Cardiac contractility is regulated by several neural, hormonal, paracrine, and autocrine factors. Amongst these, signaling through β-adrenergic and serotonin receptors generates the second messenger cyclic AMP (cAMP), whereas activation of natriuretic peptide receptors and soluble guanylyl cyclases generates cyclic GMP (cGMP). Both cyclic nucleotides regulate cardiac contractility through several mechanisms. Phosphodiesterases (PDEs) are enzymes that degrade cAMP and cGMP and therefore determine the dynamics of their downstream effects. In addition, the intracellular localization of the different PDEs may contribute to regulation of compartmented signaling of cAMP and cGMP. In this review, we will focus on the role of PDEs in regulating contractility and evaluate changes in heart failure. Phosphodiesterases (PDEs) are the enzymes that degrade both cyclic adenosine 3′,5′-monophosphate (cAMP) and cyclic guanosine 3′,5′-monophosphate (cGMP) to nucleoside phosphates . PDEs are categorized in 11 families with subfamilies and splice variants ,3. All PIn this review, we focus on the role of PDEs in regulating cardiac contractility . After c2+ channels (LTCC) and influx of extracellular Ca2+, which in turn triggers the opening of ryanodine receptors (RyR) in the sarcoplasmic reticulum (SR) that release Ca2+ into the cytosol. Intracellular Ca2+ binds to TnC, leading to a conformational change and binding to TnI; this in turn moves its inhibitory domain away from actin and promotes the displacement of tropomyosin, leaving the actin surface free to bind the myosin heads. Together, these conformational changes lead to formation of cross-bridges along the myosin and actin myofilaments. When the cross-bridges are formed, ATP binds to myosin. Its hydrolysis brings the myosin heads to produce the power stroke, and the myofilaments will slide past each other. This triggers muscle shortening and ultimately contraction of the heart, called systole. Following contraction, cytosolic Ca2+ is efficiently removed and the cytosolic Ca2+ levels drop, due primarily to the activity of the sarcoplasmatic reticulum Ca2+-ATPase (SERCA) and the sarcolemmal Na+/Ca2+ exchanger, but also the sarcolemmal ATPase and the mitochondrial Ca2+ uniporter. As calcium levels drop, calcium bound to TnC is released, and TnI and tropomyosin are returned to a closed state that prevents actin from interacting with myosin. Thus, relaxation of the heart (diastole) can occur [2+ removal as well as the contractile proteins themselves are regulated by, e.g., cAMP and cGMP, mainly by phosphorylation of proteins involved in Ca2+ handling, such as LTCC, RyR, and the SERCA-inhibiting protein phospholamban (PLB), and sarcomeric proteins, such as TnI, myosin binding protein C (MyBP-C), and titin. Effects that enhance contractility, i.e., the ability of the heart to contract, are called (positive) inotropic effects, whereas effects that enhance cardiac relaxation are called (positive) lusitropic effects.Cardiomyocytes are the key players in cardiac contraction and relaxation. They contain contractile structures composed of the myofilaments, myosin and actin, organized in sarcomeric units. Along the myofilaments, actin monomers are localized with tropomyosin and the complex of troponin T, troponin I, and troponin C  . Electrian occur . The effs. Gi-coupled GPCRs, on the other hand, activate the inhibitory G protein Gi that inhibits AC activity. Cyclic AMP activates several effector proteins, such as protein kinase A (PKA), exchange proteins directly activated by cAMP (Epac1 and Epac2), cyclic nucleotide-gated ion channels (CNGCs), and Popeye-domain-containing proteins, which regulate a vast array of downstream processes. PKA, as the most important cAMP-activated effector in regulation of contractility, is spatially localized and regulated by a family of scaffolding proteins termed A Kinase Anchoring Proteins (AKAPs). AKAPs anchor PKA to its target and the complexes often include PDEs responsible for regulating local cAMP levels, and phosphatases to tightly regulate PKA-mediated phosphorylation [Cyclic AMP is a second messenger synthetized by adenylyl cyclase (AC) that can be activated in response to stimulation of G-protein-coupled receptors (GPCRs) coupled to the AC-stimulating G protein Grylation ,10,11,123-AR [Cyclic GMP is synthesized by soluble guanylyl cyclase (sGC), activated by nitric oxide (NO), which is in turn synthesized by nitric oxide synthases (NOS); particulate guanylyl cyclases (GCs) can also synthesize cGMP when activated by the natriuretic peptides (NP) atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) . Increas3-AR . In orde3-AR ,16.2+ into the SR. Phosphorylation of TnI, on the other hand, reduces the sensitivity of the myofilaments for binding Ca2+, thus allowing faster Ca2+ dissociation. Together, this results in a faster relaxation (lusitropic response) [2+ is not limiting, as under cAMP stimulation, PLB phosphorylation will also contribute to a positive inotropic effect through increasing SR Ca2+ content. PKG can also phosphorylate PLB and TnI, causing a LR similar to PKA phosphorylation [2+ availability due to phosphorylation of PLB and LTCC, combined with TnI phosphorylation, PKG activation is mostly associated with a negative inotropic response (NIR) [2+ may be limiting due to lack of LTCC stimulation, and PLB phosphorylation will mainly contribute to a NIR [Cyclic AMP and cGMP activation of PKA and PKG, respectively, modulates contractility through phosphorylation of several proteins. PKA phosphorylation of LTCC and RyR increases their open probability and therefore increases cytosolic calcium levels. Together, this generates a positive inotropic response (PIR). Cyclic AMP also mediates a lusitropic response (LR) through PKA phosphorylation of PLB and TnI. Phosphorylation of PLB removes its inhibitory tone on the SERCA pump, which results in increased pumping of Caesponse) . In siturylation ,22,23,24se (NIR) ,27,28,29se (NIR) ,31. Thus1 did not, despite both increasing cAMP [1 was found only in the soluble fraction [Intracellular signaling mediated by cyclic nucleotides (CNs) is organized in spatial-temporal nanodomains defined by localization and activities of receptors, adenylyl or guanylyl cyclases, protein kinases and PDEs, and this is defined as compartmentation. The first evidence for compartmentation of second messenger signaling in the heart was published in the 1970s and early 1980s for cAMP, where it was observed that isoproterenol (Iso) stimulation of β-ARs increased contractility, while stimulation with the prostaglandin PgEing cAMP ,33,34. Ifraction ,36.1-adrenergic receptor (β1-AR) is the main β-AR in cardiomyocytes. Stimulation of these receptors activates Gs that activates ACs, of which AC5 and AC6 are the main isoforms expressed in the heart [2-ARs represent a smaller portion of ARs in the heart and also activate Gs (as β1-AR). Whereas β2-AR stimulation can trigger a PIR, its ability to trigger a LR was disputed [2-ARs to activate both Gs and Gi but may also be related to the receptor localization. In this regard, β2-ARs are found in the T-tubules, and thereby specifically regulate LTCC current [1-AR stimulation produces cAMP both in the T-tubules and at the cell crest [2-AR signaling seems very restricted, while the β1-AR-stimulated cAMP can diffuse more throughout the cell and reach multiple targets. Similar to β-AR signaling, the serotonin 5-HT4 receptors can also give an increase in cAMP and a PIR, at least in failing hearts [The GPCR βhe heart . Increashe heart . β2-ARs disputed , althougdisputed . The red current , while βll crest ,46,47,48g hearts ,51,52,53Cyclic AMP compartments relevant for contractility are regulated by several PDEs. Amongst the cAMP-selective PDEs in the heart, PDE4 is the main PDE, whereas little is known about PDE8.m (1.2–10 µM). It comprises a family of 20 isoforms encoded by PDE4A, B, C, and D genes, of which all except PDE4C are found in the heart [1/β2-AR mediated contractility, while inhibition of PDE3 did. However, also in human failing myocardium, whereas PDE4 inhibition alone had no effect, a role of PDE4 inhibition in enhancement of a 5-HT4-mediated positive inotropic response to serotonin was revealed in the presence of PDE3 inhibition [PDE4 is a cAMP-selective PDE with a low Km .2–10 µM.he heart . The N-the heart ,58,59,60he heart . Howeverhe heart ,62,63,64he heart  or carvehe heart , showed hibition . On a si1-AR-mediated PIR and LR, has been reported in both atria and ventricle [2-AR-mediated contractility, there are regional differences in the heart, where PDE4 regulates contractility in the base but not the apex of the ventricle. This could be attributed to several factors, including more regular T-tubular organization and higher PDE4 activity at the base of the heart [2-AR-stimulated cAMP near PLB, whereas PDE2 and PDE3 did [1-AR is regulated by PDE4 near PLB. One study has also reported localization of PDE4 near PLB in human failing myocardium [In rodents, PDE4 is the main PDE responsible for hydrolysis of cAMP, when measuring cAMP levels, and together with PDE3 regulates β-AR-stimulated contractility ,68,69,70entricle ,71,72. Fhe heart . In one he heart . In anotPDE3 did . Taken tocardium .2+ leak and in heart failure (HF) reduced local PDE4 gives increased phosphorylation of RyR and greater Ca2+ efflux from the SR [2+ by being part of a complex with SERCA, PKA, and PLB [2+ entrance in the cell when phosphorylated by PKA [PDE4D3 forms a signalosome together with PKA and RyR regulating the SR-Cam the SR . Moreove and PLB . PDE4B id by PKA ,79. Reced by PKA . This cod by PKA .1-AR-, β2-AR-, or 5-HT4-mediated contractility was not enhanced by inhibiting PDE4 alone, despite increased levels of cAMP. Rather, simultaneous inhibition of both PDE3 and PDE4 was needed to obtain an increased contractility, both in normal and failing hearts [4 receptor stimulation, inhibition of both PDE3 and PDE4 significantly increased cAMP levels in porcine left atrium [In some reports on human, porcine, and rodent cardiac ventricle, βg hearts ,82,83,84t atrium  and in ht atrium . Togethet atrium .