Department of Physiology and Biophysics and Department of Medicine, Division of Cardiology, Cardiovascular Research Laboratories, David Geffen School of Medicine at University of California, Los Angeles, California 90095
IN 2003, the field of cardiac cell signaling celebrates over a decade of achievements detailing the importance of protein kinase C (PKC) family of kinases. Among them, a critical accomplishment has been identification and characterization of extracellular stimuli for PKC isozymes in the heart, including those that are hormonal, neuronal, and mechanical in nature (6, 7, 16). In particular, tremendous progress has been made in understanding how these stimuli act as upstream triggers to provoke the activation of PKC in cardiac cells (6, 7). Moreover, considerable evidence supports the concept that PKC functions in an isoform-dependent manner and that the activation of individual isozymes, not the activity of the entire family of kinases, mediates the manifestation of cardiac phenotypes (5, 7, 10, 13). Therefore, delineation of isozyme-selective downstream targets of PKC, and characterization of molecules that function in accord with PKC activation, will offer mechanistic insight into the pathogenesis of cardiac diseases and aid in the development of novel pharmacological interventions for effective treatment (5, 10, 12, 2023).
Recently, with the establishment of PKC isozymes as essential molecules for the transduction of extracellular signals, increasing effort has been devoted to the downstream signaling events that these kinases orchestrate. A number of investigations have embarked on the search of cellular molecules that are targets of PKC activation (14, 79, 1114, 1622). Indeed, there are disparities with respect to the molecular makeup of the downstream realm of individual PKC isozymes, which may be due in part to differences in species, cell models, and experimental conditions. Not-withstanding these differences, most studies support the notion that each isoform governs a myriad of downstream signaling events and that activation of an individual isozyme can lead to distinct cardiac phenotypes. In particular, it appears that some of the downstream signaling elements are commonly shared by all isozymes, whereas others are uniquely modulated in an isozyme-specific fashion; nevertheless, all are essential to mediate the biological functions of PKC.
At least two classes of molecular target of PKCs are emerging from these studies. The first contains those that bear consensus domain(s) of unique amino acid sequences that are targets for PKC phosphorylation; thus activation and/or regulation of these molecules involves direct protein-protein interactions between the PKC isozyme and the substrate, and often subsequent posttranslational modification of the substrate by PKC. In contrast to the binding partners depicted in the first class, the second class comprises proteins that are physically distant from PKCs. Accordingly, signaling transduction from PKC to these molecules is usually not associated with PKC phosphorylation-triggered chemical modifications; instead, activation of the second class of molecular targets usually requires ancillary molecules and/or other signaling mediators that themselves are direct substrates of PKCs. Although many myocardial molecules harbor PKC phosphorylation consensus sequences, only a notable few have been shown experimentally to be targets of direct phosphorylation in vivo. Examples include troponin I (4), which impacts contractile function, and the Lck typrosine kinase, which functions as a PKC binding partner in the genesis of cardioprotection against ischemic insults (14). However, the majority of PKC isozyme-activated molecular targets identified thus far belong to the repertoire of the second class. This rather long list includes MAPKs (2, 3, 6, 11, 17), the protective kinase Akt (17, 23), the L-type calcium channel (1), caveolae proteins (15), and many signaling (2, 3, 6, 8, 11), mitochondrial-associated (8, 9), and transcription- and translation-related proteins (8, 20).
It is important to note that the successful development of two key technologies, namely, cardiac-targeted transgenesis and adenovirus-mediated gene transfer, have empowered the investigation of isozyme-selective responses of the PKC family of kinases. These approaches have been critical to the success of these studies in the past decade.
The study by Porter et al., which is the current article in focus (Ref. 15, see p. C39 in this issue), exemplifies the strategy of adenovirus-mediated gene transfer and elegantly characterizes the effect of PKC isozyme activation on specific downstream signaling events. Using a model that has been previously established to study PKC isozymes and cardiac cell function, they investigated the role of individual PKC isozymes in modulating sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2) gene expression in cardiac cells. Although activation of PKC has been widely documented to induce cardiac hypertrophy in an isoform-dependent fashion (7), the manner in which this activation is coupled with diversified downstream signaling events of PKC remains unclear. Thus one of the central questions is to identify and characterize downstream targets of PKC isoforms that facilitate their isoform-specific action. The authors approached this question by examining whether there is a distinct effect of individual PKC isoforms on the expression of SERCA2; their results offer new insights regarding mechanisms of PKC signaling as well as the biology of cardiac hypertrophy.
