Phosphoregulation on mitochondria: Integration of cell and organelle responses

Abstract Mitochondria are highly integrated organelles that are crucial to cell adaptation and mitigating adverse physiology. Recent studies demonstrate that fundamental signal transduction pathways incorporate mitochondrial substrates into their biological programs. Reversible phosphorylation is emerging as a useful mechanism to modulate mitochondrial function in accordance with cellular changes. Critical serine/threonine protein kinases, such as the c‐Jun N‐terminal kinase (JNK), protein kinase A (PKA), PTEN‐induced kinase‐1 (PINK1), and AMP‐dependent protein kinase (AMPK), readily translocate to the outer mitochondrial membrane (OMM), the interface of mitochondria‐cell communication. OMM protein kinases phosphorylate diverse mitochondrial substrates that have discrete effects on organelle dynamics, protein import, respiratory complex activity, antioxidant capacity, and apoptosis. OMM phosphorylation events can be tempered through the actions of local protein phosphatases, such as mitogen‐activated protein kinase phosphatase‐1 (MKP‐1) and protein phosphatase 2A (PP2A), to regulate the extent and duration of signaling. The central mediators of OMM signal transduction are the scaffold proteins because the relative abundance of these accessory proteins determines the magnitude and duration of a signaling event on the mitochondrial surface, which dictates the biological outcome of a local signal transduction pathway. The concentrations of scaffold proteins, such as A‐kinase anchoring proteins (AKAPs) and Sab (or SH3 binding protein 5—SH3BP5), have been shown to influence neuronal survival and vulnerability, respectively, in models of Parkinson's disease (PD), highlighting the importance of OMM signaling to health and disease. Despite recent progress, much remains to be discovered concerning the mechanisms of OMM signaling. Nonetheless, enhancing beneficial OMM signaling events and inhibiting detrimental protein‐protein interactions on the mitochondrial surface may represent highly selective approaches to restore mitochondrial health and homeostasis and mitigate organelle dysfunction in conditions such as PD.


| BACKG ROU N D
Mitochondria are highly integrated organelles responsible for regulating cellular bioenergetics and viability in a continually changing environment. This extensive level of integration requires intricate and precise systems for communication with other cellular compartments. As the cellular environment changes, mitochondria must adjust their function, and cells must be able to detect, respond, and modulate mitochondria rapidly to adapt. Mitochondria have evolved sophisticated signaling mechanisms with cells that involve gases, ions, hormones, metabolites, and proteins. 1 Thus, mitochondria are crucial hubs for receiving, integrating, and transmitting signals within cells. 2 Environmental, cellular, and organellebased messengers impact all areas of mitochondrial physiology including genome integrity, bioenergetics, translation, and protein import ( Figure 1). 3 The quickness necessitated for accurately adjusting mitochondrial function to physiological perturbations implies that post-translational modifications (PTMs) of local proteins are used before the induction of transcriptional programs. Reversible phosphorylation has emerged as a prominent mechanism for rapidly regulating mitochondrial protein function; in fact, recent proteomic studies indicate that ~40% of the organelle proteome is phosphorylated. [4][5][6][7][8] Additionally, over 30 protein kinases and phosphatases are reported to migrate to mitochondria or have mitochondrial substrates. 6,[8][9][10][11] These phosphorylation events affect metabolism, mitochondrial dynamics, organelle function, and apoptosis demonstrating the importance of coordinated mitochondria-cell communication.
The outer mitochondrial membrane (OMM) is the interface between mitochondria and the rest of the cell. Despite that most mitochondrial proteins reside within the inner mitochondrial membrane (IMM), the OMM hosts significant elements of mitochondrial physiology including organelle dynamics, protein import, and apoptosis.
Consequently, protein kinases and phosphatases on the OMM are appropriately positioned to convey signals to and from mitochondria to directly manipulate mitochondrial form and function. The importance of OMM signaling is supported by numerous studies demonstrating that cytosolic kinases migrate to the organelle surface and modify local substrates to affect mitochondrial physiology. 8,9,11 Neurons have a particular reliance on mitochondria for energy production, calcium buffering, and managing ionic changes related to synaptic transmission. 12 Furthermore, dysfunction of OMM signaling components can contribute to the pathophysiology of neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD). 9,13 In this review, we will identify the kinases and phosphatases found on the OMM (summarized in Table 1) of neurons and other CNS cells.
We will present the molecular architecture required for each signaling protein and discuss the effects of specific substrate phosphorylation events including the impacts on organelle and neuronal physiology.
We will highlight how perturbations in kinase and phosphatase activities can contribute to the pathophysiology of neurological diseases.
F I G U R E 1 Mitochondrial signaling mechanisms regulate organelle, cell, and organisms physiology. Mitochondria are influenced by the intracellular and extracellular stimuli such as ions, metabolites, and molecules in the environment like oxygen and pesticides. However, the impact of signaling cascades in response to indirect actions of second messengers or stressors is emerging as significant manipulators of physiology. Mitochondrial signaling and second messengers have been shown to affect mitochondrial processes ranging from local events such as bioenergetics, mitochondrial dynamics, and proteins import to controlling transcriptional programs such as mitochondrial biogenesis, gene expression, and redox homeostasis. Of course, mitochondria are crucial to cell viability because the organelles are home to cell death machinery. Mitochondrial signaling can also transcend the cell through metabolites, second messengers, and even extracellular vesicles to impact metabolism, immune responses, inter-tissue signaling, and the microbiota within an organism. Mitochondrial signaling has emerged as a critical component to human health and disease. The stimuli and transcriptional programs converge on signaling proteins on the outer mitochondrial membrane (OMM), which are uniquely positioned to receive and convey signals from both cell and organelle. Discrete OMM signaling events then coordinate mitochondrial and cellular responses to adapt organelle, cell, and organismal physiology to the current environment

