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REPORT
1Departments of Physiology and Medicine/Cardiology, Cardiovascular Research Laboratories, David Geffen School of Medicine at University of California Los Angeles, Los Angeles, California 90095; and 2Division of Cardiology, University of Louisville, Louisville, Kentucky 40292
Submitted 10 May 2004 ; accepted in final form 1 June 2004
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ABSTRACT |
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is a central regulator of protective signal transduction in the heart. However, the signaling modules through which PKC- exerts its protective effects have only begun to be understood. We have identified a novel participant in the PKC- signaling system in cardioprotection, the nonreceptor tyrosine kinase Bmx. Functional proteomic analyses of PKC- signaling complexes identified Bmx as a member of these complexes. Subsequent studies in rabbits have indicated that Bmx is activated by nitric oxide (NO) in the heart, concomitant with the late phase of NO donor-induced protection, and provide the first analysis of Bmx expression/distribution in the setting of cardioprotection. In addition, increased expression of Bmx induced by NO donors was blocked by the same mechanism that blocked cardioprotection: inhibition of PKC with chelerythrine. These findings indicate that a novel type of PKC-tyrosine kinase module (involving Bmx) is formed in the heart and may be involved in pharmacological cardioprotection by NO donors.
myocardial ischemia; preconditioning; signaling complex; signaling module; nitric oxide
Proteomic analysis of myocardial PKC- complexes identified bone marrow tyrosine kinase gene in chromosome X (Bmx, also known as Etk), a member of the Tec/Btk family of nonreceptor tyrosine kinases, as a member of the PKC- subproteome (10). Previous studies have implicated a role for the Tec family in proliferative and senescent cellular processes and, in particular, in mediation of signal transduction by cytokines, growth factors, and G protein-coupled receptors (8, 12). Although one study documented expression of Bmx in the endocardium and the macrovascular endothelium (3), virtually nothing is known regarding the functional signaling role(s) of Bmx in the heart. Specifically, it is completely unknown 1) whether Bmx is activated by nitric oxide (NO) in the myocardium; 2) whether other nonreceptor tyrosine kinases, such as Bmx, form modules with PKC- in the heart; and 3) whether Bmx participates in cardioprotective signaling. To answer these questions, the expression and activation of Bmx were examined in a well-characterized rabbit model of NO donor-induced late preconditioning. The data indicate that Bmx was activated during protection and was resident in the PKC- complex, suggesting that this molecule may form a novel tyrosine kinase module with PKC-. Given the known cardioprotective role of NO, these data have important implications regarding the ability of this ubiquitous signaling molecule to activate Tec kinases in the heart. Furthermore, the findings suggest a broader role for PKC-nonreceptor tyrosine kinase modules in the myocardium, which includes members outside the Src family.
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METHODS |
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Conscious rabbit model of diethylenetriamine-NO-induced late cardioprotection.
A well-established conscious rabbit model of NO-induced cardioprotection was used as previously described (9, 15). Briefly, male New Zealand White rabbits (2.0–2.5 kg, 3–4 mo of age) were intravenously administered the NO donor diethylenetriamine (DETA)/NO (0.1 mg/kg once every 25 min for 75 min; total dose of 0.4 mg/kg). This dose of DETA/NO has been shown to activate PKC- and to induce a late phase of cardioprotection against infarction and stunning 24 h later (9, 15). The PKC inhibitor chelerythrine (CHE; 5 mg/kg) was given 5 min before the first DETA/NO injection; this CHE dose has been shown to abolish NO-induced activation of PKC- and cardioprotection (9, 15). Hearts were harvested either 30 min or 24 h after the last DETA/NO injection and and stored at –80°C until biochemical analyses were conducted.
Immunoprecipitation and Western blot analysis.
Myocardial lysates were fractionated by differential centrifugation. Immunoprecipitation, SDS-PAGE, and immunoblotting were performed as previously described (9, 11, 15). Antibodies to PKC- and Bmx and horseradish peroxidase-conjugated secondary antibodies were obtained from BD Transduction Laboratories. Values are means ± SE. Groups were compared using Student's t-test for unpaired data. P < 0.05 was considered significant.
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RESULTS AND DISCUSSION |
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signaling in a rabbit model of NO-induced cardioprotection. Taken with our previous study using a transgenic model of cardioprotection in the mouse (10), this finding suggests that a cardioprotective role for Bmx may transcend model and species.
The present investigation demonstrates for the first time that exogenous NO donors activate Bmx in the myocardium, as evidenced by recruitment of Bmx to the particulate fraction. Importantly, the NO donor activated and increased the expression of Bmx concomitant with the late phase of cardioprotection afforded by the drug. Furthermore, the NO donor induced increased expression of Bmx via a PKC-dependent mechanism. These data suggest that a novel class of tyrosine kinases may form modules with PKC- during cardioprotection.