Two important, novel findings in this study are: 1) activation of
PKC and PKC
, at levels that did not impact cardiac cell size, was
shown to significantly downregulate SERCA2 gene expression; and 2)
activation of PKC
, at levels that have been previously documented to
induce cardiac cell hypertrophy, did not trigger altered SERCA2 gene
expression. Together, these data indicate that PKC isoforms may target
distinct downstream signaling events, as illustrated by their regulation of
SERCA2 gene expression.
As we move forward in our search for isozyme-selective molecular targets of PKC, we are presented with new challenges. There is increasing consensus that the activation of molecular targets of PKC is profoundly influenced by at least three fundamental factors: location (i.e., the subcellular compartment at which the isozyme resides), time (i.e., the temporal profile that the isozyme displays), and quantity (the amplitude of expression that the isozyme exhibits). Success in addressing those issues will require experimental strategies that are effective in defining the cellular location where a particular PKC isozyme is expressed and activated, will necessitate approaches that tightly regulate the temporal expression of PKC isozymes, and will demand methodologies that are capable of monitoring the level of expression with a greater degree of precision. The rapid development of several technologies, such as conditional and tissue-specific transgenesis and the use of organelle-targeting sequences to enable subcellular compartment specific expression, will aid our efforts in this direction. It may take considerable time before complete success in such efforts is achieved; nevertheless, the outcomes are likely to be extraordinary, especially in terms of precisely defining isozyme-selective molecular targets in a subcellular compartment-, time-, and quantity-specific fashion. These lines of information will enable the construction of cellular maps portraying all molecular substrates and downstream targets of PKC in an isozyme-specific manner, thus providing a holistic view of PKC signaling subproteomes.
ACKNOWLEDGMENTS
I thank Drs. T. M. Vondriska, J. D. Molkentin, and P. A. Insel for helpful discussions and advice on this editorial.
This work was supported in part by National Heart, Lung, and Blood Institute Grants R01-HL-63901 and R01-HL-65431 (both to P. Ping) and by the Laubisch Endowment.
Address for reprint requests and other correspondence: P. Ping, Dept. of
Medicine, Division of Cardiology, David Geffen School of Medicine at Univ. of
California, Los Angeles, Suite 1609/1619, MRL Bldg., 675 CE Young Drive, Los
Angeles, CA 90095-1760 (E-mail:
peipeiping{at}earthlink.net
or
peipeiping{at}hotmail.com).
REFERENCES
1. Alden KJ,
Goldspink PH, Ruch SW, Buttrick PM, and Garcia J. Enhancement of L-type
Ca2+ current from neonatal mouse ventricular myocytes by
constitutively active PKC-II. Am J Physiol Cell
Physiol 282:
C768C774, 2002.
2. Braz JC, Bueno
OF, De Windt LJ, and Molkentin JD. PKC regulates the hypertrophic
growth of cardiomyocytes through extracellular signal-regulated kinase1/2
(ERK1/2). J Cell Biol 156:
905919, 2002.
3. Bueno OF and
Molkentin JD. Involvement of extracellular signal-regulated kinases 1/2 in
cardiac hypertrophy and cell death. Circ Res
91: 776781,
2002.
4. Burkart EM,
Sumandea MP, Kobayashi T, Nili M, Martin AF, Homsher E, and Solaro RJ.
Phosphorylation or glutamic acid substitution at protein kinase C sites on
cardiac troponin I differentially depresses myofilament tension and shortening
velocity. J Biol Chem 278:
1126511272, 2003.
5. Chen L, Hahn H,
Wu G, Chen CH, Liron T, Schechtman D, Cavallaro G, Banci L, Guo Y, Bolli R,
Dorn GW 2nd, and Mochly-Rosen D. Opposing cardioprotective actions and
parallel hypertrophic effects of PKC and
PKC. Proc
Natl Acad Sci USA 98:
1111411119, 2001.
6. Clerk A,
Bogoyevitch MA, Anderson MB, and Sugden PH. Differential activation of
protein kinase C isoforms by endothelin and phenylephrine and subsequent
stimulation of p42 and p44 mitogen activated protein kinases in ventricular
myocytes cultured from neonatal rat hearts. J Biol
Chem 269:
3284832857, 1994.
7. Clerk A and
Sugden PH. Untangling the web: specific signaling from PKC isoforms to
MAPK cascades. Circ Res 89:
847849, 2001.
