The TOMM machinery is a molecular switch in PINK1 and PARK2/PARKIN-dependent mitochondrial clearance

Loss-of-function mutations in PARK2/PARKIN and PINK1 cause early-onset autosomal recessive Parkinson disease (PD). The cytosolic E3 ubiquitin-protein ligase PARK2 cooperates with the mitochondrial kinase PINK1 to maintain mitochondrial quality. A loss of mitochondrial transmembrane potential (ΔΨ) leads to the PINK1-dependent recruitment of PARK2 to the outer mitochondrial membrane (OMM), followed by the ubiquitination and proteasome-dependent degradation of OMM proteins, and by the autophagy-dependent clearance of mitochondrial remnants. We showed here that blockade of mitochondrial protein import triggers the recruitment of PARK2, by PINK1, to the TOMM machinery. PD-causing PARK2 mutations weakened or disrupted the molecular interaction between PARK2 and specific TOMM subunits: the surface receptor, TOMM70A, and the channel protein, TOMM40. The downregulation of TOMM40 or its associated core subunit, TOMM22, was sufficient to trigger OMM protein clearance in the absence of PINK1 or PARK2. However, PARK2 was required to promote the degradation of whole organelles by autophagy. Furthermore, the overproduction of TOMM22 or TOMM40 reversed mitochondrial clearance promoted by PINK1 and PARK2 after ΔΨ loss. These results indicated that the TOMM machinery is a key molecular switch in the mitochondrial clearance program controlled by the PINK1-PARK2 pathway. Loss of functional coupling between mitochondrial protein import and the neuroprotective degradation of dysfunctional mitochondria may therefore be a primary pathogenic mechanism in autosomal recessive PD.     

The accumulation of misfolded proteins in the mitochondrial matrix is sensed by PINK1 to induce PARK2/Parkin-mediated mitophagy of polarized mitochondria

Defective mitochondria exert deleterious effects on host cells. To manage this risk, mitochondria display several lines of quality control mechanisms: mitochondria-specific chaperones and proteases protect against misfolded proteins at the molecular level, and fission/fusion and mitophagy segregate and eliminate damage at the organelle level. An increase in unfolded proteins in mitochondria activates a mitochondrial unfolded protein response (UPR^mt) to increase chaperone production, while the mitochondrial kinase PINK1 and the E3 ubiquitin ligase PARK2/Parkin, whose mutations cause familial Parkinson disease, remove depolarized mitochondria through mitophagy. It is unclear, however, if there is a connection between those different levels of quality control (QC). Here, we show that the expression of unfolded proteins in the matrix causes the accumulation of PINK1 on energetically healthy mitochondria, resulting in mitochondrial translocation of PARK2, mitophagy and subsequent reduction of unfolded protein load. Also, PINK1 accumulation is greatly enhanced by the knockdown of the LONP1 protease. We suggest that the accumulation of unfolded proteins in mitochondria is a physiological trigger of mitophagy.     

PINK1-PRKN/PARK2 pathway of mitophagy is activated to protect against renal ischemia-reperfusion injury

Damaged or dysfunctional mitochondria are toxic to the cell by producing reactive oxygen species and releasing cell death factors. Therefore, timely removal of these organelles is critical to cellular homeostasis and viability. Mitophagy is the mechanism of selective degradation of mitochondria via autophagy. The significance of mitophagy in kidney diseases, including ischemic acute kidney injury (AKI), has yet to be established, and the involved pathway of mitophagy remains poorly understood. Here, we show that mitophagy is induced in renal proximal tubular cells in both in vitro and in vivo models of ischemic AKI. Mitophagy under these conditions is abrogated by Pink1 and Park2 deficiency, supporting a critical role of the PINK1-PARK2 pathway in tubular cell mitophagy. Moreover, ischemic AKI is aggravated in pink1 andpark2 single- as well as double-knockout mice. Mechanistically, Pink1 and Park2 deficiency enhances mitochondrial damage, reactive oxygen species production, and inflammatory response. Taken together, these results indicate that PINK1-PARK2-mediated mitophagy plays an important role in mitochondrial quality control, tubular cell survival, and renal function during AKI.     