| OMM PROTEIN KINA S E S
Protein kinases influence mitochondrial protein function through phosphorylation. Intriguingly, most kinase activity within mitochondria can be attributed to tyrosine kinases such as Src. 14,15 Alternatively, much of the phosphorylation on the mitochondrial surface is associated with serine/threonine (Ser/Thr) protein kinases. 8 Accordingly, the OMM protein kinases are almost exclusively Ser/Thr kinases, which we will discuss below.

| c-Jun N-terminal kinase
The most well-characterized MAPK with OMM localization is the c-Jun N-terminal kinase (JNK). There are three JNK isoforms (JNK1, JNK2, and JNK3) that are expressed in the brain, and studies from mice suggest that JNK isoforms account for most of the proline-directed phosphorylation in the brain. 17 26,31,32 Disrupting the interaction between JNK and Sab is neuroprotective in rodent models of PD and cerebral ischemia. 23,33 Mitochondrial JNK activity impacts apoptosis, metabolism, and organelle dynamics ( Figure 2); perhaps, the most widely identified JNK mitochondrial substrates are proteins from the Bcl-2 superfamily. 34  In addition to regulating cell viability, JNK signaling is associated with bioenergetics in the brain. The first evidence of the regulation of mitochondrial metabolism by local JNK signaling came from studies in the aging mouse brain. 52 to enhanced ROS production. The direct inhibition of both PDH and complex I by JNK is problematic given that the substrates are within the mitochondria, and JNK had been shown to be present F I G U R E 2 c-Jun N-terminal kinase (JNK) on the OMM impacts apoptosis and bioenergetics. JNK signaling has long been linked to cell death and metabolism, and only recently was it realized that mitochondrial JNK signaling on the OMM scaffold protein Sab was critical to the induction of apoptosis through the manipulation of proteins within the Bcl-2 superfamily. Following activation, JNK translocates to mitochondria. JNK interacts with and phosphorylates the OMM scaffold protein Sab most likely through an interaction with the KIM2 motif (K2) because the KIM1 motif (K1) may be on the inner leaf of the OMM. In cases of neurotoxin exposure, ischemia, and cytotoxic stress, mitochondrial JNK activity leads to amplification of ROS production, organelle dysfunction, and cell death. Mitochondrial JNK can impair complex I by a yet-to-be-described mechanism to impair mitochondrial metabolism and engage ROS production. However, this may be facilitated by signaling on the coiled-coil motifs (CC1/CC2) of Sab's SH3 domain. Mitochondrial JNK can initiate apoptosis through phosphorylation of Bcl-2 on Ser70 inducing its emigration from mitochondria. JNK activity on mitochondria has also been demonstrated to impact mitochondrial dynamics through phosphorylation of Mfn2, which leads to its degradation and mitochondrial fission. JNK also influences mitochondrial metabolism directly through the inhibition of PDH via phosphorylation of the Eα1 subunit. In addition to Bcl-2, JNK can also influence the activities of BH3-only proteins such as Bim to induce apoptosis, and mitochondrial JNK has been shown to phosphorylate Bax to induce permeabilization of the OMM. The local activities of mitochondrial JNK suggest it could be a significant physiological player in organelle health on the mitochondrial surface. Therefore, JNK phosphorylates the substrates either before or during protein import, unless indirect mechanisms may be responsible for the inhibition. This appears to be the case for complex I, as Win  to ubiquitination and degradation by the proteasome. 56 Diminished smARF levels raised the steady-state levels of p62, a mediator of autophagic-lysosomal degradation, which was more readily degraded in the absence of JNK2. 56 Ablation of JNK2 produced higher levels of basal mitophagy and autophagy and was typified by high smARF and low p62 (a result of increased autophagy) levels. 56 Mitochondrial JNK activity has also been linked to the turnover of mitofusin-2 (Mfn2) but not Mfn1. JNK was shown to phosphorylate Mfn2 on Ser27, which promoted the ubiquitination and proteasomal degradation of Mfn2. 57 The loss of Mfn2 contributed to mitochondrial fragmentation in human U2OS osteosarcoma cells.
Additionally, the loss of Mfn2 was associated with induction of apoptosis to genotoxic stress induced by doxorubicin. 57 Therefore, it is possible that local JNK signaling can contribute to the turnover of stressed mitochondria by enhancing fission and degradation of problematic organelles. Collectively, mitochondrial JNK is a crucial regulator of mitochondrial form and function as well as cellular viability in the CNS.