8. Edmondson RD,
Vondriska TM, Biederman KJ, Zhang J, Jones RC, Zheng Y, Allen DL, Xiu JX,
Cardwell EM, Pisano MR, and Ping P. Protein kinase C signaling
complexes include metabolism- and transcription/translation-related proteins:
complimentary separation techniques with LC/MS/MS. Mol Cell
Proteomics 1:
421433, 2002.
9. Fryer RM, Hsu
AK, Wang Y, Henry M, Eells J, and Gross GJ. PKC inhibition does not
block preconditioning-induced preservation in mitochondrial ATP synthesis and
infarct size reduction in rats. Basic Res Cardiol
97: 4754,
2002.[ISI][Medline]
10. Hahn HS,
Yussman MG, Toyokawa T, Marreez Y, Barrett TJ, Hilty KC, Osinska H, Robbins J,
and Dorn GW 2nd. Ischemic protection and myofibrillar cardiomyopathy:
dose-dependent effects of in vivo PKC inhibition. Circ
Res 91:
741748, 2002.
11. Heidkamp MC,
Bayer AL, Martin JL, and Samarel AM. Differential activation of
mitogen-activated protein kinase cascades and apoptosis by protein kinase
C and
in neonatal rat ventricular myocytes. Circ
Res 89:
882890, 2001.
12. Kerkela R,
Ilves M, Pikkarainen S, Tokola H, Ronkainen J, Vuolteenaho O, Leppaluoto J,
and Ruskoaho H. Identification of PKC isoform-specific effects in
cardiac myocytes using antisense phosphorothioate oligonucleotides.
Mol Pharmacol 62:
14821491, 2002.
13. Mochly-Rosen D and Gordon AS. Anchoring proteins for protein kinase C: a means for
iscozyme selectivity. FASEB J
12: 3542,
1998.
14. Ping P, Song C,
Zhang J, Guo Y, Cao X, Li RC, Wu W, Vondriska TM, Pass JM, Tang XL, Pierce WM,
and Bolli R. Formation of protein kinase C-Lck signaling modules
confers cardioprotection. J Clin Invest
109: 499507,
2002.
15. Porter MJ, Heidkamp MC, Scully BT, Patel N, Martin JL, and Samarel AM. Isoenzyme-selective regulation of SERCA2 gene expression by protein kinase C in neonatal rat ventricular myocytes. Am J Physiol Cell Physiol 285: C39C47, 2003.
16. Rybin VO, Xu X,
and Steinberg SF. Activated protein kinase C isoforms target to
cardiomyocyte caveolae: stimulation of local protein phosphorylation.
Circ Res 84:
980988, 1999.
17. Saurin AT,
Martin JL, Heads RJ, Foley C, Mockridge JW, Wright MJ, Wang Y, and Marber
MS. The role of differential activation of p38-mitogen-activated protein
kinase in preconditioned ventricular myocytes. FASEB J
14: 22372246,
2000.
18. Shizukuda Y and
Buttrick PM. Protein kinase C- modulates thromboxane
A2-mediated apoptosis in adult ventricular myocytes via Akt.
Am J Physiol Heart Circ Physiol
282: H320H327,
2002.
19. Steer SA,
Wirsig KC, Creer MH, Ford DA, and McHowat J. Regulation of
membrane-associated iPLA2 activity by a novel PKC isoform in
ventricular myocytes. Am J Physiol Cell Physiol
283: C1621C1626,
2002.
20. Trejo J,
Massamiri T, Deng T, Dewji NN, Bayney RM, and Brown JH. A direct role for
protein kinase C and the transcription factor Jun/AP-1 in the regulation of
the Alzheimer's -amyloid precursor protein gene. J Biol
Chem 269:
2168221690, 1994.
21. Vondriska TM,
Klein KB, and Ping P. Use of functional proteomics to investigate PKC
-mediated cardioprotection: the signaling module hypothesis.
Am J Physiol Heart Circ Physiol
280: H1434H1441,
2001.
22. Ytrehus K, Liu
Y, and Downey JM. Preconditioning protects ischemic rabbit heart by
protein kinase C activation. Am J Physiol Heart Circ
Physiol 266:
H1145H1152, 1994.
23. Zhou HZ,
Karliner JS, and Gray MO. Moderate alcohol consumption induces sustained
cardiac protection by activating PKC- and Akt. Am J Physiol
Heart Circ Physiol 283:
H165H174, 2002.
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