Translocation of Bmx is known to be indicative of activation, and association of Bmx with the membrane has been shown to occur through its lipid-binding plekstrin-homology domain (8, 12). To determine whether Bmx participates in protective signaling systems of the heart, the subcellular distribution of this kinase was examined in the normal heart and after treatment with cardioprotective doses of the NO donor DETA/NO. Under basal conditions, the vast majority (90 ± 9%; Fig. 1B) of Bmx resided in the cytosol of the rabbit heart, in agreement with previous findings in other cell types (8, 12). No translocation of Bmx was observed 30 min after administration of the NO donor (i.e., during the "trigger phase" of NO-induced protection), akin to what was observed for Src tyrosine kinase at this time (15). In contrast to Src, however, the NO donor induced a significant increase in total expression of Bmx (128 ± 2% of control; Fig. 1A), concomitant with redistribution of Bmx to the particulate fraction 24 h later (38 ± 3% in DETA/NO group vs. 10 ± 1% in control group, P > 0.05; Fig. 1B). This finding is particularly interesting, because translocation of Bmx, indicative of its activation (12), occurred at the time at which the heart was protected against ischemic insult: 24 h after NO donor administration (i.e., during the "mediator phase" of NO-induced protection).
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-mediated cardioprotection in the mouse, Bmx was found to localize to PKC- complexes (as detected by immunoprecipitation for PKC- followed by immunoblotting for Bmx) in the particulate, but not the cytosolic, fraction of the rabbit myocardium (Fig. 1C).
It is important to note that although the -isoform of PKC has been shown to be protective in the mouse, studies from other species have implicated different isoforms of PKC in cardioprotection, for example, the 
-isoform in dogs (7) and pigs (13) and the 
-isoform in some forms of protection in rats (5). Indeed, the species dependence of PKC isoform-specific signaling to Bmx and other Tec family kinases in the myocardium will be a key issue to resolve in future studies. At this point, the downstream tasks regulated by Bmx in cardioprotection are completely unknown. Previous studies have shown that Bmx activates STAT and NF-
B in noncardiac cells (8, 12). Therefore, these transcription factors, both of which have been shown to be necessary for cardioprotection, may be modulated by Bmx-dependent mechanisms in the heart. Furthermore, cross talk between Src and Bmx pathways and the role of Src in activation of Bmx [which has been documented in noncardiac cells (8, 12)] remain to be determined in the setting of cardioprotection.
It has not escaped our attention that increased expression of a second, higher-molecular-weight species of Bmx is observed in the protected myocardium at 24 h (Fig. 1A). The possibility exists that this second, higher-molecular-weight band is a posttranslationally modified species of Bmx. This modified species may represent an active, double tyrosine-phosphorylated form of Bmx, which is in agreement with previous investigations suggesting dual tyrosine phosphorylation as a mechanism to activate Tec kinases (8). A second possibility would be a heretofore unrecognized serine/threonine phosphorylation of Bmx (in support of which, Bmx contains 9 putative PKC phosphorylation sites). The potential role of posttranslational modifications of Bmx in cardioprotective signal transduction is the focus of ongoing studies.
The present study is the first, to our knowledge, to report NO-dependent activation of the nonreceptor tyrosine kinase Bmx in the heart and to implicate a role for this kinase in cardioprotection. These findings are potentially important with regard to a greater understanding of PKC-tyrosine kinase modules in the heart.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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in opioid-initiated cardioprotection. Am J Physiol Heart Circ Physiol 280: H1346–H1353, 2001. in activation of ecto-5'-nucleotidase in the preconditioned canine myocardium. Biochem Biophys Res Commun 239: 171–175, 1997.[CrossRef][ISI][Medline]
signaling complexes in the normal heart and during cardioprotection. Circ Res 88: 59–62, 2001.-Src modules direct signal transduction in nitric oxide-induced cardioprotection: complex formation as a means for cardioprotective signaling. Circ Res 88: 1306–1313, 2001.This article has been cited by other articles:
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  T. Tu, D. Thotala, L. Geng, D. E. Hallahan, and C. D. Willey Bone Marrow X Kinase-Mediated Signal Transduction in Irradiated Vascular Endothelium Cancer Res., April 15, 2008; 68(8): 2861 - 2869. [Abstract] [Full Text] [PDF]
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 C. H. Chau, C. A. Clavijo, H.-T. Deng, Q. Zhang, K.-J. Kim, Y. Qiu, A. D. Le, and D. K. Ann Etk/Bmx mediates expression of stress-induced adaptive genes VEGF, PAI-1, and iNOS via multiple signaling cascades in different cell systems Am J Physiol Cell Physiol, August 1, 2005; 289(2): C444 - C454. [Abstract] [Full Text] [PDF]
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 P. Mathur, S. Kaga, L. Zhan, D. K. Das, and N. Maulik Potential candidates for ischemic preconditioning-associated vascular growth pathways revealed by antibody array Am J Physiol Heart Circ Physiol, June 1, 2005; 288(6): H3006 - H3010. [Abstract] [Full Text] [PDF]
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