated PKC-tyrosine kinase modules in the activation of this molecule (21). In noncardiac cells, tyrosine kinase-induced activation of the NF-B pathway has been reported (10, 25), and specific activation of Src tyrosine kinase, a known protective member of PKC- complexes, has been shown to promote nuclear activation of NF-B (8). Despite this, the mechanism of NF-B activation in the heart, and in particular, during cardioprotection, remains completely unknown.
The finding that the IKK-IB- pathway was involved in NF-B activation in the present study was not surprising, as this is a well-characterized mechanism of NF-B activation (20, 27). However, the finding that this pathway appears to be a target of PKC signaling during ischemic PC holds important significance for these ubiquitously expressed molecules. In other words, the findings of the present study have broad implications for signal transduction by PKCs in other physiological conditions that involve temporal regulation of protein expression and, specifically, transcriptional regulation of target genes. These findings expand on the mechanism by which ischemic PC-induced PKC-tyrosine kinase modules lead to increased DNA binding activity by NF-B to affect expression of protective proteins. They provide a functional link between PKC-tyrosine kinase modules and regulation of NF-B activation in a cardioprotective setting and indicate that these tasks executed by PKC-tyrosine kinase modules may involve regulation of IB- and its specific kinases, IKKs.
Mechanism of IB- induction by ischemic PC. Multiple studies indicated that IKK-associated modification of IB- is a conserved mechanism of NF-B activation—findings that were supported by the present study in the setting of ischemic PC. Furthermore, this modification of IB- occurred on a previously characterized serine residue, known to be a regulatory site for IB- activity.
Interestingly, the present study also indicates that IB- is activated during cardioprotection by a dual phosphorylation event: one serine directed and the other tyrosine directed. Previous studies indicated that serine and tyrosine phosphorylations are mechanisms to regulate activity of IB-. However, this study is the first to demonstrate that a physiological stimulus (ischemic PC) induces these posttranslational modifications as part of a signaling mechanism to reduce infarct size. It will be interesting to determine whether this activation pathway is conserved in response to other cardioprotective modalities (such as pharmacological agents or transgenic approaches). Furthermore, the data herein provide a rationale for determining the necessity of either or both of these posttranslational modifications of IB- on the activation of NF-B in this model and on the phenotype of cardioprotection in general.
IB- degradation occurs via a ubiquitin-dependent proteasome mechanism (26). Interestingly, it has also been reported that cardiac-specific overexpression of a phosphoserine-resistant mutant of IB- resulted in abrogation of myocardial NF-B activation in response to TNF- and LPS stimulation (5). These studies indicate that on stimulation with TNF- or LPS, a major pathway for NF-B activation in the heart may be through serine phosphorylation, and subsequent degradation, of IB-. Surprisingly, although serine phosphorylation of IB- was observed during ischemic PC, no alteration in IB- protein expression was detected. It is interesting to speculate that the ratio of serine to tyrosine phosphorylation may determine the degree to which IB- protein levels change. Consequently, the fact that tyrosine phosphorylation of IB- was also enhanced during ischemic PC may explain why IB- protein expression was unaltered in this model.
Role of tyrosine kinase modules in PKC signaling system. There is increasing evidence for functional coupling of PKC to tyrosine kinases in the heart. These modules have been demonstrated to be critical signaling components in the networks responsible for cardioprotection by ischemic PC, nitric oxide donors, and PKC- transgenesis, suggesting a conserved role for this signaling interaction (21, 29). The present study provides additional evidence suggesting the importance of PKC-tyrosine kinase signaling tasks in the heart, in particular, to elicit activation of IB- and NF-B.
The data also indicate that although both posttranslational modifications of IB- occur in response to ischemic PC and appear to be dependent on activation of PKC, the upregulation of IKK activity does not appear to be tyrosine kinase dependent (Figs. 3 and 4). This finding has critical conceptual importance with regard to the hierarchical arrangement of signaling within the PKC subproteome. Although PKC-tyrosine kinase modules are required for activation of NF-B and for tyrosine and serine phosphorylation of IB-, they appear to act through separate, but converging, mechanisms to achieve these end points. LD-A administration, shown previously to block cardioprotection (6), was sufficient to inhibit ischemic PC-induced IB- phosphorylation but not IKK activation. Intriguing, however, was the finding that LD-A (or Che) was sufficient to block serine and tyrosine phosphorylation in the model. A possible explanation is that PKC-tyrosine kinase modules act in a nonlinear fashion, a mechanism previously highlighted in an elegant study by Heusch and colleagues (28), in which these modules are necessary for IB- and NF-B activation (21) but not for increased IKK activity. It is also conceivable that protein phosphate activity regulates the phosphorylation of IB- during this process, whereby an increase in IKK activity could occur without a simultaneous increase in IB- phosphorylation, as is observed after LD-A administration in this model. Future studies will be required to fully elucidate this signaling response. These studies underscore the importance of specific posttranslational modifications that occur to engender cardioprotection and support a potential role of these signaling events to impact cardiac phenotype.