The interaction of CK2alpha and CK2beta, the subunits of protein kinase CK2, requires CK2beta in a preformed conformation and is enthalpically driven

The protein kinase CK2 (former name: "casein kinase 2") predominantly occurs as a heterotetrameric holoenzyme composed of two catalytic chains (CK2alpha) and two noncatalytic subunits (CK2beta). The CK2beta subunits form a stable dimer to which the CK2alpha monomers are attached independently. In contrast to the cyclins in the case of the cyclin-dependent kinases CK2beta is no on-switch of CK2alpha; rather the formation of the CK2 holoenzyme is accompanied with an overall change of the enzyme's profile including a modulation of the substrate specificity, an increase of the thermostability, and an allocation of docking sites for membranes and other proteins. In this study we used C-terminal deletion variants of human CK2alpha and CK2beta that were enzymologically fully competent and in particular able to form a heterotetrameric holoenzyme. With differential scanning calorimetry (DSC) we confirmed the strong thermostabilization effect of CK2alpha on CK2beta with an upshift of the CK2alpha melting temperature of more than 9 degrees . Using isothermal titration calorimetry (ITC) we measured a dissociation constant of 12.6 nM. This high affinity between CK2alpha and CK2beta is mainly caused by enthalpic rather than entropic contributions. Finally, we determined a crystal structure of the CK2beta construct to 2.8 A resolution and revealed by structural comparisons with the CK2 holoenzyme structure that the CK2beta conformation is largely conserved upon association with CK2alpha, whereas the latter undergoes significant structural adaptations of its backbone.     

miR‐181a regulates p62/SQSTM1, parkin,and protein DJ‐1 promoting mitochondrial dynamics in skeletal muscle aging

One of the key mechanisms underlying skeletal muscle functional deterioration during aging is disrupted mitochondrial dynamics. Regulation of mitochondrial dynamics is essential to maintain a healthy mitochondrial population and prevent the accumulation of damaged mitochondria; however, the regulatory mechanisms are poorly understood. We demonstrated loss of mitochondrial content and disrupted mitochondrial dynamics in muscle during aging concomitant with dysregulation of miR‐181a target interactions. Using functional approaches and mito‐QC assay, we have established that miR‐181a is an endogenous regulator of mitochondrial dynamics through concerted regulation of Park2, p62/SQSTM1, and DJ‐1 in vitro. Downregulation of miR‐181a with age was associated with an accumulation of autophagy‐related proteins and abnormal mitochondria. Restoring miR‐181a levels in old mice prevented accumulation of p62, DJ‐1, and PARK2, and improved mitochondrial quality and muscle function. These results provide physiological evidence for the potential of microRNA‐based interventions for age‐related muscle atrophy and of wider significance for diseases with disrupted mitochondrial dynamics.     

Phosphorylation of recombinant human spermidine/spermine N(1)-acetyltransferase by CK1 and modulation of its binding to mitochondria: a comparison with CK2.

Cytosolic spermidine/spermine acetyltransferase (SSAT) catalyzes the acetylation of the N(1)-propylamino groups of spermine and spermidine. The enzyme has a very short half-life and is rapidly induced by various stimuli. Once acetylated, these polyamines are subjected to the action of polyamine oxidase, which, besides initiating polyamine catabolism, may produce reactive oxygen species that in turn trigger modifications in subcellular compartments such as mitochondria. The present work evaluates the ability of the cAMP-independent Ser/Thr-protein kinase CK1 to phosphorylate SSAT. Results demonstrate that SSAT is phosphorylated by CK1, in sites distinct from those phosphorylated by CK2. Moreover, both phosphorylation processes are involved in the uptake of SSAT into rat liver mitochondria. Although CK2 is less effective than CK1 in phosphorylating SSAT, CK2 phosphorylation is much more powerful in preventing binding of SSAT to mitochondrial structures. These results suggest the involvement of CK1- and CK2-mediated SSAT phosphorylation in regulating the contents of polyamines and SSAT itself within subcellular compartments and implicate SSAT and polyamines as indirect modulators of progression through the cell cycle.     

Persistent nuclear accumulation of protein kinase CK2 during the G1-phase of the cell cycle does not depend on the ERK1/2 pathway but requires active protein synthesis

Protein kinase CK2 and phosphorylated ERK1/2 accumulated in nucleus after serum stimulation of quiescent HepG2 cells. Nonetheless, phospho-ERK1/2 accumulated mainly in the nuclease-extracted fraction (NE) whereas the increases in nuclear CK2 (either CK2alpha or CK2beta) occurred initially in the nuclease-resistant fraction (NR). Transient decreases in CK2 were observed in cytoplasm and NE in the first 3h but thereafter they either reverted (cytoplasm) or increased above the control (NE). CK2 levels in both NE and NR were high in cells arrested at G1/S. Maximal nuclear accumulation of CK2 was blocked by cycloheximide but little affected by PD98059, SB203580 or apigenin, all of which affected nuclear phopho-ERK1/2. Thus, nuclear accumulation of CK2 during G1 phase is independent of ERK1/2 pathway. Although this process may initially relay on intracellular redistribution of the preexisting enzyme, active protein synthesis is required to attain maximal nuclear CK2 levels.     

