*12.2.1. Protein kinase A*

PKA is activated by a signaling pathway originating from G-protein receptors, which produces cAMP. The association of PKA with mammalian mitochondria was documented four decades ago [86]. Mitochondrial PKA is present in the outer and inner mitochondrial membranes as well as in the mitochondrial matrix [28, 87]. Several mitochondrial substrates of PKA with distinct tissue-specific responses have been identified. During myocardial I/R, PKA is activated by increased levels of ROS and translocates to mitochondria [88]. The increase in activity of mitochondrial PKA is independent of mitochondrial levels of cAMP and is due to increased sequestration of the catalytic α-subunit in the mitochondrial matrix [88]. The presence of the catalytic α-subunit of PKA results in phosphorylation of subunits I (on S115 and S116), IVi1 (on T52), and Vb (on S40) of complex IV, followed by their degradation and the loss of activity [38, 40, 88]. Because the identified phosphorylation sites on complex IV are not within PKA consensus sites, it was proposed that PKA activation plays an indirect role in the loss of complex IV activity during myocardial ischemia [39]. These changes are sufficient to disrupt the function of mitochondrial respirasome and increase production of ROS [88]. PKA-mediated phosphorylation regulates ischemia-induced dysfunction of complex IV [38, 45, 88]. Ca2+ influx activated by PKA in cardiomyocytes induces mitochondrial permeability transition, which stimulates caspase-9 and apoptosis [89]. Inhibition of PKA prevents the loss of activity of complex IV during cardiac I/R, promotes postischemic cardiac contractile recovery, and decreases the infarct size in the ischemic heart [38, 45, 90]. Thus, PKA inhibitors could serve as candidates for cardioprotective agents [87, 89]. PKA also phosphorylates NDUFS4 and NDUFA10 subunits of complex I and this phosphorylation stimulates the activity of complex I and complex I-driven respiration [28, 29, 91]. Finally, Hsp20, which localizes to mitochondria and is expressed at high levels in cardiac, skeletal, and vascular smooth muscle, is regulated by the β-adrenergic/PKA signaling pathway [92]. PKA-mediated phosphorylation of Hsp20 on S16 increases in ischaemic myocardium and is cardioprotective [92].

and formation of pores that mediate the release of pro-apoptotic AIF, Smac, and cytochrome c from the mitochondria [81]. Smac is phosphorylated by JNK3, which decreases its pro-apoptotic actions [81]. Phosphorylation of Y97 and Y48 on cytochrome c regulates its capacity to form the apoptosome and activate caspase-9 [81]. Thus, phosphorylation of specific amino acids on a mitochondrial protein determines the fate of mitochondria by protecting their

**12. Role of protein kinases in regulating mitochondrial functions in** 

GSK3-β, PINK1, and tyrosine kinases, which suggests their involvement in I/R injury.

**12.2. Serine/threonine kinases in regulation of mitochondrial functions in ischemia/**

PKA is activated by a signaling pathway originating from G-protein receptors, which produces cAMP. The association of PKA with mammalian mitochondria was documented four

**12.1. Protein kinases regulating large dehydrogenase complexes**

glucose, such as neuronal and cardiac tissues.

**reperfusion**

*12.2.1. Protein kinase A*

Ischemia rapidly changes activities of different protein kinases including the calcium/calmodulin-dependent protein kinase II (CaMK-II), PKA, protein kinase B (PKB/Akt), PKC, Raf-1, ERK1/2, c-jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38 MAPK),

A specific family of protein kinases is exclusively present in the mitochondrial matrix of eukaryotic cells. These kinases have different sequences from the cytosolic protein kinases and they phosphorylate and inactivate large enzymatic complexes in the mitochondrial matrix, the branched-chain α-ketoacid dehydrogenase complex and pyruvate dehydrogenase complex, PDC. Perturbations in PDC activity result in energy deficits, neuronal dysfunction, and brain injury, such as those observed in stroke. Phosphorylation of the E1 α-subunit of PDC by PDK isozymes inhibits, whereas dephosphorylation increases PDC activity [82]. Cardiac and cerebral ischemia has no effect on the activity of PDC; however, reperfusion results in a fast decline in PDC activity in the brain [83]. Increased expression of PDK2 following traumatic brain injury may maintain E1 in the hyperphosphorylated (inactive) state, which impairs glucose oxidation after stroke and traumatic brain injuries [83]. Further, reperfusion-induced oxidative stress activates PKC-δ, which translocates to mitochondria and activates PDK2 leading to inhibition of E1 activity [84]. The inhibition of PDK during reperfusion restores PDC activity and decreases brain injury, demonstrating that phosphorylation by PDK2 mediates inhibition of PDC activity [85]. Infusion of the specific peptide inhibitor of PKC-δ, Tat-δV1-1, prevents the translocation of PKC-δ to mitochondria and maintains PDC activity [84]. Isozymes of PDK are considered attractive targets for therapies to improve PDC activity to diminish the detrimental effects of I/R in tissues that depend on metabolism of

integrity and functions or inducing MPT and apoptosis.

