Pivotal role of AKAP121 in mitochondrial physiology

Abstract

In this Perspective, we discuss some recent developments in the study of the mitochondrial scaffolding protein AKAP121 (also known as AKAP1, or AKAP149 as the human homolog), with an emphasis on its role in mitochondrial physiology. AKAP121 has been identified to function as a key regulatory molecule in several mitochondrial events including oxidative phosphorylation, the control of membrane potential, fission-induced apoptosis, maintenance of mitochondrial Ca²⁺ homeostasis, and the phosphorylation of various mitochondrial respiratory chain substrate molecules. Furthermore, we discuss the role of hypoxia in prompting cellular stress and damage, which has been demonstrated to mediate the proteosomal degradation of AKAP121, leading to an increase in reactive oxgyen species production, mitochondrial dysfunction, and ultimately cell death.

mitochondria are critical to cell function and survival, as they are vital in the maintenance and regulation of cellular homeostasis (4, 13). One of the key regulatory molecules responsible for the control of this organelle is the A kinase anchor protein 121 (AKAP121) (2). Acting as a scaffold protein, AKAP121 functions to anchor protein kinase A (PKA) and other signaling molecules onto the outer mitochondrial membrane (13). By performing this function, AKAP121 allows for the phosphorylation of specific key substrate molecules in the vicinity of the mitochondrial membrane, and it therefore achieves effective spatial and temporal control over their phosphorylation state (13).

Because of its recent identification as an essential regulator of mitochondrial respiration, the role that AKAP121 plays in mitochondrial dysfunction has led to an emergence of new research studies on this topic. In this issue of the American Journal of Cell Physiology, we highlight some recent findings in the fields relating hypoxia, mitochondrial dysfunction, and AKAP121 activity.

Several Critical Mitochondrial Functions Are Heavily Dependent On AKAP121

In addition to binding and anchoring PKA to the outer mitochondrial membrane, AKAP121 functions similarly in bringing various other signaling molecules from the cytosol and plasma membrane to the area directly surrounding the organelle, commonly referred to as the mitochondrial microenvironment (8, 9). Two such molecules transported to this mitochondrial microenvironment include SRC (constitutes a large family of protein tyrosine kinases) and protein tyrosine phosphatase D1 (PTPD1) (3, 12). Following the assembly and activation of these two proteins into the PTPD1/SRC complex, AKAP121 specifically binds to PTPD1, allowing SRC to phosphorylate mitochondrial membrane-specific proteins (Fig. 1) (9). Through this signaling pathway, AKAP121 was found to regulate mitochondrial membrane potential, oxidative phosphorylation, and the phosphorylation of several substrate molecules belonging to the mitochondrial respiratory chain (9). The mechanism by which the PTPD1/SRC complex phosphorylates proteins within the mitochondrial matrix, however, is yet to be identified (9).

Fig. 1.

Following a positive feedback loop involving cAMP/PKA and A kinase anchor protein 121 (AKAP121) formation, AKAP121 binds to and anchors PKA, as well as the protein tyrosine phosphatase D1 (PTPD1)/SRC complex, to the outer mitochondrial membrane (OMM) (2, 9, 13). Through a PKA-independent pathway, AKAP121 is able to successfully mitigate the interaction between dynamin-related protein 1 (DRP1) and fission protein 1 homolog (FIS1), which would otherwise lead to FIS1/DRP1-induced fission events (8). Through the PKA-dependent inhibitory phosphorylation of DRP1, AKAP121 also averts FIS1/DRP1-induced fission events by preventing the binding of DRP1 to FIS1 on the OMM (8). AKAP121-bound PKA also functions to phosphorylate several other key proteins located within the mitochondria, as well as those bound to and around the OMM, many of which may play roles in mitochondrial respiration and generation of mitochondrial membrane potential (9). During hypoxic conditions, SIAH2 binds AKAP121, causing the SIAH2/AKAP121 complex to enter the ubiquitin/proteasome pathway. Its degradation through this pathway leads to significantly decreased mitochondrial activity and ultimately cell death (2, 13).