−/− cardiomyocytes have larger Ca2+ transients, increased LTCC current, and increased cardiomyocyte contractility upon β-AR activation, but also larger Ca2+ spark frequency in the absence of receptor activation [1-AR caused a widespread cAMP signal, whereas β2-AR caused a much more localized cAMP signal [2-AR cAMP signal more similar to that of β1-AR. However, the difference was insensitive to PDE inhibition by IBMX, which at the time would indicate that PDEs were not involved. However, an intriguing possibility is that PDE8 could be involved in the difference, and that PDE8 inhibition, maybe in addition to IBMX, would make the β2-AR signal more like the β1-AR signal.PDE8 is a cAMP-selective phosphodiesterase and has two isoforms: PDE8A and PDE8B . The regtivation . This suP signal . If thisOne of the first indications of cGMP compartmentation was seen in the early 1990s when cGMP from NO was found to be in both the soluble and the particulate fraction, while cGMP from the pGC was present only in the particulate fraction . In rat The PDEs that selectively hydrolyze cGMP in the heart are PDE5 and PDE9.PDE5 selectively degrades cGMP and has three isoforms (PDE5A1–3) expressed in human hearts . The reg2-AR-mediated contractility after PDE5 inhibition [3-AR activation, at caveolae-enriched membrane fractions in T-tubules. This cGMP pool was then responsible for a decrease in cAMP levels through PDE2 activation [3-ARs were found to elicit a NIR and positive LR via eNOS and cGMP [Inhibiting PDE5 does not affect basal contractility. However, when activating all three β-ARs, PDE5 inhibition (by sildenafil) reduced contractility in normal mouse heart involving increase in NOS3-NO-sGC-originated cGMP ,103, as hibition . In additivation . These dand cGMP ,110. In and cGMP .m for cGMP (0.70–0.17 µM) and it was recently discovered to be expressed in the heart, localized to the SR , and regulating cGMP generated after GC-A stimulation, but not from stimulating sGC [PDE9A is the PDE with the lowest Kting sGC ,105. Morting sGC . We are Although the cAMP- and cGMP-pathways modulate contractility differently, as described above, increased concentrations of cGMP can also modulate cAMP levels, by affecting the activity of certain PDEs. Cyclic GMP binds to a GAF domain on PDE2 to increase the enzymatic activity, and thereby reducing cAMP responses, whereas at PDE3 cGMP is hydrolyzed slowly and this inhibits cAMP degradation, increasing cAMP-mediated responses. Thereby, PDEs that can hydrolyze both cAMP and cGMP are responsible for so-called cross-talk between the two signaling pathways. The compartments relevant in contractility that involve such cross-talk are not easy to differentiate from the specific cAMP/cGMP compartments described above. Further investigation with focus on local cyclic nucleotide fluctuations and on how subcellular compartmentation occurs, will help to resolve this.The dual-substrate PDEs also have important roles towards the two single pathways, and in the next section, we describe how PDE1, PDE2, and PDE3 contribute to modulate contractility by regulating either cAMP or cGMP separately, but also how they modulate contractility by creating cGMP/cAMP cross-talk.2+/calmodulin (CaM)-dependent enzymes and their regulatory domain contains two CaM binding sites, two phosphorylation sites, and an inhibitory region, which retains the enzyme in its inactive configuration when the calcium levels are low [2+/CaM and therefore its activation [m than for cAMP, while PDE1C has equal affinity [The PDE1 family includes the isozymes PDE1A, PDE1B, and PDE1C . These a are low ,114. CaMtivation ,116. PDEaffinity . Among taffinity ,118,119.affinity .Most studies report data on the functional role of PDE1 in rodent hearts and do not investigate its specific effect on contractility. Recently, the group of David Kass has shown the role of PDE1C in rabbit and dog hearts, which, similar to human hearts, express predominantly this isoform and show positive inotropic and chronotropic effects after inhibiting PDE1. These effects are due to PDE1C activity over a soluble AC-cAMP nanodomain that regulates LTCC current via PKA, but does not involve modulation of SERCA current, phosphorylation of PLB, TnI, and myosin binding protein C, like in the β-AR-mediated contractility ,121.max and high Km for both cAMP and cGMP [PDE2 is a dual substrate phosphodiesterase with similar high Vand cGMP ,123. Theand cGMP ,125,126.and cGMP  and in cand cGMP .The regulatory region contains two GAF domains, A and B . Cyclic In rat and mouse cardiomyocytes PDE2 contributes to a low fraction of the total cellular cAMP-PDE activity ,110, whiTotal cGMP levels seem to be mainly controlled by PDE2 both in normal  and fail1- and β2-AR activation [3-ARs trigger NO release and consequent cGMP augmentation and that these pools of cGMP are responsible for PDE2 activation and thereafter reduction in β1- and/or β2-AR-stimulated cAMP levels and PIR [3-ARs in the caveolae and the related cGMP pools in the T-tubules, which are locally regulated mostly by PDE2 and PDE5 [Considering that PDE2 is a dual substrate PDE, and that cGMP can bind to the PDE2-GAF domain and increase its enzymatic activity, the role of PDE2 in cGMP/cAMP cross-talk has been investigated. Specifically, cGMP-increase after sGC stimulation reduces LTCC current through activation of PDE2 and this is mediated by a decrease in cAMP and PKA phosphorylation of LTCC ,137,138.tivation , and suctivation . Mongill and PIR . Supportand PDE5 , and stiand PDE5 .1- and β2-AR compartments, revealing an ANP-induced increase of the positive inotropic effect of β-adrenergic stimulation in a mild hypertrophic model through PDE3 inhibition [1-AR microdomain and increased around the β2-AR microdomain, therefore, ANP-stimulated cGMP will switch from reducing cAMP levels via PDE2 activation  to increase cAMP levels via PDE3 inhibition (in a disease model) relatively to the β1-AR microdomain [CNP can also, through cGMP-mediated activation of PDE2, decrease cAMP levels and thereby decrease β-AR-stimulated LTCC current ,140. Evehibition . In thisrodomain .m cAMP 0.16 µM; Km cGMP 0.09 µM), while the Vmax is >10-fold higher for cAMP. Hence, when cGMP binds, it is hydrolyzed slowly and this inhibits cAMP degradation [2+ re-uptake in the SR [PDE3 has two isoforms, PDE3A and PDE3B, both expressed in the heart ,143,144.radation . The N-tradation . The latradation ,148,149.n the SR ,151.4 receptor-mediated PIR and LR [PDE3 is responsible for hydrolyzing global cellular cAMP levels in hearts from normal and HF animals, to a lower extent than PDE4, which is the main cAMP-PDE in rodents ,68,74. AR and LR ,52,98 anR and LR ,155.2-AR signaling in caveolin-enriched compartments such as T-tubules [2-AR-cAMP at the plasma membrane was reduced, with no changes in global expression, but with clear reduction in the caveolin-rich fractions [4-receptor-induced cAMP levels, calcium, PIR and LR, compared to inhibiting either PDE alone [Using targeted FRET-based biosensors, PDE3 subcellular activity was found to prevail around cAMP nanodomains defined by a PKA RI-targeted biosensor  and regu-tubules . Howeverractions . Similarractions . TherefoDE alone ,84,156.With regard to cGMP, PDE3 regulates global cellular cGMP levels and cytosolic cGMP measured by a non-targeted biosensor  and cont1-AR and β2-AR in normal and failing rat left ventricle [4 receptor-induced PIR was also shown to be increased by CNP-induced PDE3 inhibition in both failing rat left ventricle and in porcine left atrium [4-mediated PIR (through PDE3 inhibition), but also reduced β1-mediated IR [1-mediated cAMP and IR by NO/sGC has been shown by others as discussed above under “PDE2 and cGMP/cAMP cross-talk relevant for contractility” [1-AR and 5-HT4 receptors may indicate different compartmentation of PDEs in their signaling pathways. As also addressed above, ANP potentiated the β-AR-stimulated PIR, presumably through PDE3 inhibition in a mild hypertrophic model, but not in Sham [1-AR-dependent contractility, neither in a chronic HF model nor in sham-operated animals [The natriuretic peptide CNP can modulate contractility by affecting cAMP signaling through cGMP-mediated inhibition of PDE3, causing a PIR mediated by βentricle ,62,98. 