CK2 Activation Research Articles

Hyperphosphorylation of fetal liver IGFBP-1 precedes slowing of fetal growth in nutrient-restricted baboons and may be a mechanism underlying IUGR.

In cultured fetal liver cells, insulin-like growth factor (IGF) binding protein (IGFBP)-1 hyperphosphorylation in response to hypoxia and amino acid deprivation is mediated by inhibition of mechanistic target of rapamycin (mTOR) and activation of amino acid response (AAR) signaling and casein kinase (CK)2. We hypothesized that fetal liver mTOR inhibition, activation of AAR and CK2, and IGFBP-1 hyperphosphorylation occur before development of intrauterine growth restriction (IUGR). Pregnant baboons were fed a control (C) or a maternal nutrient restriction (MNR; 70% calories of control) diet starting at gestational day (GD) 30 (term GD 185). Umbilical blood and fetal liver tissue were obtained at GD 120 (C, n = 7; MNR, n = 10) and 165 (C, n = 7; MNR, n = 8). Fetal weights were unchanged at GD 120 but decreased at GD 165 in the MNR group (-13%, P = 0.03). IGFBP-1 phosphorylation, as determined by parallel reaction monitoring mass spectrometry (PRM-MS), immunohistochemistry, and/or Western blot, was enhanced in MNR fetal liver and umbilical plasma at GD 120 and 165. IGF-I receptor autophosphorylationTyr1135 (-64%, P = 0.05) was reduced in MNR fetal liver at GD 120. Furthermore, fetal liver CK2 (α/α'/β) expression, CK2β colocalization, proximity with IGFBP-1, and CK2 autophosphorylationTyr182 were greater at GD 120 and 165 in MNR vs. C. Additionally, mTOR complex (mTORC)1 (p-P70S6KThr389, -52%, P = 0.05) and mTORC2 (p-AktSer473, -56%, P < 0.001) activity were decreased and AAR was activated (p-GCN2Thr898, +117%, P = 0.02; p-eIF2αSer51, +294%, P = 0.002; p-ERKThr202, +111%, P = 0.03) in MNR liver at GD 120. Our data suggest that fetal liver IGFBP-1 hyperphosphorylation, mediated by mTOR inhibition and both AAR and CK2 activation, is a key link between restricted nutrient and oxygen availability and the development of IUGR.

Inhibition of decidual IGF-1 signaling in response to hypoxia and leucine deprivation is mediated by mTOR and AAR pathways and increased IGFBP-1 phosphorylation

Decidual mechanistic target of rapamycin (mTOR) is inhibited, amino acid response (AAR) and protein kinase CK2 are activated, and IGF (insulin-like growth factor) binding protein (IGFBP)-1 is hyperphosphorylated in human intrauterine growth restriction (IUGR). Using decidualized human immortalized endometrial stromal cells (HIESC), we hypothesized that hypoxia and leucine deprivation causing inhibition of decidual IGF-1 signaling is mediated by mTOR, AAR, CK2 and IGFBP-1 phosphorylation. Mass spectrometry demonstrated that hypoxia (1% O2) or rapamycin increased IGFBP-1 phosphorylation singly at Ser101/119/169 (confirmed using immunoblotting) and dually at pSer169 + 174. Hypoxia resulted in mTOR inhibition, AAR and CK2 activation, and decreased IGF-1 bioactivity, with no additional changes with rapamycin + hypoxia. Rapamycin and/or hypoxia promoted colocalization of IGFBP-1 and CK2 (dual-immunofluorescence and proximity ligation assay). Leucine deprivation showed similar outcomes. Changes in IGFBP-1 phosphorylation regulated by mTOR/AAR signaling and CK2 may represent a novel mechanism linking oxygen and nutrient availability to IGF-1 signaling in the decidua.

Protein kinase CK2 phosphorylation of SAPS3 subunit increases PP6 phosphatase activity with Aurora A kinase.

Protein Ser/Thr phosphatase-6 (PP6) regulates pathways for activation of NF-kB, YAP1 and Aurora A kinase (AURKA). PP6 is a heterotrimer comprised of a catalytic subunit, one of three different SAPS subunits and one of three different ankyrin-repeat ANKRD subunits. Here, we show FLAG-PP6C expressed in cells preferentially binds endogenous SAPS3, and the complex is active with the chemical substrate DiFMUP. SAPS3 has multiple acidic sequence motifs recognized by protein kinase CK2 (CK2) and SAPS3 is phosphorylated by purified CK2, without affecting its associated PP6 phosphatase activity. However, HA3-SAPS3-PP6 phosphatase activity using pT288 AURKA as substrate is significantly increased by phosphorylation with CK2. The substitution of Ala in nine putative phosphorylation sites in SAPS3 was required to prevent CK2 activation of the phosphatase. Different CK2 chemical inhibitors equally increased phosphorylation of endogenous AURKA in living cells, consistent with reduction in PP6 activity. CRISPR/Cas9 deletion or siRNA knockdown of SAPS3 resulted in highly activated endogenous AURKA, and a high proportion of cells with abnormal nuclei. Activation of PP6 by CK2 can form a feedback loop with bistable changes in substrates.