|
DISCLOSURES |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
|---|
|
FOOTNOTES |
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* J. Zhang and P. Ping contributed equally to this study. 
|
REFERENCES |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
|---|
B- proteolysis by site-specific, signal-induced phosphorylation. Science 267: 1485–1488, 1995.B activation in mice by transdominant expression of a mutant IB-. J Mol Cell Cardiol 33: 161–173, 2001.[Web of Science][Medline]
signaling complexes include metabolism- and transcription/translation-related proteins: complimentary separation techniques with LC/MS/MS. Mol Cell Proteomics 1: 421–433, 2002.B. J Immunol 152: 2710–2719, 1994.[Abstract]
-protein kinase C antagonist inhibits protection of cardiac myocytes from hypoxia-induced cell death. J Biol Chem 272: 30945–30951, 1997.B- activates NF-B without proteolytic degradation of IB-. Cell 86: 787–798, 1996.[Web of Science][Medline]
B activity. Annu Rev Immunol 18: 621–663, 2000.[Web of Science][Medline]
with a lipid raft-associated inhibitor of B factor kinase (IKK) complex plays a role in the activation of the NF-B cascade by TCR and CD28. J Immunol 165: 6933–6940, 2000. during in vivo myocardial ischemia. Am J Physiol Heart Circ Physiol 280: H1264–H1271, 2001. modulates NF-B and AP-1 via mitogen-activated protein kinases in adult rabbit cardiomyocytes. Am J Physiol Heart Circ Physiol 279: H1679–H1689, 2000.B-inducing kinase activates IKK- by phosphorylation of Ser-176. Proc Natl Acad Sci USA 95: 3792–3797, 1998.B. Requirement for p56 Lck and ZAP-70 protein tyrosine kinases. Eur J Biochem 268: 1505–1515, 2001.
B in tyrosine kinase signaling of p38 MAP kinase regulation of myocardial adaptation to ischemia. FEBS Lett 429: 365–369, 1998.[Web of Science][Medline]
B kinases essential for NFB activation. Science 278: 860–866, 1997.B kinase and by two upstream kinases, NF-B-inducing kinase and mitogen-activated protein kinase/ERK kinase kinase-1. Proc Natl Acad Sci USA 95: 3537–3542, 1998.-Lck signaling modules confers cardioprotection. J Clin Invest 109: 499–507, 2002.[Web of Science][Medline]
B- in NF-B activation by an oxidative stress. J Immunol 164: 4292–4300, 2000.B. Annu Rev Cell Biol 10: 405–455, 1994.[Web of Science][Medline]
B through a pathway that includes the NF-B-inducing kinase and the IB kinases IKK and IKK. Proc Natl Acad Sci USA 95: 10106–10111, 1998.B plays an essential role in the late phase of ischemic preconditioning in conscious rabbits. Circ Res 84: 1095–1109, 1999.B kinase complex (IKK) contains two kinase subunits, IKK and IKK, necessary for IB phosphorylation and NF-B activation. Cell 91: 243–252, 1997.[Web of Science][Medline]
-FB and iNOS. Circ Res 89: 915–922, 2001.This article has been cited by other articles:
|
|
 |
|
  Y. Higuchi, T. O. Chan, M. A. Brown, J. Zhang, B. R. DeGeorge Jr., H. Funakoshi, G. Gibson, C. F. McTiernan, T. Kubota, W. K. Jones, et al. Cardioprotection afforded by NF-{kappa}B ablation is associated with activation of Akt in mice overexpressing TNF-{alpha} Am J Physiol Heart Circ Physiol, February 1, 2006; 290(2): H590 - H598. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Brown, M. McGuinness, T. Wright, X. Ren, Y. Wang, G. P. Boivin, H. Hahn, A. M. Feldman, and W. K. Jones Cardiac-specific blockade of NF-{kappa}B in cardiac pathophysiology: differences between acute and chronic stimuli in vivo Am J Physiol Heart Circ Physiol, July 1, 2005; 289(1): H466 - H476. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Murphy Primary and Secondary Signaling Pathways in Early Preconditioning That Converge on the Mitochondria to Produce Cardioprotection Circ. Res., January 9, 2004; 94(1): 7 - 16. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