5t atrium ,81. In Hiated IR . The redctility” ,134,139. in Sham . However animals ,98.PDE10 is a dual PDE discovered in 1999 ,158,159 In HF the heart fails to provide adequate blood flow/pressure to meet the body’s demands and this is associated with alterations in the cardiac structure, function, rhythm, and conduction. In HF, the body responds by activating compensatory mechanisms, such as salt and water retention, increase in noradrenaline and adrenaline (sympathetic activation), and increase in several hormones, such as angiotensin II, aldosterone, endothelin, and natriuretic peptides. Some of these changes will in the short term improve cardiac contractility and cardiac output, but in the long term some of these will worsen the prognosis ,161. Of Despite the fact that cAMP and cGMP have many similarities in the signaling pathway and have similar protein targets, they can mediate different and sometimes opposite effects on contractility in the heart, as described above. In HF there is increased adrenergic activity stimulating β-ARs and increased natriuretic peptides. Long-term stimulation of β-AR is detrimental in chronic heart failure and β-blockers are a cornerstone in HF management ,163. IncPDE2 expression increases in HF ,180. HowIn heart failure, alteration in PDE3 expression is not that clear. Some studies show a reduction in both humans and animal models ,183,184,In HF expression of both PDE4A and PDE4B, as well as PDE4 activity, is reduced and accompanied by heart dysfunction ,184,186.In a recent study, PDE4B overexpression both decreased contractility and lusitropic responses through reduced phosphorylation of PLB, TnI, and MyBPC . In addiPDE5 activity is enhanced in HF, both in humans and in animal models ,208,209 The expression of PDE9 in the heart is low, but it is upregulated in human left ventricular biopsies from HFpEF and aortic stenosis patients and in animals with HFrEF . Inhibitm. However, inhibition of PDE2 increased cell shortening at basal conditions [For many years, studies on the intracellular signaling modulating contractility have focused on measuring global cAMP levels in relation to a specific functional response. However, we now understand more of the organization of the compartmented signaling networks relaying responses, from GPCR activation to cAMP signaling in subcellular nanodomains . Conseqnditions . Similarnditions , but whenditions . Targetinditions . These oLocalization of signaling in subcellular compartments could also explain the discrepancy between global cAMP concentrations at basal conditions, which are around 1 µM, and PKA affinity for cAMP, which is ~100 nM ,224,225.Since PDE inhibitors were first used for treatment of HF, many advances have been made in understanding the role of PDEs in cardiac contractility. However, there are some ongoing challenges to consider. For example, a large number of studies are conducted using animal models where PDE activity and expression can be different from those in humans, and thus caution is needed when interpreting the data. As we appreciate more the importance of signaling compartmentation, localization of specific PDEs is also of relevance in modulation of contractility.Real-time measurements of cAMP and cGMP using targeted biosensors has been pivotal in understanding how PDEs contribute to mediate contractility at the subcellular level and helped to uncover signaling compartmentation. Yet, there are still some questions and challenges remaining: how do PDEs efficiently degrade cAMP/cGMP to protect protein kinase from unwanted cellular activation? How does stimulation of a specific receptor trigger the activation of certain downstream targets and in some compartments rather than others? Moreover, how are the compartments involving cGMP/cAMP cross-talk organized vs. the single cAMP/cGMP compartments?Using targeted biosensors combined with in silico modeling will help to understand the compartments in a cell, which modulators are involved, the organization of cAMP/cGMP local nanodomains and their size, and how these are affected in cardiac dysfunction.Heart failure has been associated with changes in either expression or localization of PDEs. In addition, there is evidence that some PDEs can behave in a redundant manner. How one PDE may compensate for changes in the activity of another PDE during signaling modulation or whether this redundancy changes during HF, remains to be fully elucidated.


Serine 129 (S129) phosphorylation of α-synuclein (αSyn) is a central feature of Lewy body (LB) disease pathology. Although the neighboring tyrosine residues Y125, Y133, and Y136 are also phosphorylation sites, little is known regarding potential roles of phosphorylation cross-talk between these sites and its involvement in the pathogenesis of LB disease. Here, we found that αSyn aggregates are predominantly phosphorylated at Y136 in the Lewy body dementia brain, which is mediated by unexpected kinase activity of Casein kinase 2 (CK2). Aggregate formation with S129 and Y136 phosphorylation of recombinant αSyn (r-αSyn) were induced by CK2 but abolished by replacement of S129 with alanine (S129A) in vitro. Mutation of Y136 to alanine (Y136A) promoted aggregate formation and S129 phosphorylation of r-αSyn by CK2 in vitro. Introduction of Y136A r-αSyn oligomers into cultured cells exhibited increased levels of aggregates with S129 phosphorylation compared to wild-type r-αSyn oligomers. In addition, aggregate formation with S129 phosphorylation induced by introduction of wild-type r-αSyn oligomers was significantly attenuated by CK2 inhibition, which resulted in an unexpected increase in Y136 phosphorylation in cultured cells. Our findings suggest the involvement of CK2-related αSyn Y136 phosphorylation in the pathogenesis of LB disease and its potential as a therapeutic target.The online version contains supplementary material available at 10.1186/s40478-021-01281-9. Lewy body (LB) diseases, including Lewy body dementia (LBD) and Parkinson’s disease (PD), are progressive diseases characterized by extensive accumulation of intracellular proteinaceous inclusions composed mainly of aggregated α-synuclein (αSyn) in the brain called LB. Although the pathological mechanisms of LB disease have not been fully elucidated, accumulation of LB, i.e., αSyn aggregates, is thought to play a significant role.vivo studies yielded contrasting results regarding the significance of S129 phosphorylation (pS129) for LB formation, showing facilitatory ). The 96-well plates were covered with sealing tape  and incubated at 40 °C in a plate reader  with intermittent shaking, consisting of 30 s of double orbital shaking at 500 rpm and no shaking for 30 s. ThT fluorescence intensity on the bottom of the plates was measured every 10 min to monitor the kinetics of amyloid fibril formation using monochromators with excitation and emission wavelengths of 450 ± 10 and 480 ± 10 nm, respectively. Lag phase was defined as the time required to reach fluorescence greater than or equal to the mean fluorescence intensity for all samples within the first 24 h of reaction plus 4 × the standard deviation.r-αSyn (40 μg) was prepared in 96-well optical black-bottomed plates  in 100 μL of reaction buffer  was incubated in 20 mM Tris–HCl, pH 7.5, containing 50 mM KCl, and 10 mM MgCl2 in DMEM/Ham’s F12 medium  containing 15% fetal bovine serum , penicillin–streptomycin , and MEM Non-essential Amino Acids Solution . Introduction of r-αSyn seeds and/or pcDNA3.1 plasmid encoding human WT or Y136A mutant αSyn into cells was performed using Lipofectamine™ LTX (Invitrogen) according to the manufacturer’s instructions. Lipofectamine/r-αSyn seeds and/or plasmid complexes were prepared in Optimem  by mixing Lipofectamine LTX reagent in the presence or absence of r-αSyn seeds (4 μg) or plasmid (5 μg). Cells were cultured to 40–50% confluence in 6-well plates and treated with the complexes. For CK2 inhibitor treatment, cells were incubated with 10 or 100 nM 4,5,6,7-tetrabromobenzotriazole  for 1 h before addition of the complexes. cells were incubated for 3 d after introduction of r-αSyn seeds and/or plasmid.Human neuroblastoma SH-SY5Y cells were maintained at 37 °C in 5% COCells were washed with PBS and harvested, and cellular proteins were extracted with Triton-DOC lysis buffer. The total protein concentration of the lysates was measured using a BCA protein assay kit .t test was used for comparisons between two groups. One-way analysis of variance (ANOVA) followed by the Tukey–Kramer test was used for comparisons among more than two groups. In all analyses, P < 0.05 was taken to indicate statistical significance.Statistical analyses were performed with OriginPro 2015 (OriginLab). The 2-tailed Student’s Brain lysates from LBD patients were analyzed by SDS-PAGE followed by immunoblotting with anti-αSyn antibody, and all exhibited a band at approximately 20 kDa with another band just below 20 kDa likely corresponding to full-length and cleaved αSyn, respectively Fig. b. All ly2 of tissue. There were no significant differences in the numbers of pY125-αSyn and pY133-αSyn deposits between the two types of LBD  seeding assay . We confTo examine whether CK2 is involved in aggregate formation and C-terminal phosphorylation of αSyn in cells, we investigated the effects of the CK2 inhibitor, 4,5,6,7-tetrabromobenzotriazole (TBB), in SH-SY5Y cells exposed to agitated WT r-αSyn. TBB significantly reduced the levels of accumulation of αSyn > 25 kDa in a dose-dependent manner in cells transfected with agitated WT r-αSyn Fig. a, b. TBBThe results of the present study showed that insoluble αSyn is highly phosphorylated at both Y136 and S129 in the LBD brain. In addition, pY136-αSyn, which was presumed to be oligomeric and/or ubiquitinated, was found to be constitutively expressed in the brain regardless of the presence or absence of neocortical LB. Both pY125-αSyn and pY133-αSyn were almost undetectable by immunoblotting and were detected at low levels by immunohistochemical analysis, indicating that phosphorylated αSyn is present in relatively small amounts in the brain. Moreover, Y125 and Y133 phosphorylation were unaffected by the formation of insoluble αSyn aggregates in the brain. The level of pY125 was reported to be higher in the control than LBD brain by immunoblotting, and was shown to be reduced in the aging human brain . Both coSeveral protein kinases have been suggested to be responsible for S129 phosphorylation of αSyn, including CK1, G protein-coupled receptor kinases (GRKs), and polo-like kinases (PLKs). We also showed that CK2 phosphorylated αSyn at S129 in vitro, which was strongly correlated with αSyn aggregate formation consistent with our previous report , 33, 37.42− group and may therefore lower the threshold for tyrosine phosphorylation by CK2. It has been reported that most of the phosphorylation sites are located in intrinsically disordered regions [The substrate specificity of CK2 is determined by one or more negatively charged residues, i.e., aspartic acid (D)/glutamic acid (E), surrounding the phosphorylatable serine (S) and threonine (T) residues. The minimum consensus sequence