**ischemia/reperfusion**

190 Mitochondrial Diseases

In contrast, the activation of PKA during liver IR is cAMP-dependent and plays a protective role against liver injury [93]. The cAMP that activates mitochondrial PKA does not originate from membrane-bound adenylyl cyclase activated by the Gα protein, but is produced inside mitochondria by the carbon dioxide/bicarbonate-regulated soluble adenylyl cyclase in response to metabolically generated carbon dioxide [93]. Mitochondrial PKA regulates mitochondrial biogenesis, normalizes ROS production, and activates complex IV [46]. Inhibition of PKA exacerbates hepatocellular damage, whereas increasing cAMP levels to activate PKA protects the ischemic liver from injury [93]. PKA activation diminishes neutrophil and macrophage infiltration into ischemic liver tissue, reduces production of tumor necrosis factor α, interleukin (IL)-6, and IL-12 by macrophages, increases IL-10 expression, and prevents hepatocyte death [93]. Thus, PKA activation reduces the inflammatory response associated with reperfusion after liver ischemia. Because ischemia is an inherent component of liver transplantation, activation of PKA was proposed as a rationale for novel therapies to combat I/R injury and protect transplants [94]. However, increased cAMP levels in the liver *in vivo* inhibit complex IV due to phosphorylation of Y304 [41]. Because PKA is not a tyrosine kinase, it was proposed that PKA activates a downstream tyrosine kinase, which phosphorylates and inactivates complex IV [41]. Therefore, attempts aimed at activating PKA to protect the liver against ischemia should be treated with great caution.

PKA dysregulation has been implicated in several neurodegenerative disorders. PKAdependent pathways in different regions of the brain play a role in pathogenesis and cognitive decline in Alzheimer and Parkinson's diseases [94]. The acute phase of cerebral ischemia is accompanied by decreases in affinity of PKA for cAMP and PKA activity [94]. In contrast, the peri-ischemic and less-injured areas exhibit increased PKA-mediated phosphorylation compared to the ischemic core, which suggests that active PKA is associated with the survival of neuronal tissue [94]. Derangement of cAMP-dependent signal transduction is associated with ischemic neuronal damage and activation of the PKA is important for neuronal survival in acute cerebral ischemia [94]. Phosphorylation of Bad at S155 by mitochondria-anchored PKA leads to cytosolic sequestration of Bad and blocks its mitochondrial translocation, the release of cytochrome c, and neuronal apoptosis [77]. Also, PKA-mediated phosphorylation of Drp1 at S637 blocks its translocation to mitochondria and their fission and promotes mitochondrial fusion. In hippocampal neurons, expression of a constitutively active catalytic subunit of PKA targeted to the mitochondrion promotes the fusion of mitochondria into networks [95]. Finally, PKA suppresses autophagy and mitochondrial degradation in neurons by phosphorylating and inhibiting the microtubule-associated protein 1A/1B-light chain 3 (LC3) and increases neurite outgrowth [94, 96].

membrane receptors or those generated by metabolic stress have a rapid effect on Akt activation status in mitochondria. Akt could serve as a therapeutic target promoting mitochondrial functions in ischemic organs. However, the application is complicated by the fact that Akt

Protein Kinases and Regulation of Mitochondrial Function in Ischemia/Reperfusion Injury

http://dx.doi.org/10.5772/intechopen.71094

193

PKC plays a key role in mediating I/R injury in the brain, heart, and kidneys; however, elucidation of this role is complicated due to the presence of 11 distinct PKC isozymes, which have unique tissue and cellular localizations and often play quite opposite roles in ischemic injury even within the same organ. Alterations in PKC activity or subcellular localization during ischemia occur in the brain, heart, liver, and kidney [25, 104–106]. Major isozymes of PKC (α, β, γ, δ, ε) translocate to mitochondria in response to ischemia and/or reperfusion and oxidative stress, and have been implicated in regulating mitochondrial functions in ischemic and postischemic tissues [104]. The classical PKC isozymes (α, β, γ) are dependent on Ca2+ and 1,2-diacylglycerol, and ischemia increases the levels of both activators through the stimulation of phospholipase C. PKC-α activation has been implicated in ischemia-induced heart failure and cerebral barrier breakdown after ischemic stroke [107, 108]. PKC-α inhibition or deletion protects the heart from decompensation and cardiomyopathy and attenuates cerebral barrier breakdown after ischemia [107, 108]. In contrast, PKC-α translocation to mitochondria is protective against mitochondrial dysfunction, ATP deficits, and cell death caused by ischemia,

hypoxia, and oxidative stress in RPTC [109]. We have shown that ATP synthase (F<sup>0</sup>

activation of PKC-α exerts differential effects in different types of cells and tissues.