| Extracellular regulated kinase
The extracellular regulated kinase (ERK) exists as numerous isoforms in the brain with ERK1/2 being the most well-characterized species of the family. Similar to JNK, ERK1/2 activity on the mitochondria has been reported in the hippocampus, 60 implicated in PD, 61  ERK activity, along with protein kinase A (PKA), is required for maximal steroid production in Leydig cells. 80 These studies collectively implicate local ERK signaling in the regulation of mitochondrial form and function.

| P38 kinases
The p38 kinases, in particular p38α (MAPK14), have been implicated in the pathogenesis of neurodegenerative, and pharmacological inhibitors of p38 have been shown to protect against neuronal loss in preclinical models of neurodegenerative disorders in the CNS. 81,82 The basis for these studies stems from the involvement of p38 in apoptosis. The general effect of p38 signaling affects the levels of Bcl-2 (decrease) and Bax (increase) levels on mitochondria through transcriptional and post-transcriptional means contributing to potentiating cells toward apoptosis. 83 However, p38 direct effects on Bcl-2 superfamily proteins remain elusive. Nonetheless, p38 has been shown to migrate to mitochondria follow stress (such as ischemia or oxidative stress) in cardiac tissue. 84

| PROTEIN K INA S E A , G , C ( AG C K INA S E S)
The AGC kinase family has two members present on the OMM that can influence organelle physiology: PKA and isoforms of PKC. 87 PKA is likely the most well-investigated cytosolic kinase on the mitochondrial surface, as it is well documented to play a role in sustaining organelle health. 88 Meanwhile, the role of PKC on mitochondria is more complicated with distinct variants contributing to the divergent aspects and outcomes of mitochondrial function and health. 89,90

| Protein kinase A (PKA)
Mitochondrial PKA has long been associated with mitochondria through its interaction with scaffold proteins of the A-kinase anchoring proteins (AKAP) family in particularly AKAP-1 (or D-AKAP as often used for the mitochondrial PKA scaffold). 91,92 PKA has been documented to affect numerous mitochondrial proteins ranging from bioenergetics to dynamics to redox homeostasis and cell survival. 93 The substrates for mitochondrial PKA have been identified both on and within mitochondria suggesting the ability of mitochondrial PKA to transcend the OMM. Mitochondrial PKA signaling is thought to be mainly beneficial to the organelle facilitating essential functions such as protein import while impeding processes such as fission and apoptosis. 94 Increasing the levels of mitochondrial PKA through genetic and pharmacological manipulation has been shown to be neuroprotective in preclinical models of neurodegenerative diseases, specifically PD. 95 These results indicate that diminished PKA signaling is a component of neurodegenerative disorders and restoring local PKA activity could be a means to impede the pathogenesis of these conditions.