is S/T-X-X-D/E, where X can be any amino acid. The most crucial acidic residue position for susceptibility to phosphorylation by CK2 is n + 3 followed by n + 1 . In the  regions , and tha regions . Indeed, regions . It has  regions . TherefoPreventing phosphorylation of Y136 by Y136A mutation facilitated aggregate formation and S129 phosphorylation of r-αSyn and αSyn in cultured cells. Y136 may also be one of the major phosphorylatable sites for CK2 in negatively charged αSyn aggregates formed with increased pS129 and to protect against further S129 phosphorylation and αSyn aggregate formation by undergoing phosphorylation instead of S129. TBB significantly inhibited S129 phosphorylation and suppressed αSyn aggregate formation in cultured cells. These results suggested that CK2 is the main protein kinase for S129 phosphorylation of αSyn and that phosphorylation of S129 by CK2 is closely related to the formation of αSyn aggregates in SH-SY5Y cells. Therefore, CK2 may be a therapeutic target for LB disease. Unexpectedly, phosphorylation of Y136 in αSyn aggregates was significantly increased by TBB in cultured cells, suggesting that CK2 plays an inhibitory role against Y136 phosphorylation in cells. As CK2 phosphorylates hundreds of physiological substrates to control various cellular processes . TherefoThe findings of the present study provide the first evidence that CK2 phosphorylates Y136 in αSyn aggregates by mediating S129 phosphorylation as a dual-specificity kinase and that pY136 has a protective effect against αSyn aggregation. Although the primary kinase for pY136 in vivo is not yet clear, this study suggested that CK2 is a candidate kinase responsible for pY136 and that it participates in regulating αSyn aggregate formation. Further studies are necessary to elucidate the potential roles of the interactions between CK2 and αSyn C-terminal phosphorylation, their involvement in the pathogenesis of LB diseases, and their potential for the development of novel therapeutic strategies.Additional file1. Supplementary fgures.


Opioid-associated overdoses and deaths due to respiratory depression are a major public health problem in the US and other Western countries. In the past decade, much research effort has been directed towards the development of G-protein-biased µ-opioid receptor (MOP) agonists as a possible means to circumvent this problem. The bias hypothesis proposes that G-protein signaling mediates analgesia, whereas ß-arrestin signaling mediates respiratory depression. SR-17018 was initially reported as a highly biased µ-opioid with an extremely wide therapeutic window. It was later shown that SR-17018 can also reverse morphine tolerance and prevent withdrawal via a hitherto unknown mechanism of action. Here, we examined the temporal dynamics of SR-17018-induced MOP phosphorylation and dephosphorylation. Exposure of MOP to saturating concentrations of SR-17018 for extended periods of time stimulated a MOP phosphorylation pattern that was indistinguishable from that induced by the full agonist DAMGO. Unlike DAMGO-induced MOP phosphorylation, which is reversible within minutes after agonist washout, SR-17018-induced MOP phosphorylation persisted for hours under otherwise identical conditions. Such delayed MOP dephosphorylation kinetics were also found for the partial agonist buprenorphine. However, buprenorphine, SR-17018-induced MOP phosphorylation was fully reversible when naloxone was included in the washout solution. SR-17018 exhibits a qualitative and temporal MOP phosphorylation profile that is strikingly different from any other known biased, partial, or full MOP agonist. We conclude that detailed analysis of receptor phosphorylation may provide novel insights into previously unappreciated pharmacological properties of newly synthesized MOP ligands. Opioids are the most effective drugs for the treatment of severe pain. However, their clinical use in acute and chronic pain is limited by severe adverse side effects such as respiratory depression, constipation, dependence, and development of tolerance ,2. CurreSR-17018 is one of the most recently described G-protein-biased agonists ,4. SchmiIn contrast, a more recent study by Gillis et al. (2020) showed that SR-17018 consistently exhibited low intrinsic efficacy across a variety of assays and showed no statistically significant bias towards or away from any G-protein activation. Furthermore, similar kinetics were observed between antinociception and respiratory depressant effects. Similar in vitro results were obtained with the partial agonist buprenorphine, albeit with an increased therapeutic window regarding respiratory depression ,6,7. In 375. Recent phosphorylation studies with SR-17018 have reported an unusual phosphorylation pattern which is limited to Ser375 during the first 20 min of stimulation, corresponding to a low-efficacy agonist [In the past decade, we have shown that the phosphorylation barcode of the MOP carboxyl-terminal tail is dependent on agonist efficacy and indicative of ß-arrestin recruitment and receptor internalization ,10. High agonist . In contGiven its unusual pharmacological profile and unknown mechanism of action, we performed a series of MOP phosphorylation and dephosphorylation experiments in vitro and compared the effects of SR-17018 with the partial agonist buprenorphine.SR-17018 was developed as a G-protein-biased MOP agonist, but exhibits a number of pharmacological effects which cannot be explained by the biased signaling hypothesis. To better understand SR-17018 ligand properties, we performed a series of MOP phosphorylation and dephosphorylation experiments comparing SR-17018 to the low-efficacy agonist buprenorphine and the full agonist DAMGO as internal standard.375 phosphorylation under otherwise identical conditions.First, we evaluated dose- and time-dependent MOP phosphorylation induced by DAMGO, SR-17018 or buprenorphine . As showAs depicted in 375 phosphorylation was also resistant to PBS washout  was purchased from Sigma Aldrich . SR-17018 was obtained from MedChemExpress , buprenorphine from Indivior , and naloxone from Ratiopharm . PierceTM HA epitope tag antibody was obtained from Thermo Scientific . The rabbit polyclonal phosphosite-specific µ-opioid receptor antibodies anti-pT370 (7TM0319B), anti-pT376 (7TM0319D), anti-pT379 (7TM0319E), anti-pS375 (7TM0319C) and anti-HA antibody (7TM000HA) were obtained from 7TM Antibodies  [[D-Ala70 7TM031B, anti-pGermany) ,14,15. T2. Cells were stably transfected with mouse MOP-HA and the assays performed have been extensively characterized in previous publications [HEK293 cells were originally obtained from the German Resource Centre for Biological Material  and grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, and 100 U/mL penicillin/streptomycin and cultured in a humidified atmosphere containing 5% COications .l-lysine-coated 60 mm dishes and grown to 90% confluency. After agonist stimulation, cells were lysed in RIPA buffer  containing protease and phosphatase inhibitors . When indicated, cells were washed three times with either PBS buffer (PBS washout) or PBS supplemented with 10 µM naloxone . After removal of agonist, the cells were incubated in the absence of agonist at 37 °C as indicated and lysed in RIPA buffer containing protease and phosphatase inhibitors, as described previously. The assays were performed at both physiological temperature (37 °C) and at room temperature (22 °C) to slow down the cellular processes if indicated. PierceTM HA epitope tag antibody beads  were used to enrich HA-tagged MOP. The samples were washed several times afterwards. To elute proteins from the beads, the samples were incubated in SDS sample buffer for 25 min at 43 °C. Supernatants were separated from the beads, loaded onto 8% SDS polyacrylamide gels, and then immunoblotted onto nitrocellulose membranes afterwards. After blocking, membranes were incubated with anti-pT370 (7TM0319B), anti-pS375 (7TM0319C), anti-pT376 (7TM0319D), or anti-pT379 (7TM0319E) antibody overnight at 4 °C . Membranes were incubated in HRP-linked secondary antibody for 2 h, followed by detection using a chemiluminescence system . Blots were subsequently stripped and incubated again with the phosphorylation-independent anti-HA antibody to confirm equal loading of the gels. Protein bands on Western blots were exposed to X-ray films.HEK293 cells stably expressing HA-MOP were seeded onto poly-l-lysine coated 24-well plates overnight. On the next day, cells were pre-incubated with anti-HA antibody for 2 h at 4 °C. Cells were then transferred to 37 °C, exposed to 10 µM agonist for 30 min at 37 °C and fixed with 4% paraformaldehyde and 0.2% picric acid in phosphate buffer (pH 6.9) for 30 min at room temperature (RT). After washing the coverslips with PBS w/o Ca2+/Mg2+ buffer several times, cells were blocked with phosphate buffer containing 3% NGS for 2 h and were then incubated with Alexa488-conjugated secondary antibody (1:2000)  overnight at 4 °C. On the next day, cells were washed several times with PBS w/o Ca2+/Mg2+ and specimens were mounted with Roti®-MountFluorCare DAPI  and examined using a Zeiss LSM510 META laser scanning confocal microscope . HEK293 cells stably expressing HA-MOP were seeded onto poly-The authors declare that all data supporting the findings of this study are presented within the paper and its supporting information files. The data that support the findings of this study are available from the authors upon reasonable request. Together, the present study reveals a mechanism of action for SR-17018 that is clearly different from any other known MOP agonist. Our findings also demonstrate that newly synthesized compounds should be fully characterized, including detailed analysis of their receptor phosphorylation kinetics, before classification as biased, partial, or full agonists.


