**11.2. Pyruvate dehydrogenase complex (PDC)**

PDC is composed of multiple copies of three distinct enzymes: (1) pyruvate dehydrogenase (E1), dihydrolipoyl transacetylase (E2), and dihydrolipoyl dehydrogenase (E3), which form a large complex. PDC is the rate-limiting enzyme in the oxidative metabolism of all carbohydrates and its activity is tightly regulated by multiple mechanisms including phosphorylation. Phosphorylation of the PDC by the pyruvate dehydrogenase kinase (PDK) inactivates the complex [64]. The α-subunit of the E1 is phosphorylated at multiple sites, but the most known are phosphorylations of three distinct serines [64]. Although phosphorylation at any of these serines is sufficient to inhibit the activity of PDC, the S293 residue has the highest affinity for phosphate, and phosphorylation of this serine has the greatest impact on the inhibition of activity of PDC [64, 65]. I/R-induced injury in cardiac tissue is associated with a 4–5 fold decrease in the phosphorylation of S293, which results in the activation of PDC [64]. PDC activation protects against ischemic injury and improves cardiac efficiency and contractile capacity in the postischemic heart [65].

#### **11.3. Proteins involved in mitochondrial dynamics**

target of ischemia [5, 53]. The four human isoforms, ANT1, 2, 3, and 4, are phosphorylated on tyrosines [49]. Phosphorylation of ANT on Y194 and Y190 alters the activity of ADP/ATP translocase and the transport of both nucleotides in the brain [49]. Phosphorylation of ANT1 at Y194 in cardiac tissue is diminished by I/R but maintained by pre- and post-conditioning, which suggests that this phosphorylation plays a protective role against ischemia in the heart and could improve tolerance against injury [54]. Although this phosphorylation improves respiration and mitochondrial function, it is not known whether phosphorylation of Y194 is sufficient for protection of cardiomyocytes from I/R-induced injury [54]. A binding of phosphorylated (inactive) glycogen synthase kinase-3β (GSK-3β) to ANT was also shown, but it is

VDAC, a channel protein localized to the outer mitochondrial membrane, conducts a variety

is a key regulator of energy metabolism [56]. VDAC forms a complex with ANT, which facilitates influx of ADP into mitochondria and the efflux of ATP to the cytoplasm [56–58]. VDAC1 closure leads to hyperpolarization of mitochondria, disruption of ADP/ATP exchange, decrease in ATP synthesis, and metabolic dysfunction, and rupture of mitochondria [56–58]. The four known VDAC isoforms present in the outer mitochondrial membrane are phosphorylated on multiple serines and threonines [21, 22]. A total of 19 distinct phosphorylations were identified in VDAC isoforms [21, 65]. These phosphorylations are mediated by different kinases including PKA, PKC, tyrosine kinase, hexokinase, GSK-3β, Akt, JNK3, and p38 [59]. The phosphorylation of neuronal and hepatic VDAC1 by PKA and ischemia-activated GSK-3β and JNK induces its closure, disrupts formation of the complex with ANT, and decreases ATP synthesis [59]. In contrast, phosphorylation of cardiac VDAC1 by protein kinase C-ε (PKC-ε) promotes formation of the VDAC-ANT complex, prevents the opening of MPTP, and protects mitochondrial integrity after I/R injury [60]. Phosphorylation of VDAC1 by never-in-mitosis A related kinase 1 (Nek1) on S193 closes the channel in RPTC, blocks release of cytochrome c,

Phosphoproteomic analyses revealed that most enzymes of the TCA cycle are phosphorylated/dephosphorylated [62]. These include aconitase, isocitrate and oxo-ketoglutarate dehydrogenases, succinyl-coenzyme A synthetase, succinate dehydrogenase, fumarate hydratase, and mitochondrial malate dehydrogenase [62, 63]. Although the functional role of these phosphorylations and the protein kinases that mediate them are not yet known, data suggest that

PDC is composed of multiple copies of three distinct enzymes: (1) pyruvate dehydrogenase (E1), dihydrolipoyl transacetylase (E2), and dihydrolipoyl dehydrogenase (E3), which form

/NADH, ADP, and ATP) and ions across the outer membrane and

unclear whether GSK-3β directly phosphorylates ANT [55].

**11. Voltage-dependent anion channel (VDAC)**

of small metabolites (NAD+

188 Mitochondrial Diseases

and prevents cell death by apoptosis [61]

**11.1. Phosphorylation of enzymes of the tricarboxylic acid cycle (TCA)**

phosphorylation regulates the activity of these enzymes.

**11.2. Pyruvate dehydrogenase complex (PDC)**

Mitochondrial morphology is dynamically changed by the balance between fusion and fission (fragmentation). Phosphorylations of GTPases, Drp1, Mfn1/2, and Opa1 regulate mitochondrial fission and fusion [66]. Phosphorylation of fission-inducing Drp1 on S616 and S637 can be mediated by cyclin-dependent kinase (cdk)-1, cdk-5, PKA, PKC-δ, and extracellular signal regulated kinase-2 (ERK-2) [66–71]. Phosphorylation of Drp1 at S616 by cdk-1, PKC-δ, and ERK-2 promotes Drp1 translocation to mitochondria and fission whereas phosphorylation of S637 by PKA inhibits Drp1 and mitochondrial fission [67–71]. We have demonstrated that activation of PKC-ε induces the translocation of Drp1 to mitochondria, mitochondrial fission and apoptosis in non-injured RPTC [72]. Drp1-dependent mitochondrial fission is associated with mitochondrial outer membrane permeabilization and apoptosis, whereas elongation of the mitochondria through fusion promotes ATP synthesis and prevents mitochondrial autophagy. When phosphorylated by ERK, Mfn1 has decreased ability to oligomerize and tether mitochondria, which inhibits their fusion, recruitment of Bak to the mitochondria, and apoptosis [73]. Mfn2 is phosphorylated on T111 and S442 by PTEN-induced putative kinase protein 1 (PINK1), which recruits the protein Parkin to depolarized mitochondria and eliminates them by mitophagy [74]. Disruption in the balance between fission and fusion occurs in pathological conditions including I/R, which increases phosphorylation of Drp1, its translocation to mitochondria, and mitochondrial fission in the heart and brain [75]. Inhibition of Drp1 protects the heart and brain against ischemia and has been proposed as a therapeutic target following cardiac arrest [76].

#### **11.4. Proteins of the intrinsic apoptotic cascade**

Mitochondrial proteins involved in the intrinsic apoptosis include Bcl-2 family members, AIF, Smac/DIABLO, cytochrome c, and Omi/Htra2. With an exception of one, all are regulated by phosphorylation [20]. Phosphorylation of Bcl-2 at S70 is required to prevent permeabilization of the mitochondrial outer membrane and for Bcl-2's anti-apoptotic activity [77]. Several protein kinases serve as Bcl-2 kinases to inhibit (JNK, p38, and GSK-3) or activate (Akt, PKA, and PKC-α) Bcl-2 [77]. Cardiac and neuronal tissues are protected against I/R when Bcl-2 is active [78–80]. Ischemia-induced acute kidney injury upregulates Bcl-2 and Bcl-XL in the distal and Bax in the proximal tubules [80]. Pro-apoptotic Bax, Bak, Bad, and Bid are phosphorylated on serines and tyrosines, which controls their insertion into the outer mitochondrial membrane 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 integrity and functions or inducing MPT and apoptosis.

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

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

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of Hsp20 on S16 increases in ischaemic myocardium and is cardioprotective [92].

against ischemia should be treated with great caution.

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

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