In a study conducted by Livigni et al. (9), human embryonic kidney cells were transfected using AKAP121 and PTPD1 expression vectors. The authors found cytochrome-c oxidase (COX) to be a specific downstream phosphorylation target of the PTPD1/SRC complex (9). Furthermore, via semiquantitative PCR analysis, the same study found that, through an unknown mechanism, AKAP121 activity promoted the mitochondrial DNA (mDNA) accumulation of two key mitochondrial genes, NADH dehydrogenase and cytochrome B (9). Using similar methodologies, downstream targets of AKAP121 and its bound macromolecules may be identified.

Scorziello et al. (14) revealed a direct connection between AKAP121 and mitochondrial calcium (Ca²⁺) ion transport in neuronal cells. Through the colocalization of the mitochondrial Na⁺/Ca²⁺ exchanger isoform 3 (mNCX3) with AKAP121, activation of mNCX3 led to an increase in the maintenance of mitochondrial Ca²⁺ homeostasis. This mechanism accordingly resulted in a boosted mitochondrial metabolism and consequently, increased cellular survival. Whether mNCX3 translocates to the inner mitochondrial membrane or mitochondrial matrix following the colocalization with AKAP121 remains unknown (14). The connection of AKAP121 to these key mitochondrial functions underlines its importance in mitochondrial control (14).

AKAP121 Levels Are Controlled Via an Ubiquitin/Proteasome Pathway

The upregulation of AKAP121 takes place through a positive feedback loop in which the increased activity of the cAMP/PKA pathway induces expression of AKAP121 (2). As stated previously, this increase in AKAP121 levels enhances several of the critical mitochondrial pathways responsible for cell survival. To uncover the pathway by which AKAP121 was downregulated, and to determine the causes and effects of such decreased activity, Carlucci et al. (2) conducted further investigations, which have led to some novel findings.

The authors found AKAP121 to be posttranslationally degraded by the ubiquitin/proteasome pathway. In response to hypoxic (low oxygen) conditions, seven in absentia homolog 2 (siah2), an E3-ubiquitin ligase, was found to bind AKAP121 and tag it for rapid degradation via the ubiquitin/proteasome pathway (Fig. 1) (2). Owing to the involvement of AKAP121 in several vital mitochondrial functions, Carlucci et al. found that degradation of AKAP121 by the SIAH2-mediated pathway led to significantly reduced mitochondrial activity and signaling. In addition, the loss of AKAP121 occurred rapidly in a non-cell specific manner, beginning only 2–4 hours following hypoxic exposure. This finding brings forward a new mechanism by which hypoxia diminishes oxidative metabolism, leading to cellular stress, damage, and necrosis (2). Further studies may focus on identifying other potential mechanisms of AKAP121 degradation in addition to the SIAH2-mediated pathway (2).

Mitochondrial Fission-Induced Apoptosis Begins With AKAP121

Mitochondrial fission and fusion are absolutely vital for allowing mitochondria to rapidly respond to metabolic and environmental stress. These mitochondrial fusion events are known to have key implications in the maintenance of mitochondrial DNA (mDNA) and the various mitochondrial membrane machineries (16, 17). Mitochondrial fission events however, are vital in the removal of dysfunctional mitochondria, and in the facilitation of apoptosis during extreme stress conditions.

Kim et al. (8) found that through the PKA-dependent inhibitory phosphorylation of dynamin-related protein 1 (DRP1) and the PKA-independent control of fission protein 1 homolog (FIS1) complex formation with DRP1, AKAP121 is able to successfully control these mitochondrial fission events (8). Furthermore, building upon previous studies similar to the one conducted by Carlucci et al., Kim et al. uncovered a direct connection between the FIS1/DRP1 complex activity and the hypoxia-SIAH2-proteosome pathway. During hypoxic conditions, an increase in SIAH2 activity led to the degradation and associated decrease in levels of AKAP121 (8). This decreased availability of AKAP121 to control FIS1/DRP1-mediated fission resulted in inhibited oxidative phosphorylation, ultimately leading to cellular damage and cell death. However, such fission events caused by the hypoxia-induced, SIAH2-mediated proteosomal degradation of AKAP121 have also been linked to a cellular adaptation to hypoxia, resulting in cell survival (8). Overall, these findings suggest that AKAP121 performs a key role in regulating fission-related cellular apoptosis and adaptation to hypoxia. Further investigation should pinpoint key protein kinases involved in the modulation of FIS1/DRP1 interactions as well as identify the different mechanisms by which AKAP121 interactions with mitochondrial substrates can be facilitated (8).