The protein kinase CK2 (CK2) family encompasses a small number of acidophilic serine/threonine kinases that phosphorylate substrates involved in numerous biological processes including apoptosis, cell proliferation, and the DNA damage response. CK2 has also been implicated in many human malignancies and other disorders including Alzheimer′s and Parkinson’s diseases, and COVID-19. Interestingly, no single mechanism describes how CK2 is regulated, including activation by external proteins or domains, phosphorylation, or dimerization. Furthermore, the kinase has an elongated activation loop that locks the kinase into an active conformation, leading CK2 to be labelled a constitutively active kinase. This presents an interesting paradox that remains unanswered: how can a constitutively active kinase regulate biological processes that require careful control? Here, we highlight a selection of studies where CK2 activity is regulated at the substrate level, and discuss them based on the regulatory mechanism. Overall, this review describes numerous biological processes where CK2 activity is regulated, highlighting how a constitutively active kinase can still control numerous cellular activities. It is also evident that more research is required to fully elucidate the mechanisms that regulate CK2 and what causes aberrant CK2 signaling in disease. Three mechanisms can generally explain how kinase activity is regulated based on how the kinase active sites are exposed. The first involves activation by external proteins or domains, where the kinase conformation changes upon binding to other molecules such as proteins or second messengers such as cyclic adenosine monophosphate (cAMP), thus exposing the active site. An example of a kinase that subscribes to this mode of regulation is cyclin-dependent kinase (CDK) 2 (CDK2). The second common regulatory mechanism of kinase activity applies to AGC-family kinases like protein kinase B (PKB/AKT). Here, phosphorylation of conserved residues in both the kinase activation segment and a conserved hydrophobic region towards the C-terminal are required to form the active conformation. The third kinase activation mode involves kinase dimerization, where activation occurs through allosteric interactions with another domain of the same kinase. An example of this involves checkpoint kinase 2 (CHK2), where dimerization enables reciprocal phosphorylation of the kinase activation loop, promoting the active conformation .Of the 518 protein kinases identified in the human genome, some that have gained considerable attention are from the protein kinase CK2 family . CK2 was first discovered in 1954 by Burnett and Kennedy as a “mitochondrial enzyme” from rat liver extracts that utilized adenosine triphosphate (ATP) to phosphorylate purified casein [The CK2 family is comprised of two catalytic subunits (⍺ and ⍺′) and the regulatory β subunit that generally assemble into tetrameric complexes with two catalytic and two regulatory subunits. Within the human genome, CK2⍺, CK2⍺′, and CK2β are encoded by the CSNK2A1, CSNK2A2, and CSNK2B genes, respectively. CK2⍺ and CK2⍺′ catalyze the transfer of the gamma-phosphate from ATP or guanosine triphosphate (GTP) and share 90% sequence identity, diverging in their C-termini . InteresCK2⍺, CK2⍺′, and CK2β are present in numerous cellular compartments, including the nucleus, cytoplasm, plasma membrane , mitochoCK2 exists in different forms, both as tetrameric assemblies and as free subunits. The most common is the CK2⍺ holoenzyme, a tetrameric complex composed of two CK2⍺ subunits connected by a dimer of CK2β subunits. Although less prevalent, CK2 is also present as a tetrameric holoenzyme with two CK2⍺′ subunits or with one CK2⍺ and one CK2⍺’ subunit. CK2⍺, CK2⍺′, and CK2β monomers also exist within cells . CK2 subCK2 is implicated in numerous human diseases such as Alzheimer’s , ParkinsIn 2016, a noteworthy study that involved 4102 patients with neurodevelopmental disorders was published. While no studies to date have identified any gain-of-function mutations in any CK2 subunits, five unrelated female children in this study that presented with similar neurodegenerative symptoms, including developmental delay and intellectual disability behavioural problems, speech problems, and dysmorphic facial features had germline mutations in the CK2⍺ gene. Whole-exome sequencing revealed that of these five individuals, two had missense mutations within the activation segment of the kinase (D175G and K198R), another two with missense mutations within the ATP/GTP binding loop , and the fifth patient had an c.824 + 2T>C splice variant. All five of these mutations were predicted in silico to be deleterious and disease causing . This diIn 2019, the novel coronavirus disease 2019 (COVID-19) pandemic caused by the SARS-CoV-2 virus quickly spread throughout the world, leading to intense effort to understand, diagnose, and treat the disease. Interestingly, phosphoproteomic analysis revealed that SARS-CoV-2 infection strongly affected CK2 as well as other p38 pathway-associated kinases. In addition to phosphorylating SARS-CoV-2 proteins, CK2⍺′ and CK2β physically interact with the viral nucleocapsid protein and co-localize at virus-induced filopodial protrusions. This interaction leads to increased phosphorylation of several proteins, including histone deacetylase 2 (HDAC2), high mobility group protein HMG-I/HMG-Y (HMGA1), non-histone chromosomal protein HMG-14 (HMGN1), signal transducer and activator of transcription 1 (STAT1), and catenin alpha-1 (CTNNA), and suggested that SARS-CoV-2 may regulate CK2-mediated cytoskeletal organization. An in vitro infection assay further probed the relationship between CK2 and SARS-CoV-2, where Vero E6 cells were pre-treated with the antiviral remdesivir, followed by SARS-CoV-2 infection. Forty-eight hours post-infection, cells were treated with 0.1-100 µM of Silmitasertib, resulting in a significantly reduced viral titer compared to a dimethyl sulfoxide (DMSO) control . To builIt is evident that dysregulated CK2 activity is frequently detected in many human diseases, but how does this occur? To answer this question, we must first understand how CK2 is regulated.One of the most interesting structural features of catalytic CK2 subunits involves its activation loop. Traditionally, activation loops are dynamic regions that control kinase activation; however, in CK2, this domain is 30 residues longer than what is seen in cyclin-dependent kinases, resulting in additional interactions with the N-terminal lobe of the kinase. These interactions have two effects on CK2: the first being to seemingly enhance catalytic activity as disruption of the N-terminal segment increases the Km of ATP from ~10 µM to greater than 500 µM. Second, CK2 is locked in its “active” conformation, resulting in the kinase being constitutively active .Given the ubiquitous expression of CK2 in cells, it is unsurprising that the kinase has been implicated in numerous fundamental biological processes, including apoptosis, cell growth and proliferation, transcription, translation, circadian rhythm, and the DNA damage response. However, what is more paradoxical is how a constitutively active kinase can play such an essential regulatory role in these biological functions.Careful regulation of cellular processes is essential, so mechanisms that regulate CK2 activity must exist to prevent aberrant signaling. Here, we summarize a selection of the expansive published literature that highlights specific biological pathways where CK2 is regulated, either through intrinsic (self) regulation, or through extrinsic regulation by proteins, stressors, oligonucleotides, and other molecules. Many of these examples can be categorized into one of the three modes of kinase regulation that were discussed previously.CK2 can self-regulate its activity through autophosphorylation that can occur either in free catalytic subunits or within the holoenzyme. Within the free catalytic subunits, this occurs intermolecularly at Y182 on CK2⍺ and Y183 on CK2⍺′, resulting in increased catalytic activity through modulating interactions between the residue and the N-terminal of the kinase. Interestingly, holoenzyme-formation inhibits this autophosphorylation, so it can only occur to catalytic subunits not complexed with CK2β . Second,CK2 can also intrinsically regulate its expression and activity via protein–protein interactions. On a transcriptional level, a negative transcriptional feedback loop exists with CK2⍺. After translation of CK2⍺, it complexes with CK2β to form the holoenzyme, which can then phosphorylate its transcription factors Sp1, Ets-1, and NF-kB, resulting in decreased expression . An elegStudies have shown that CK2 also forms oligomeric superstructures that inhibit its activity. Here, CK2β initiates holoenzyme aggregation when the acidic loop on one tetramer binds to a polybasic region proximal to the catalytic substrate pocket on CK2⍺/CK2⍺′ in another tetramer, occluding the pocket and decreasing catalytic activity. Upon substrate binding to the acidic loop, the inter-tetramer interaction is released, opening the catalytic pocket and enabling phosphorylation of the substrate ,42,43,44Overall, these