| Protein kinase C (PKC)
Protein kinase C isoforms are emerging as potential regulators of mitochondrial function in the areas of metabolism and apoptosis [137][138][139] ; however, a complicating factor could be that distinct activities of PKC isoform could have discrete impacts on mitochondrial physiology. To date, three isoforms of PKC, PKCα, 140 PKCδ, and PKCε, 141 have shown mitochondrial localization. PKCδ is associated more with neurodegeneration and neuroinflammation, 142,143 while PKCε has been demonstrated to be more protective. 144 PKCδ has been shown to undergo cleavage by caspase activity in response to stress, and the cleaved species of PKCδ contributes to stress-induced mitochondrial dysfunction. PKCδ-induced mitochondrial dysfunction is also associated with dopaminergic neurotoxicity in preclinical PD models. 143,145 Although the outcomes of mitochondrial PKCδ signaling affect apoptosis, respiration, membrane potential, ROS production, antioxidant capacity, and organelle dynamics, [146][147][148] only Drp-1 has been shown to interact with PKCδ and contribute to organelle fragmentation. 149 Further investigation is needed to elaborate on PKCδ functions at mitochondria.
PKCε also demonstrates mitochondrial localization in response to cerebral ischemia and increases sirtuin 5 (Sirt5) levels. 150 Outside of this report, most of the work has been performed in non-CNS tissues. PKCε has been shown to phosphorylate complex IV and increase its activity in the heart and kidney in response to stress. 138,151 Also, PKCε has been shown to phosphorylate and regulate cardiac sodium channels in response to metabolic changes.
Concerning the CNS, PKCε phosphorylation has been shown to increase the activity of endothelin converting enzyme (ECE), which is responsible for amyloid beta clearance, via an N-terminal modification site. 152 This would indicate that PKCε may be downregulated in Alzheimer's disease, 153 and restoring PKCε activity could be neuroprotective similar to how PKCε activity is observed in cardiac tissue.

| PARKIN SON ' S D IS E A S E RELE VANT PROTEIN K INA S E S
Protein kinases have been implicated in the pathogenesis of

| Leucine-rich repeat kinase 2 (LRRK2)
A protein kinase implicated in familial and sporadic cases of PD is leucine-rich repeat kinase 2 (LRRK2), wherein mutations in LRRK2 have been shown to increase kinase activity. 154  Early studies found that expression of the most common clinical LRRK2 mutant G2019S could impact mitochondrial morphology increasing mitochondrial fission. The enhanced fission was found to be a result of interaction between LRRK2:G2019S and Drp-1. 160 The recruitment of Drp-1 to mitochondrial and subsequent fission in PD patient-derived fibroblasts and human SH-SY5Y cells was found to be dependent upon LRRK2 kinase activity. 161

| OTHER K INA S E S A SSO CIATED WITH OMM
As mentioned earlier, the occurrences of cytosolic protein kinases are increasing and more OMM kinases are being found through disease-specific proteomic studies. These include protein kinases that are implicated in cellular homeostasis, such as AMP-dependent protein kinase (AMPK), casein kinases (CK1 and CK2), and mammalian target of rapamycin (mTOR). Intriguingly, kinases implicated in the cell cycle have also emerged as OMM signaling agents. The appearance of these kinases on the OMM is further evidence of the extensive integration of organelle and cell physiologies and quality control.