Binding of polylysine to protein kinase CK2, measured by Surface Plasmon Resonance

The interaction between protein kinase CK2 and polylysine has been studied by Surface Plasmon Resonance (SPR). The binding process has a very low energy of activation, it is irreversible, and too slow as to explain the enzyme activity stimulation as a direct consequence of the polylysine binding. The polylysine interaction with a peptide substrate and with casein are faster, and in agreement with a substrate-mediated mechanism of activity stimulation. After several hours of incubation, the binding of polylysine to CK2 produces the loss of enzymatic activity.     

Assurance of mitochondrial integrity and mammalian longevity by the p62-Keap1-Nrf2-Nqo1 cascade

Sqstm1/p62 functions in the non-canonical activation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2). However, its physiological relevance is not certain. Here, we show that p62(-/-) mice exhibited an accelerated presentation of ageing phenotypes, and tissues from these mice created a pro-oxidative environment owing to compromised mitochondrial electron transport. Accordingly, mitochondrial function rapidly declined with age in p62(-/-) mice. In addition, p62 enhanced basal Nrf2 activity, conferring a higher steady-state expression of NAD(P)H dehydrogenase, quinone 1 (Nqo1) to maintain mitochondrial membrane potential and, thereby, restrict excess oxidant generation. Together, the p62-Nrf2-Nqo1 cascade functions to assure mammalian longevity by stabilizing mitochondrial integrity.     

Multiple forms of protein kinase CK2 present in leukemic cells: In vitro study of its origin by proteolysis

Human recombinant CK2 subunits were incubated for different times with the two main cytosolic proteases m-calpain and 20 S proteasome. Both, m-calpain in a calcium dependent manner and the 20 S proteasome, were able to degrade CK2 subunits in vitro. In both cases, CK2 was more resistant to these proteases than CK2. When these proteases were assayed on the reconstituted (22 holoenzyme, a 37 kDa -band, analogous to that observed in AML extracts, was generated which was resistant to further degradation. No degradation was observed when the 26 S proteasome was assayed on free subunits. Studies with CK2 deletion mutants showed that m-calpain and the 20 S proteasome acted on the C-terminus end of CK2. These results pointed to cytosolic proteases as agents involved in the control of the amount of free CK2 subunits within the cell, which becomes evident when CK2 is overexpressed as in AML cells.     

Association of protein kinase CK2 with eukaryotic translation initiation factor eIF-2 and with grp94/endoplasmin

Protein kinase CK2 forms complexes with some protein substrates what may be relevant for the physiological control of this protein kinase. In previous studies in rat liver cytosol we had detected that the trimeric form of eukaryotic translation initiation factor 2 (eIF-2) co-eluted with protein kinase CK2. We have now observed that the ratio between eIF-2 and cytosolic CK2 contents in testis, liver and brain is quite similar, being eIF-2 levels about 5-fold higher than those of CK2. Furthermore eIF-2 was present in liver samples immunoprecipitated with anti-CK2/ antibodies, confirming the existence of complexes containing both proteins. Nonetheless, these complexes would represent only a fraction of total cytosolic CK2 and eIF-2.We had also observed that rat liver membrane glycoproteins obtained through chromatography on wheat-germ lectin-Sepharose contain CK2 activity which copurifies with grp94/endoplasmin. We have now confirmed that this activity was due to the presence of protein kinase CK2 as evidenced by immunodetection with antibodies against CK2/. The fractions enriched in grp94/endoplasmin and CK2 also contained another 55-kDa polypeptide (p55) phosphorylated by CK2 which has been identified as calreticulin by N-terminal sequencing. Calreticulin and grp94/endoplasmin could be partially resolved from CK2 by chromatography on heparin-agarose and almost completely on ConA-Sepharose. However, phosphorylation of immunoprecipitated grp94/endoplasmin was enhanced by its preincubation with purified CK2 prior to immunoprecipitation, what confirms the easy reassociation between these proteins.The association of protein kinase CK2 with eIF-2 and with grp94/endoplasmin may serve to locate the enzyme in the cellular machinery involved in protein synthesis and folding, and reinforces the possible involvement of CK2 in these processes.     

