Protein Kinase C (PKC) is a group of enzymes that control the function of other proteins through the phosphorylation of serine/threonine on these proteins. Numerous studies have shown that PKC isoforms differ in their primary structure, subcellular localization, tissue distribution and biological function. It is localized in cytosol in an inactive conformation and is translocated to different subcelluar organelles including plasma membrane, Golgi complex and endoplasmic reticulum (ER) upon activation. In ischemic preconditioning, it is the key mediator since different signaling pathways converge at PKC and it mediates distinct cellular functions by phosphorylating specific downstream target proteins. However, little is known about adenosine- mediated PKC activation in the heart though adenosine is one of the three triggers in ischemic preconditioning. Therefore, understanding the PKC translocation by adenosine receptor can provide useful insights into the protective mechanism of PKC in ischemic preconditioned hearts. Accordingly, the present investigation was performed to determine adenosine receptor-mediated PKC translocation to different subcellular locations in the heart.
We demonstrated that activation of adenosine A1 receptors induced a rapid association of PKC¿¿¿¿¿ or PKCδ with caveolin-3 in adult rat cardiac myocytes. Specifically, we found that activation of adenosine A1 receptors with CCPA induced the selective translocation of PKC¿¿¿¿¿ and PKCδ (but not PKCα, PKCβ and PKCζ) from the cytosol to the membrane. Notably, we showed that activated PKC¿¿¿¿¿ and PKCδ were targeted to the caveolin-rich plasma membrane microdomains. We have also shown that PKC¿¿¿¿¿ and PKCδ colocalize to and associate with caveolin-3. Taken together, we demonstrated that activation of adenosine A1 receptors promotes targeting of novel PKC isoforms, PKC¿¿¿¿¿ and PKCδ to caveolin-rich plasma membrane microdomains (Chapter 2).
Identification of downstream effectors on the plasma membrane will lead to a better understanding of cardiac protection in the adenosine receptor-mediated PKC signaling in the heart. Using the patch clamp technique, we provide the first evidence that Kir2.1 channels are negatively regulated by caveolin-3, both in HEK293T cells and in rat cardiac myocytes. Specifically, we show that Kir2.1 channel activity is significantly inhibited by expression of caveolin-3 while its expression on the cell plasma membrane is not changed. Further, we demonstrate that caveolin-3 scaffolding domain peptide blocks the caveolin-3 mediated suppression of Kir2.1 current. Moreover, our data indicate that caveolin-3 is critical in PKC induced IK1 inhibition in the heart (Chapter 3).
There is ample evidence that PKCε resides in cardiac mitochondria. However, the signals that promote translocation of PKCε are largely unknown. We found that adenosine receptor activation induced a rapid association of PKCε with TOM70, which is dependent on HSP90. Specifically, we showed that activation of adenosine receptors induced a selective translocation of PKCε (but not PKCδ) from the cytosol to mitochondria. We also showed that adenosine-mediated PKCε translocation to mitochondria was significantly reduced by inhibiting HSP90 function or suppressing HSP90 expression. Further, our data indicate that HSP90 is critical for association of PKCε and TOM70. We demonstrate for the first time that adenosine receptor activation induces selective translocation of PKCε to mitochondria. This translocation process is associated with the mitochondrial import machinery TOM70 and is dependent on HSP90 function (Chapter 4).
Collectively, our data demonstrate the molecular mechanism underlying adenosine-mediated targeting of PKC isoforms to different subcellular locations, which consequently regulates the downstream of signaling such as Kir2.1 channel on the plasma membrane. Thus, the work presented in this thesis is not only important for a better understanding of ischemic preconditioning, but also for designing a strategy for clinical application of preconditioning or postconditioning biology.