| AMP-dependent protein kinase (AMPK)
A recent addition to the OMM signaling landscape is the AMPdependent protein kinase (AMPK), which was identified in two independent reports. 120

| Cyclin-dependent kinase 1 (Cdk1)
Cyclin-dependent kinase 1 (Cdk1) complexed with cyclin B is known to impact mitochondrial substrates during mitosis. Cdk1 was first reported to impact mitochondrial proteins in 2007 when Taguchi and colleagues discovered that Cdk1/cyclin B could phosphorylate Drp-1 on Ser585 (Ser616 human) and promoting mitochondrial fission prior to cell division. 195 It was later demonstrated that Cdk1/cyclin B could impact matrix proteins as well including components of respiratory complex I. The phospho-manipulation of the complex I subunits ultimately promoted the respiration necessary to produce the bioenergetic currency necessary for cell division. 196 Both the regulation of mitochondrial fission and respiratory chain by Cdk1/cyclin B have been linked to oncogenesis and therapeutic resistance in brain tumors as well as other types of cancer. 197 Collectively, mitochondrial signaling by Cdk1/cyclin B is an excellent example of the extensive coordination between mitochondrial and cellular programming.

| Cyclin-dependent kinase 5 (Cdk5)
Another example of a Cdk exhibiting OMM signaling capabilities is cyclin-dependent kinase 5 (Cdk5). Like Cdk1, Cdk5 can phosphorylate Drp1 on Ser616 in mature rat neurons, the same site as Cdk1 in mitotic cells, inhibiting mitochondrial fission. 198 Cdk5 activity was associated with elongated mitochondria during the maturation of cultured cortical neurons. 198 Intriguingly, Cdk5-mediated phosphorylation of Drp1 is linked to oncogenic potential in brain tumor initiating cells, in which enhanced Drp1 activity correlated with a poor glioblastoma prognosis. 199 In another study,

| Cyclin-dependent kinase 11 (Cdk11)
Similar to Cdk5, cyclin-dependent kinase 11 (Cdk11) has been shown by colocalization studies to have subpopulation on mitochondria. 203,204 After the identification of Cdk11 on mitochondria, there have been no follow-up studies to identify substrates.
Intriguing the localization of Cdk11 preceded the induction of apoptosis leading investigators to surmise that Cdk11 may have a role in the induction of cell death. 203,204 Cdk11 represents an interesting OMM kinase for study, as research related to mitochondria. Cdk11 could shed light on the relationship between organelle physiology and the cell cycle.

| Casein kinase I (CK1)
Both casein kinase I and II (CK1 and CK2) have been described to have mitochondrial influence and localization. 124,205,206 CK1 and CK2 are commonly viewed as Ser/Thr kinases that influence the activity of transcription factors to influence developmental signaling (ie, Wnt pathway) and circadian rhythms. However, CK1 and CK2 have been implicated in the regulation of mitochondrial function.
CK1 is involved the inhibition of Fas-mediated apoptosis through the phosphorylation of Bid on Ser64 and Ser66. 207,208 Phosphorylation by CK1 prevents the cleavage of Bid by caspase 8 limiting the release of cytochrome c from mitochondria. 207,208 While specific CK1 targets on mitochondria have yet to be identified, CK1 has been colocalized with mitochondria, and further studies may unearth CK1 substrates on mitochondria.

| Casein kinase II (CK2)
The role of CK2 on mitochondria is more extensive than CK1 where in addition to apoptosis CK2 plays a significant role in the regulation of organelle protein import 122,123 and quality control. 206 Concerning apoptosis, CK2 is known to phosphorylate

| Mammalian target of rapamycin (mTOR)
The mTOR signaling pathway has long been implicated in sensing mitochondrial substrates like branched amino acids; moreover, mTOR has been linked to bioenergetics and mitochondrial protein synthesis for some time. 212,213 The localization of mTOR signaling components on lysosomes and in proximity to mitochondrial-associated membranes (MAMs) meant it was only a matter of time before mitochondrial mTOR substrates would be discovered. 214

| OMM PROTEIN PHOS PHATA S E S
Compared to the breadth of studies on OMM protein kinases, far less is known regarding the activities and substrates of protein phosphatases on the mitochondrial surface. Because a number of the phosphorylation events ascribed to protein kinases on the OMM are reversible, it is likely that some phosphatases would be required to offset and regulation these phosphorylation events to adjust and maintain organelle physiology. 8,11 We will describe the actions of some of the known protein phosphatases on mitochondria below.