studies reveal distinct complementary mechanisms by which CK2 can regulate its own activity.CK2 is subject to several post-translational modifications that have been associated with an increase in activity. For example, CK2⍺ has been shown to be phosphorylated by AKT at T13 within its N-terminal segment, which has been associated with enhanced ribosomal DNA (rDNA) synthesis through increased CK2-mediated phosphorylation of transcription intermediary factor 1-alpha (TIF-1⍺) . FollowiInterestingly, a significant number CK2 post-translational modifications occur during mitosis. First, studies have shown that CDK1 phosphorylates CK2⍺ at T344, T360, S362, and S370. This phosphorylation is strongest during prophase and metaphase before decreasing during anaphase and almost undetectable during telophase and cytokinesis. Furthermore, this phosphorylation seemingly regulates CK2 activity as phosphomimetic mutations of these residues promote cell death by mitotic catastrophe, and phosphoablative mutations cause cell cycle arrest after significant spindle damage . CK2β isLastly, within the transforming growth factor beta (TGFβ)-mediated activation of the epithelial-to-mesenchymal transition pathway, CK2β is phosphorylated at an unknown residue by the kinase domain of TGFβ receptor (TGFBR) 1 kinase, targeting the protein for degradation. The resulting CK2⍺:CK2β subunit abundance imbalance results in activation of CK2 .While phosphorylation accounts for most CK2⍺ post-translational modifications, CK2 is also subject to other post-translational modifications including acetylation at lysine 102 that has been shown to increase its kinase activity ,56 Figu.Post-translational modifications have also been shown to decrease CK2 activity. First, CK2⍺ S347  is O-GlcNAc glycosylated. This glycosylation antagonizes CDK1-mediated phosphorylation of CK2 and increases its susceptibility to proteasomal degradation . FurtherAltogether, CK2 is sensitive to numerous post-translational modifications that can regulate its activity. Protein–protein interactions have been shown to regulate many roles of CK2 . During Protein–protein interactions also regulate the roles of CK2 in cell morphology and cytoskeletal organization. For example, the scaffold protein casein kinase 2-interacting protein 1 (CKIP-1) binds with CK2⍺ (but not CK2⍺′) at the plasma membrane. Localization of CK2, CKIP-1 and PAK1 to the membrane has been reported to allow phosphorylation and activation of PAK1 ,71,72,73CK2 is an essential regulator of cell cycle progression, and there are instances of regulation via protein–protein interactions. For example, CK2 has been shown to increase the stability of the tumour suppressor p53, which promotes cell cycle arrest following DNA damage after UV irradiation. Here, CK2 must first associate with the FACT complex (hSpt16 and SSRP1) before p53 phosphorylation can occur . Further2+/calmodulin-dependent protein kinase 2 (CaMKII). Then, activated CaMKII creates a complex with CK2 and glutamate receptor ionotropic, NMDA 2B (GluN2B), resulting in CK2-mediated phosphorylation of GluN2B, disrupting its interaction with scaffolding membrane-associated guanylate kinase (MAGUK) proteins, leading to its subsequent shutting away from synapses [Protein–protein interactions have also been shown to regulate the neurological functions of CK2. Studies have shown that following activation of the extracellular domain of L1 cell adhesion molecule (L1CAM) during neuritogenesis, its intracellular domain binds CK2⍺, resulting in phosphorylation of CK2⍺ at T360 and S362 by an unknown kinase and CK2-mediated inhibition of the tumour suppressors phosphatase and tensen homolog (PTEN) and p53 as well as increased neuronal growth . CK2 is synapses .Outside of its functions in specific biological pathways, CK2 has been associated with multifaceted stress-response signalling pathways regulated by the chaperone proteins heat shock protein 90 (HSP90) and p38. During heat shock, HSP90 increases CK2 activity by binding CK2⍺ and protecting it from aggregation and inactivation through the formation of stable complexes. Treatment with heparin inhibits this interaction, suggesting that the HSP90:CK2⍺ interaction may occur at the heparin-binding sites on CK2⍺ . A poten2+ channel Cav1.2 via phosphorylation at T1704. AT2 controls CK2 here. Specifically, the protein binds to the AT1 receptor which then interacts with β-arrestin 2, which stimulates the kinase SFK, leading to phosphorylation of T88 on p27, preventing it from inhibiting CK2 phosphorylation of Cav1.2 [A component of healthy blood flow in humans involves regulating blood pressure through the renin–angiotensin–aldosterone system (RAAS). Here, the liver produces the precursor protein angiotensinogen and is cleaved by renin to produce angiotensin I (AT1). AT1 is then converted into angiotensin II (AT2) by angiotensin-converting enzymes, which, through multiple pathways, causes vasoconstriction, increased blood volume, and sympathetic nervous system activation . Small mf Cav1.2 .T cells are an integral component of adaptive immunity, and their production is dependent on the activation of ERK in a CK2-dependent manner. However, CK2 requires activation by CD5 before this can occur. Studies have shown that CD5-increased CK2 activity is 9-fold higher than its activity without any CD5 interaction . ActivatWhile hemoglobin is essential for oxygen transport in mammals, it can also contribute to disease. For example, during hemolytic anemia, hemoglobin is released from red blood cells and can cause oxidative stress. However, to prevent this, free hemoglobin binds to haptoglobin, allowing removal by macrophages after interacting with scavenger receptor cysteine-rich type 1 protein M130 (CD163) . CD163 iThe transcription factor SRY-box transcription factor (SOX) 2 (SOX2) is associated with abnormal cell proliferation in esophageal cancer. Studies have shown that SOX2 increases the expression of the microRNA (miRNA) miR-30e, which decreases the expression of ubiquitin carboxyl-terminal hydrolase 4 (USP4), increasing ubiquitination and proteasomal degradation of mothers against decapentaplegic homolog 4 (SMAD4). As SMAD4 has been demonstrated to inhibit CK2, decreased SMAD4 expression would allow CK2 to activate downstream pathways that increase cell migration and proliferation .Protein–protein interactions can also inhibit the functions of CK2 in other biological contexts that will be highlighted briefly in the following discussion. While Pin1 helps localize CK2⍺ to the mitotic spindle and protects it from dephosphorylation during the cell cycle , it alsoLamin A, a prominent component of the nuclear architecture, interacts with CK2⍺ and CK2⍺′ and sequesters it to the nuclear lamina. This interaction decreases CK2 activity and induces cellular senescence . Within the NF-kB signalling pathway in hepatocellular carcinoma, tumor necrosis factor, alpha induced protein 1 (TNFAIP1) was shown to interact with CK2β, promoting its ubiquitination and degradation, suppressing CK2β-dependent NF-kB activation .The role of CK2 in vascular growth is to regulate phosphorylation of PTEN, indirectly controlling AKT1 expression and cell proliferation. However, SMAD4 inhibits CK2 to maintain homeostasis and prevent aberrant growth. Here, the TGFβ ligands bone morphogenetic protein 9/10 (BMP9/10) stimulate activin receptor-like kinase 1 (ALK1) and SMAD4, resulting in decreased CK2 expression and thus inhibiting CK2-mediated inactivation of PTEN. SMAD4 regulation of CK2 has clinical relevance as patients with hereditary hemorrhagic telangiectasia (HHT) suffer from arteriovenous malformations, which have been linked to decreased SMAD4 as this causes unregulated CK2 phosphorylation of PTEN, which is then unable to repress phosphoinositide 3-kinase (PI3K)/AKT signaling . During adipogenesis, kinesin family member 5C (KIF5C), responsible for transporting cargo along microtubules, interacts with CK2⍺′, inhibiting its nuclear translocation and kinase activity required for mitotic clonal expansion . Overall, these examples illustrate how CK2 relies on other proteins to prevent aberrant phosphorylation by regulating its accessibility to substrates until it is biologically appropriate.While protein–protein interactions encompass a large proportion of mechanisms that regulate CK2, there are multiple examples where small, endogenously produced molecules have been reported to regulate the ability of the kinase to phosphorylate its substrates.Polyamines—small, endogenously produced, basic organic compounds containing multiple amino groups separated by carbon chains—stimulate CK2 activity. Several in vitro studies have characterized this interaction between CK2 and polyamines. Spermine, the polyamine with the greatest ability to increase CK2 activity , binds tIn pancreatic cells, the transcription factor PDX-1 has a significant role in insulin expression. CK2 is involved in a feedback loop that regulates insulin production by phosphorylating PDX-1 at T231 and S232 . High exIntrauterine growth restriction (IUGR) leads to insulin-like growth factor 1 receptor (IGF-1R)-mediated fetal growth retardation through impaired oxygen and nutrient delivery. In IUGR, hypoxia