Mitochondrial Dysfunction Is Averted By AKAP121

Mitochondrial activity within the cell is heavily dependent on a variety of complex factors, all of which are greatly influenced through signaling between the various mitochondrial membranes and the mitochondrial microenvironment (15). Excessive damage to these membranes or any one of the mitochondria's key signaling pathways may result in significant loss of function in regards to oxidative phosphorylation, excessive reactive oxygen species (ROS) production, and the damage or loss of mDNA (13).

Several of the key regulatory pathways that control mitochondrial activity rely on the proper functioning of AKAP121 (13). In a study conducted by Perrino et al. (13), the authors aimed to identify the role of AKAP121 during mitochondrial respiration and ROS generation. Through downregulation of AKAP121, results confirmed disrupted mitochondrial activity and resistance to oxidative stress, highlighting the requirement for a particular level of AKAP121 to be present within the cell. Competitive displacement of AKAP121 with an inactive peptide led to increased ROS production, mitochondrial dysfunction, and ultimately cell death (5, 13). Additional pathways and substrates involved in the development of mitochondrial dysfunction following the degradation or downregulation of AKAP121 remain uncertain. Overall, these findings comprise an important series of tests that confirm the crucial role played by AKAP121 in the regulation of mitochondrial function and cell survival (13).

Localization of AKAP121 Isoforms and Their Functions

Several alternative splice forms of AKAPs exist in tissues of the heart, liver, kidney, brain, and skeletal muscle (11). Dependent on the specific N-terminal amino acids present in the spliced variants of AKAPs, these proteins cannot only scaffold different macromolecules, but also attain the ability to transfer these macromolecules to various subcellular compartments including the nucleus, plasma membrane, golgi apparatus, and endoplasmic reticulum (ER) (7, 10). Alternate splicing products S-AKAP84 and AKAP121 were found to be involved in the localization of various signaling enzymes to cytoskeletal compartments within the cell, including microtubules of the mitotic spindle apparatus present during the M phase of mitosis (1). In addition to binding PKA, further studies found AKAP100 and AKAP121 to contain a putative K homology (KH) domain, which has been proposed to bind mRNA (7). Via binding of the KH domain to the 3′-untranslated regions of transcripts involved in oxidative phosphorylation, AKAP121 may play a significant role in facilitating the import of key mitochondrial proteins (6).

CONCLUSION

AKAP121 has been clearly identified as a key protein responsible for the proper functioning and regulation of mitochondrial dynamics. Not only has AKAP121 been confirmed to function as a vital intermediary in the oxidative synthesis of ATP, regulation of membrane potential, maintenance of mitochondrial Ca²⁺ homeostasis, and phosphorylation of various mitochondrial-associated molecules, but its role in mediating fission-related apoptosis highlights this protein as an extremely interesting point of study (8, 13). Research on the degradation, downregulation, and inactive peptide replacement of this protein leads to a common conclusion of the detrimental effects that occur among cells due to decreasing levels of AKAP121. Continued studies on the adverse effects caused by AKAP121 inactivity should gravitate towards its connection to various diseases and organ systems most affected by hypoxic exposure. Additionally, because of the expression of AKAP121 in various cell and tissue types, its study may be applicable to several preexisting fields of research in which hypoxia is already being used as an experimental model, including but not limited to research dealing with respiratory disease. Future exploration of AKAP121 will undoubtedly lead to the formation of an enhanced understanding of mitochondrial responses to hypoxia and other stress-mediated conditions. Further studies should reveal whether any additional subcellular pathways mediate control over AKAP121 activity. Broadened investigation may lead to the development of novel therapeutic strategies, which may act to either overexpress AKAP121 or stabilize the AKAP121/PKA complex in stressed cells, thus preventing mitochondrial fission and apoptosis.

GRANTS

N. Kolliputi was funded by the American Heart Association National Scientist Development Grant 09SDG2260957, National Institutes of Health National Heart, Lung, and Blood Institute Grant R01 HL-105932, and the Joy McCann Culverhouse endowment to the Division of Allergy and Immunology.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

A.C. conception and design of research; A.C. and A.F. prepared figures; A.C. drafted manuscript; A.C., A.F., R.L., and N.K. edited and revised manuscript; A.C., R.L., and N.K. approved final version of manuscript.