| Mitogen-activated protein kinase phosphatase-1 (MKP-1)
MKP-1 is a dual specificity phosphatase capable of dephosphorylating Ser/Thr and Tyr residues modified by protein kinases. 224 As the name implies, the primary substrates of MKP-1 are the MAPKs. As described earlier, MAPKs (namely JNK, ERK1/2, and p38) can translocate to mitochondria and manipulate local proteins. MKP-1 was also found to migrate to neuronal mitochondria in response to treatment of cells with neuronal growth factor. 224,225 It is proposed that JNK is a substrate of MKP-1 on mitochondria based on brain-related studies [226][227][228] suggesting that MKP-1 may act to inactivation JNK signaling to prevent apoptosis. Further investigation is warranted to determine whether the phosphatase affects other MAPKs on mitochondria or has unique substrates on the OMM.

| Phosphate and tensin homolog-long (PTEN-L)
A recent report by Wang

| Protein phosphatase 1 (PP1)
The PP1 is linked to the regulation of local signaling transduction on the OMM and the function of proteins related to apoptosis. [230][231][232][233] PP1 forms a complex with PKA and AKAP1 on the mitochondrial surface, 234 and in the absence of PKA, PP1 can destabilize AKAP1 leading to its degradation and reduce potential sites for mitochondrial PKA signaling. 235 This event was linked to the induction of long-term depression through the dephosphorylation of AMPA receptor. 234 OMM PP1 has been cited as a regulator of p38 signaling on mitochondria as well. PKC was found to induce PP1 following exposure to phorbol ester leading to dephosphorylation and inactivation of p38 on the mitochondria. 231 These studies indicate that PP1 could modulate OMM signaling in a context-dependent fashion.
PP1 is associated with the dephosphorylation of Bcl-2 proteins and the induction of apoptosis. 232 PP1 dephosphorylation of Bad can release the protein from 14-3-3 and permits Bad's interaction with and inhibition of anti-apoptotic Bcl-2 proteins. 128 PP1 interaction with Bcl-2 and Bcl-xL can also impact phosphatase activity. 236 These results indicate PP1 on the OMM is a regulator of apoptotic proteins.

| OMM SC AFFOLD PROTEIN S
Scaffold proteins and adaptor proteins are crucial accessory proteins required for the coordination of signal transduction events across space and time. 240,241 The relative abundance of accessory proteins can dictate the biological outcomes of signaling events by influencing the spatial magnitude and duration of a response. 240,241 Therefore, the identification and concentrations of these elements of molecular architecture will be critical to determine the presence and impact of signaling pathways on mitochondria.

| A-kinase anchoring protein 1 (AKAP-1)
AKAPs are membrane-bound scaffolds for PKA and its related signaling components. 91  This suggests that the PKA/AKAP1 mitochondrial signaling nexus is crucial to regulating stress responses and preserving optimal organelle function in the brain.
A recent report indicates that mitochondria AKAPs are arranged into specialized membrane microdomains and this physical interaction places PKA proximal to mitochondrial substrates such as Bad. 248

| Sab (SH3-binding protein 5; SH3BP5)
As mentioned above, Sab is an OMM scaffold for the JNK and a putative substrate of p38γ on the OMM. 27 We have found that Sab mRNA and protein levels are significantly enriched in the hippocampus, substantia nigra, and cerebellum, areas of the brain vulnerable to neurotoxin stimuli. 251 Thus, we contend that the elevated Sab concentrations could lead to robust mitochondrial JNK activity contributing to apoptosis and neurodegeneration in disorders such as AD and PD. Selective disruption of the JNK-Sab interaction protects dopaminergic neurons from 6-hydroxydopamine-induced neurotoxicity. 33 Thus, the specific targeting of detrimental mitochondrial JNK signaling could be a promising target to preserve mitochondrial health in PD.