isozyme may play opposing roles at different stages of ischemic injury.

synthase [52]. Injury or inactive PKC-α disrupt, whereas active PKC-α promotes this association and increases the levels of α-, β-, and γ-subunits in injured RPTC [52]. Active PKC-α

PKC-α also reduces apoptosis by phosphorylating mitochondrial Bcl-2 on S70 [77, 110]. Thus,

PKC-γ is expressed exclusively in neurons of the brain and spinal cord and is activated rapidly during ischemia and inhibited during reperfusion [111–114]. PKC-γ plays a detrimental role in the early stages of ischemia mediating events leading to cell death. PKC-γ knockouts show smaller infarct areas after cerebral ischemia [112]. Also, PKC-γ may directly (or through the activation of a Src tyrosine kinase) phosphorylate NMDA receptors, stimulate their function, and increase the concentration of intracellular Ca2+, which results in mitochondrial dysfunction, ATP deficits, increased ROS formation, and neuronal death [112]. However, after reperfusion, PKC-γ may mediate protection against cell death [112]. Thus, the same PKC

PKC-δ is rapidly activated by signaling initiated by reperfusion. PKC-δ plays a detrimental role in ischemic stroke injury and mediates oxidative stress, cell death, and inflammation associated with reperfusion [111, 112]. Selective inhibition or deletion of PKC-δ reduces infarct size and ischemic brain injury caused by middle cerebral artery occlusion, specifically, reperfusion-induced death of parenchymal cells [115, 116]. PKC-δ inhibition activates PKB/Akt, inhibits translocation of Bad to mitochondria, and decreases apoptosis [112]. PKC-δ

F1

is a target of PKC-α, which associates with the α-, β-, and γ-subunits of the F<sup>1</sup>

promotes phosphorylation of the γ-subunit on S146 and F<sup>0</sup>

F1


domain of ATP


promotes tumor formation by blocking apoptosis.

*12.2.3. Protein kinase C*

#### *12.2.2. Protein kinase B (PKB/Akt)*

Akt can localize to the mitochondrial matrix and the inner and outer membranes of cardiac, neuronal, and kidney cells [97, 98]. Protein levels of mitochondrial Akt are regulated by a variety of extracellular signals and stresses, which induce rapid translocation of active (phosphorylated) Akt to the mitochondria [97, 98]. Mitochondrial localization of Akt is associated with cardioprotection against ischemic injury and renoprotective actions against chemical toxicity [97–100]. The cardioprotective effect of mitochondrial Akt is attributed to its action on mitoK(ATP) channels and to decreasing apoptosis [99]. Akt phosphorylates pro-apoptotic Bad on S136 and Bax on S184, and prevents MPT in neuronal cells [101]. Phosphorylated Bad associates with 14-3-3 proteins in the cytosol and cannot form complexes with mitochondrial Bcl-2/Bcl-XL to induce permeabilization of the mitochondrial outer membrane and MPT [101]. Bax phosphorylation by Akt promotes dimerization of Bax with Bcl-XL or Mcl-1 proteins, which sequesters Bax away from mitochondria and prevents their permeabilization and apoptosis [101]. Akt prevents the MPT in cardiomyocytes by phosphorylating hexokinase II, stabilizing it in the outer mitochondrial membrane, and promoting its binding to VDAC [102]. Inhibition of Akt or targeted disruption of the association of hexokinase II with mitochondria abolishes cardioprotection [102].

We have identified mitochondria as a subcellular target of protective actions of Akt against necrosis in injured RPTC [98]. Mitochondrial levels of active Akt decrease in injured RPTC and this is associated with mitochondrial dysfunction [98, 100]. Selective activation of Akt increases the levels of Akt in mitochondria, improves state 3 respiration, activities of complexes I and III and F0 F1 -ATPase (ATP synthase), and the mitochondrial membrane potential (ΔΨm), increases ATP production, and reduces ATP deficits [100]. Selective inhibition of Akt exacerbates mitochondrial dysfunction, energy deficits, and necrosis in injured RPTC [100]. These results are consistent with a report that Akt phosphorylates the β-subunit of ATP synthase and improves its activity and increases ATP production to prevent energy deficits in injured cells [97]. Also, active Akt increases activity of PDC and oxidative metabolism of carbohydrates by inhibiting phosphorylation of PDC by GSK-3β [103]. Thus, signals from cell membrane receptors or those generated by metabolic stress have a rapid effect on Akt activation status in mitochondria. Akt could serve as a therapeutic target promoting mitochondrial functions in ischemic organs. However, the application is complicated by the fact that Akt promotes tumor formation by blocking apoptosis.