or leucine deprivation activates CK2 through upregulated mTOR and general control nonderepressible 2 (GCN2) signalling, respectively, which leads to IGF-1R hyperphosphorylation and IGF-1R inhibition .One cellular priority following DNA damage is cell cycle arrest, which commonly occurs via phosphorylation of the tumour suppressor p53 . After DBioinformatic analysis of published microarrays revealed that the miRNA miR-1184 has three-fold lower expression in colon cancer tissues than in healthy cells. Subsequent evaluation revealed that CK2⍺ is a principal target of miR-1184. As CK2 has been associated with numerous proliferative and anti-apoptotic functions, it is unsurprising that increased CK2 expression resulting from reduced miR-1184-mediated inhibition is a frequent phenotype in colon cancer .Another potential regulatory interaction that decreases CK2 activity involves the negatively charged polysaccharide heparin, although the physiological relevance of CK2 inhibition by heparin remains to be established. Produced by mammalian mast cells, heparin inhibits blood clotting by interrupting the coagulation cascade through interactions with antithrombin III and is widely used in clinical settings as an anticoagulant. However, heparin also binds to some proteins that are not involved in blood clotting , includiAn interesting phenomenon that occurs with acidophilic kinases like CK2 is hierarchical phosphorylation. With hierarchical phosphorylation, when a kinase phosphorylates a residue, the addition of the negative charge primes the kinase to phosphorylate a nearby residue that now conforms to the acidic kinase consensus motif. Published examples of hierarchical phosphorylation regulating CK2 activity include the circadian rhythm protein Timeless. Phosphorylation of the protein at residues 297 and 301 by GSK-3 promotes CK2 phosphorylation at residues 305, 309, and 313 . AnotherWith the emergence and increased availability of high throughput phosphoproteomics studies, there is an increasing prevalence of studies that reveal phosphorylation of residues within peptides conforming to the CK2 consensus motif. As CK2 was not a focus of these studies, follow-up studies probing the relationship between protein and CK2 were typically not performed. As such, the mechanism of modulating CK2 activity is unknown, but still warrants inclusion here.Excitotoxic cell death after neural glutamate overstimulation activates several kinases including CK2 . AlthougIt has also been reported that CK2 is regulated by SOX4 within the β-catenin/Wnt signalling pathway. Here, SW480 cells were transfected with FLAG-tagged SOX4, then 24 h later, cells were treated with the CK2 inhibitor TBB. Subsequent Western blotting analysis revealed that β-catenin phosphorylation increased after SOX4 transfection but decreased after treatment with TBB. This suggests that SOX4 regulates CK2-mediated phosphorylation of β-catenin .Interestingly, oncogenic RAS mutations including Q61 and G12 mutations have also been shown to increase CK2⍺ expression and activity. More specifically, one study used stable isotope labeling by amino acids in cell culture (SILAC) to compare the phosphoproteomes of NRAS mutants expressed in primary human melanocytes. Motif analyses revealed that NRAS(Q61L) expression leads to increased phosphorylation of CK2⍺ and CK2⍺′ substrates . AnotherStudies have shown that photobiomodulation (using low-powered light to irradiate tissues) to induce biological effects can treat various diseases, including diabetes and various neural disorders such as Parkinson’s and severe depression . AdditioCK2-dependent phosphorylation in liver cells decreased in vivo when mice developed toxicant-associated steatohepatitis (TASH) after introducing the disease with polychlorinated-biphenyl exposure or a high-fat diet. The authors determined that the decreased CK2-dependent phosphorylation was due to attenuated expression of CK2⍺ and CK2β, which resulted in lower caspase-3 phosphorylation and thus increased cell death—which is a hallmark of TASH. Interestingly, however, expression of CK2⍺′ increased. The authors speculated that the latter observation is related to the involvement of CK2⍺′ in interleukin-6 (IL-6) and TNF inflammatory signalling, which increases in TASH .Lastly, a study that examined the role of the nongenotoxic carcinogen pregnenolone carbonitrile (PCN) in mice liver malignancy identified a novel mechanism of inhibiting CK2. PCN binds to the pregnane X receptor (PXR), which, when activated, has been associated with liver enlargement and carcinogenesis. Here, mice were fed PCN for seven days, followed by metabolomic, transcriptomic, proteomic, and phosphoproteomic analyses. The researchers discovered that 9 out of the 21 phosphosites with significantly decreased phosphorylation were CK2 substrates. Gene ontology analysis of seven of these substrates, Hsp90ab1, hepatoma-derived growth factor (HDGF), MTDH, eukaryotic initiation factor 5 beta (EIF5β), zinc finger Ran-binding domain-containing protein 2 (ZRANB2), calnexin, and nuclear ubiquitous casein and cyclin-dependent kinase substrate 1 (NUCKS1), highlighted involved in poly(A) RNA binding, suggesting that PCN may cause malignancy partly through CK2-mediated transcriptional and translational dysfunction .SDDDDD) with extracts from synchronized cells showed increased CK2 activity in nuclear, but not whole-cell, extracts [TEEE), performed with immunoprecipitates of CK2 from synchronized cell lysates followed revealed modulation of CK2 activity after release from G0 arrest. Specifically, CK2 activity increased 2.5-fold after release from G0 before slowly decreasing to G0-levels 12-h after release and then subsequently increasing slightly and plateauing for the remaining 9 h of the study [As far back as the late 1980s, there were a number of biochemical studies that yielded results suggestive of spatiotemporal regulation of CK2 activity. For example, kinase assays performed using a CK2 peptide substrate (RRADDextracts . In a sihe study . While iBased on the structures of CK2 and observations that CK2 has catalytic activity without any stimuli, it has become generally accepted that CK2 may be constitutively active. At the same time, there is a large body of evidence demonstrating that the phosphorylation of specific CK2 substrates can be regulated within cells. Moreover, there are dozens of studies, many described here, that highlight specific cases where phosphorylation of a substrate by CK2 is regulated via a variety of distinct mechanisms including post-translational modifications, protein–protein interactions, and regulatory interactions. Overall, it is important to emphasize that CK2 is responsible for regulating numerous biological processes that rely on careful control to avoid dysregulation and disease. Through the intrinsic and extrinsic regulatory mechanisms that exist, numerous patterns have emerged (summarized in Post-translational modifications and small-molecule regulators that directly interact with the kinase accentuate how CK2, like most enzymes, are precisely folded proteins susceptible to changes induced by altered residue polarity. Interestingly, three activity-increasing phosphorylation sites on CK2⍺ are clustered within its activation segment, suggesting that this region may be particularly sensitive to post-translational modifications at least under some circumstances.Second, several mechanisms that regulate CK2 activity involve protein–protein interactions that regulate substrate accessibility. Proteins can directly interact with CK2, shuttling the kinase to various cellular compartments to promote or prevent substrate phosphorylation. Alternatively, as illustrated by the case of ERK phosphorylation by CK2, there are situations where proteins that do not directly interact with CK2 can regulate its access to a phosphorylation site. Third, mechanisms have been described where levels of CK2 can be modulated. Studies have shown that the expression of CK2 subunits can be regulated through elegant transcriptional feedback loops. CK2 levels can also be regulated by the proteasome. For example, it is apparent that autophosphorylation of CK2 can confer protection from ubiquitination and degradation. For the past several decades, considerable attention within the CK2 field has been focused on identifying physiological substrates, its role in the regulation of biological processes, and its involvement in disease. Collectively, these advances have revealed the impact of CK2 as a “master regulator” involved in numerous biological processes both within normal physiological and pathophysiological settings. While it is clear that CK2 is a fascinating kinase with promise as a therapeutic target, there is a notable paradox, pertaining to how CK2 as a constitutively active kinase can be a regulatory participant in critical cellular processes, that remains to be resolved. Deciphering the precise mechanisms that regulate CK2 activity within healthy cells and how these mechanisms are perturbed in pathological processes will both more thoroughly illuminate the impact of CK2 within living systems and may hold promise for capitalizing on CK2 as a therapeutic target for a number of illnesses.