| Protein interacting with C-kinase-1 (PICK1)
PICK1 is a scaffold protein for PKCs, and PICK1 has been linked to PKAα localization on mitochondria, where the kinase protected against apoptosis induced by genotoxic stress. [252][253][254] Another report indicates that PICK1 on mitochondria can impair Parkin activity and contribute to MPTP-induced neurotoxicity. PICK1 destabilized the interaction between Parkin and UbcH7 leading to diminished E3 ubiquitin ligase activity of Parkin. PICK1 knockout mice were protected against MPTP-induced cell death. 255 This effect of PICK1 is interesting in light of the neurotoxic and neuroinflammatory activities of PKCδ and may be another mechanism by which shifts in OMM signaling can contribute to PD pathogenesis and progression. 256 Additional scaffold proteins may also be localized to the OMM, as reports indicate that 14-3-3 may have a population of mitochondrial proteins that influence organelle signaling. 257,258 Also, the growth factor receptor-bound protein 10 (Grb10) may have OMM localization. 259 We contend that the accurate and reliable identification of OMM scaffold proteins will be crucial to determining the localization and signaling pathways to the mitochondrial surface.

| CON CLUD ING REMARK S AND FUTURE DIREC TIONS
In this review, we have presented some of the emerging signaling cascades and phosphorylation events on the mitochondria surface.
OMM signaling components are appropriately positioned to impact specific aspects of mitochondrial physiology. It is likely that more rigorous studies in this area will unearth more kinases, phosphatases, and adaptor proteins. Great care should be taken in identification and validation of new signaling components on the OMM using established as well as modern proteomic-based approaches.
While the identification of OMM signaling components and signaling cascades continues to be of interest, many of the OMM kinases are beginning to demonstrate interplay on the organelle surface. Earlier, we mentioned that AMPK could stabilize PKA signaling on mitochondria to enhance ETC function. 120 However, antagonism by kinases may also occur. The best current evidence for this is that In addition to modulating protein function, it is likely that signaling cascades can target opposing pathways for degradation using by manipulating local E3-ubiquitin ligases or other turnover mechanisms (ie, autophagy). But, one can anticipate that the scaffolding and organization of these molecular pathways will be critical to this integration. This potential has been noted with AKAP1, which plays a crucial role in the integration of PKA and mTOR signaling modules. 217 It is likely that similar mechanisms exist for other signaling proteins to balance mitochondrial form and function in an everchanging cellular environment. Research oriented around these distinct interactions will be critical to understanding the complex regulation of mitochondrial function and dysfunction in disease pathogenesis, and this scenario also highlights the need to consider therapeutic regimens that impair detrimental events while restoring beneficial signaling pathways on the OMM.
It is likely that the OMM signaling pathways will be highly responsive to mitochondrial messengers, such as ROS, and OMM proteins are appropriately positioned to recognize and response to these small molecules. The response to these messengers will shape cellular responses to the organelle health status. An example of this type of integration may be best illustrated by the interplay between ROS and the members of the Bcl-2 superfamily that dictate apoptotic responses. 263 For example, Bcl-2 can "sense" the magnitude of ROS production in response to post-translational modifications, namely Ser70 phosphorylation, 264 and changes in Bcl-2 can then impact ETC function, mitochondrial antioxidant capacity, and autophagic flux. 263 The robustness of the Bcl-2 response to ROS levels can determine whether a cell undergoes apoptosis. Our work demonstrates that enhancing mitochondrial JNK signaling by increasing Sab levels amplifies ROS production and renders cells sensitive to chemical insults in part by diminishing OMM Bcl-2 levels. 31,32,250 It is established that mitochondrial ROS can trigger ASK-1 to engage sustained JNK activity 265 leading to depletion of OMM Bcl-2 by Ser70 phosphorylation and induction of apoptosis. 26 In addition to JNK, numerous OMM localized kinases and phosphatases can modulate Bcl-2 superfamily protein function, which we contend is one significant route to coordinating organelle and cellular physiology because of the importance of Bcl-2 proteins to metabolism, autophagy, and apoptosis. The actions of OMM signaling proteins in response to ROS could dictate the actions of Bcl-2 proteins within the cell. Thus, it is imperative that OMM signaling pathways converging on Bcl-2 proteins be considered with great respect toward cellular environment.
In conclusion, the signalosomes of the OMM are the gatekeepers of mitochondrial physiology and mediators of disease pathogenesis in the CNS, as such there is much promise in the elucidation of signaling relationships on the mitochondrial surface and targeting distinct OMM signaling components to combat CNS disorders.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.