Protein kinase CK2 in postsynaptic densities: phosphorylation of PSD-95/SAP90 and NMDA receptor regulation

Protein kinase CK2 (CK2) is highly expressed in rat forebrain where its function is not well understood. Subcellular distribution studies showed that the catalytic subunit of CK2 (CK2alpha) was enriched in postsynaptic densities (PSDs) by 68%. We studied the putative role of CK2 activity on N-methyl-D-aspartate receptor (NMDAR) function using isolated, patch-clamped PSDs in the presence of 2 mM extracellular Mg(2+). The usual activation by phosphorylation of the NMDARs in the presence of ATP was inhibited by the selective CK2 inhibitor 5,6-dichloro-1-beta-ribofuranosyl benzimidazole (DRB). This inhibition was voltage-dependent, i.e., 100% at positive membrane potentials, while at negative potentials, inhibition was incomplete. Endogenous CK2 substrates were characterized by their ability to use GTP as a phosphoryl donor and susceptibility to inhibition by DRB. Immunoprecipitation assays and 2D gels indicated that PSD-95/SAP90, the NMDAR scaffolding protein, was a CK2 substrate, while the NR2A/B and NR1 NMDAR subunits were not. These results suggest that postsynaptic NMDAR regulation by CK2 is mediated by indirect mechanisms possibly involving PSD-95/SAP90.     

Differential phosphorylation of Akt1 and Akt2 by protein kinase CK2 may account for isoform specific functions

Akt (also known as PKB) is a survival kinase frequently up-regulated in cancer; three isoforms of Akt exist, and among them Akt1 and Akt2 are the most widely and highly expressed. They share the same structure and activation mechanism and have many overlapping functions; nevertheless isoform-specific roles and substrates have been reported, which are expected to rely on sequence diversities. In particular, a special role in differentiating Akt1 and Akt2 isoforms has been assigned to the linker region, a short segment between the PH and the catalytic domains. We have previously found that a residue in the linker region (Ser129) is directly phosphorylated by protein kinase CK2 in Akt1; the phosphorylation of the homologous residue in Akt2 (Ser131) has never been analyzed. Here we show that Akt2, endogenously or ectopically expressed in different cell lines, is not phosphorylated on Ser131 by CK2, while in vitro recombinant Akt2 is a CK2 substrate. These data support the hypothesis that in vivo a steric hindrance occurs which prevents the access to the CK2 site. Additionally, we have found that Ser129 phosphorylation is involved in the recognition of the Akt1-specific substrate palladin; this observation provides an explanation of why Akt2, lacking Ser131 phosphorylation in the linker region, has a low efficiency in targeting palladin. CK2-dependent phosphorylation is therefore a crucial event which, discriminating between Akt1 and Akt2, can account for different substrate specificities, and, more in general, for fine tuning of Akt activity in the control of isoform-dependent processes.     

Centrosomal anchoring of the protein kinase CK1delta mediated by attachment to the large,coiled-coil scaffolding protein CG-NAP/AKAP450

Protein kinase CK1 (formerly termed casein kinase I) is ubiquitous in eukaryotic cells and comprises a family of as many as 14 isoforms (including splice variants) in mammalian cells. Mammalian CK1delta and CK1epsilon, which are highly related to each other, are enriched at the centrosomes in interphase cells and at the spindle during mitosis. In the present study we have isolated, using the yeast two-hybrid system, a 182 amino acid residue fragment of the centrosomal and golgi N-kinase anchoring protein (CG-NAP, also known as AKAP450), which specifically interacts with CK1delta and CK1epsilon, but not with other CK1 isoforms. The 182 amino acid residue CG-NAP fragment, or full length CG-NAP, co-immunoprecipitates with CK1delta and CK1epsilon from mammalian cells. Consistent with this association, endogenous CG-NAP/AKAP450 and CK1delta co-localize in cells. Moreover, when expressed in the presence of CK1delta the 182 amino acid residue CG-NAP fragment adopts the same sub-cellular localization as CK1delta. Strikingly, attachment of the CG-NAP fragment to the plasma membrane is sufficient to re-localize a significant level of CK1delta to the membrane. These findings support a model in which sub-cellular localization of CK1delta/epsilon molecules at the centrosome is mediated, at least in part, through the action of CG-NAP/AKAP450 and provide a potential mechanism by which the contribution to cell cycle progression by CK1delta/epsilon may be regulated.