Left ventricular noncompaction (LVNC) is a heterogeneous, often hereditary group of diseases, which may have diverse clinical manifestations. This article reviews the risk factors for unfavorable outcomes of LVNC in children, as well as discuss the diagnostic methods and the differences between pediatric and adult LVNC. Through a systematic review of the literature, a total of 1983 articles were outlined; 23 of them met the inclusion criteria. In echocardiography the following have been associated with adverse outcomes in children: Left ventricular ejection fraction, end-diastolic dimension, left ventricular posterior wall compaction, and decreased strains. T-wave abnormalities and increased spatial peak QRS-T angle in ECG, as well as arrhythmia, were observed in children at greater risk. Cardiac magnetic resonance is a valuable tool to identify those with systolic dysfunction and late gadolinium enhancement. Genetic testing appears to help identify children at risk, because mutations in particular genes have been associated with worse outcomes. ECG and imaging tests, such as echocardiography and magnetic resonance, help outline risk factors for unfavorable outcomes of LVNC in children and in identifying outpatients who require more attention. Refining the current diagnostic criteria is crucial to avoid inadequate restrain from physical activity. Left ventricular noncompaction (LVNC) is a heterogeneous group of diseases morphologically characterized by excessive trabeculations with concomitant deep recesses predominantly present in the left ventricle of the heart . The sigSome argue that the current definition of LVNC requires revision, because it is limited only to morphology and does not consider the function of the ventricle or the kind of genetic mutations; thus, suggestions regarding the division of LVNC into subtypes have been made ,2,5,6. TIn children, the incidence of LVNC is estimated to be 0.11 per 100,000, with the highest incidence below one year of age, and children with isolated LVNC and normal ejection fraction are usually diagnosed at an older age ,7. With This review presents current knowledge concerning the risk factors for an unfavorable outcome of LVNC in the pediatric population, and discusses the diagnostic methods and differences between pediatric and adult LVNC. To our knowledge, no systematic reviews concerning pediatric LVNC and the variety of its risk factors have been published to date.A computer search was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) scheme by two independent observers in three major databases  using the following search terms: (left ventricular noncompaction OR noncompaction OR hypertrabeculation OR LVNC OR NCLV) AND (children OR pediatric OR paediatric or neonate* OR infant* or adolescent*) AND  . ArticleCase reports, conference abstracts, meta-analyses, systematic reviews, and articles written in languages other than English were excluded from the analysis. To avoid data duplication, publications from the same centers on the same numbers of patients, or on similar numbers of patients with the same conclusions are presented in the summary tables (Tables 1 and 2) only once and were counted only once. One study was excluded, due to the inconsistency in the numerical data. Reports on both adult and pediatric populations, in which data were summed and where it was impossible to differentiate risk factors between the two groups, were not taken into consideration. Articles concerning genetic risk factors were not included in the analysis, due to the recently published meta-analysis by van Waning et al. . The selAltogether 23 out of 1983 articles were considered ; no addiFour different echocardiographic diagnostic criteria for LVNC have been used in the studies included in this review . In fourIn 14 studies of 1081 children, authors analyzed systolic dysfunction of the left ventricle as a risk factor; in 12 of the lower left ventricular ejection fraction (LVEF) or shortening fraction (FS) was associated with a worse outcome . The lowGreater left ventricular end-diastolic dimension (LVEDD) was reported as an unfavorable risk factor in children in five studies of 263 children . No correlation between outcome and NC/C ratio was found in studies by Wang et al. [The role of the noncompaction-to-compaction (NC/C) ratio in echocardiography was addressed in seven studies, with a variety of different conclusions, cut-off values for statistical significance, as well as mean NC/C ratios given ,19,21,23g et al.  , was associated with unfavorable outcomes in studies by Hirono et al. , ArunamaEchocardiography is a basic tool used in the diagnosis of LVNC. Multiple diagnostic criteria have been outlined, with those according to Jenni et al.  (presencAdult patients with LVNC and lower left ventricular ejection fraction (LVEF) are at higher risk of adverse cardiovascular events . LikewisUnlike in the adult group, in which no correlation between the severity of trabeculations and death was found, the data concerning the role of NC/C ratio are inconsistent in the pediatric population and require further research . LVPWC  . The rolThe data concerning the accuracy of echocardiography and CMR in the pediatric population are limited and inconsistent; in some studies, LVNC was diagnosed more commonly in echocardiography, in others, the superiority of CMR was outlined ,47. UndiThe segmental involvement in the pediatric population is similar to the one observed in adults; unsurprisingly, wider involvement is associated with lower ejection fraction . In the Electrocardiogram (ECG) is an easily accessible tool that is valuable not only in the diagnosis, as was indicated in a Japanese study where 42% of pediatric patients with LVNC were diagnosed, due to school ECG screening, but also in outlining higher risk patients with LVNC ,12. SomeIt is of note that the occurrence of malignant arrhythmia (such as sustained ventricular tachycardia (sVT) or ventricular fibrillation (VF)) may be an independent risk factor for unfavorable outcomes, because adult patients with LVNC after ICD implantation as a secondary prophylaxis commonly presented with normal ejection fraction . A similBecause LVNC is heterogeneous in its image, it may coexist with other types of cardiomyopathy or progress to a mixed form, which was observed in 12% of cases within two years in the study by Jefferies et al. . NeverthFurthermore, LVNC can coexist with different congenital heart diseases (CHD), varying from mild types, such as patent ductus arteriosus or septal defects to more severe, i.e., Ebstein’s anomaly or hypoplastic left heart syndrome (HLHS) . FamiliaReports concerning the effect of medical treatment on ECG and echocardiography are inconsistent. In a small study on 20 adults, 13-month treatment with β-blockers did not significantly influence the ECG or the LVEF; however, it led to a reduction in the LV mass . In scarDue to familial occurrence of LVNC in around 30% and familiar history of SCD in 18% of children with LVNC, genetic testing, and detailed familiar background may help determine higher risk patients with accidentally found LVNC ,10,60. AAmong family members of patients with different cardiomyopathies hypertrabeculation of the myocardium is the most common abnormality, which suggests that noncompaction is a morphological finding that can develop into cardiomyopathy in the future, and that the formation of LVNC is a continuous process . ConversUnequivocally, the current criteria for the diagnosis of LVNC require revision. Perhaps it would be reasonable to include abnormal ventricle function, the presence of arrhythmia, as well as genetic information into the scheme in the future. Conversely, one must keep in mind the risk of overdiagnosis, especially in the adult population . The AHAThe limitations of the study include selection bias and possible duplication of data because some reports were from the same centers, but from different years. Furthermore, due to the lack of international consensus, we included studies that used different diagnostic criteria for LVNC, which may result in an increased diversity of the groups presented. Another limitation is the lack of statistical metanalysis; heterogeneous data, as well as different cut-off values and approaches to data presentation  restricted the statistical analysis.ECG and imaging tests, such as echocardiography and magnetic resonance, help outline risk factors for unfavorable outcomes of LVNC in children, as well as dividing patients into subgroups at risk, such as the following: Those with known genetic mutations, coexisting cardiomyopathies, congenital heart diseases, decreased EF, greater LVEDD, as well as patients with ECG abnormalities and arrhythmia. It is noteworthy that some differences between adult and pediatric LVNC in terms of ECG, echocardiographic, CMR, and genetic test results can be outlined, with the latter presenting with worse outcomes when mixed cardiomyopathy traits or genetic mutations are present. Increased genetic testing will help to improve the knowledge concerning genetic variants and assist in identifying more patients at risk.Undoubtedly, the burden of pediatric LVNC prevails, and an international consensus is essential, because the current diagnostic criteria are inconsistent and do not unequivocally point outpatients who do not require special attention from health care professionals and can safely undertake physical activity.