Protein kinase CK2 activation is required for transforming growth factor β-induced epithelial-mesenchymal transition.

Transforming growth factor β (TGFβ) is overexpressed in advanced cancers and promotes tumorigenesis by inducing epithelial–mesenchymal transition (EMT), which enhances invasiveness and metastasis. Although we previously reported that EMT could be induced by increasing CK2 activity alone, it is not known whether CK2 also plays an essential role in TGFβ‐induced EMT. Therefore, in the present study, we investigated whether TGFβ signaling could activate CK2 and, if so, whether such activation is required for TGFβ‐induced EMT. We found that CK2 is activated by TGFβ treatment, and that activity peaks at 48 h after treatment. CK2 activation is dependent on TGFβ receptor (TGFBR) I kinase activity, but independent of SMAD4. Inhibition of CK2 activation through the use of either a CK2 inhibitor or shRNA against CSNK2A1 inhibited TGFβ‐induced EMT. TGFβ signaling decreased CK2β but did not affect CK2α protein levels, resulting in a quantitative imbalance between the catalytic α and regulatory β subunits, thereby increasing CK2 activity. The decrease in CK2β expression was dependent on TGFBRI kinase activity and the ubiquitin–proteasome pathway. The E3 ubiquitin ligases responsible for TGFβ‐induced CK2β degradation were found to be CHIP and WWP1. Okadaic acid (OA) pretreatment protected CK2β from TGFβ‐induced degradation, suggesting that dephosphorylation of CK2β by an OA‐sensitive phosphatase might be required for CK2 activation in TGFβ‐induced EMT. Collectively, our results suggest CK2 as a therapeutic target for the prevention of EMT and metastasis of cancers.

Site-Specific Phosphorylation of Ikaros Induced by Low-Dose Ionizing Radiation Regulates Cell Cycle Progression of B Lymphoblast Through CK2 and AKT Activation

To determine how low-dose ionizing radiation (LDIR) regulates B lympho-proliferation and its molecular mechanism related with Ikaros, transcription factor. Splenocytes and IM-9 cells were uniformly irradiated with various doses of a (137)Cs γ-source, and cell proliferation was analyzed. To determine the LDIR-specific phosphorylation of Ikaros, immunoprecipitation and Western blot analysis were performed. To investigate the physiologic function of LDIR-mediatied Ikaros phosphorylation, Ikaros mutants at phosphorylation sites were generated, and cell cycle analysis was performed. First, we found that LDIR enhances B lymphoblast proliferation in an Ikaros-dependent manner. Moreover, we found that LDIR elevates the phosphorylation level of Ikaros protein. Interestingly, we showed that CK2 and AKT are involved in LDIR-induced Ikaros phosphorylation and capable of regulating DNA binding activity of Ikaros via specific phosphorylation. Finally, we identified LDIR-specific Ikaros phosphorylation sites at S391/S393 and showed that the Ikaros phosphorylations at these sites control Ikaros's ability to regulate G1/S cell cycle progression. Low-dose ionizing radiation specifically phosphorylates Ikaros protein at Ser 391/393 residues to regulate cell cycle progression in B lymphoblast.

Increased IGFBP-1 phosphorylation in response to leucine deprivation is mediated by CK2 and PKC

Insulin-like growth factor binding protein-1 (IGFBP-1), secreted by fetal liver, is a key regulator of IGF-I bioavailability and fetal growth. IGFBP-1 phosphorylation decreases IGF-I bioavailability and diminishes its growth-promoting effects. Growth-restricted fetuses have decreased levels of circulating essential amino acids. We recently showed that IGFBP-1 hyperphosphorylation (pSer101/119/169) in response to leucine deprivation is regulated via activation of the amino acid response (AAR) in HepG2 cells. Here we investigated nutrient-sensitive protein kinases CK2/PKC/PKA in mediating IGFBP-1 phosphorylation in leucine deprivation. We demonstrated that leucine deprivation stimulated CK2 activity (enzymatic assay) and induced IGFBP-1 phosphorylation (immunoblotting/MRM-MS). Inhibition (pharmacological/siRNA) of CK2/PKC, but not PKA, prevented IGFBP-1 hyperphosphorylation in leucine deprivation. PKC inhibition also prevented leucine deprivation-stimulated CK2 activity. Functionally, leucine deprivation decreased IGF-I-induced-IGF-1R autophosphorylation when CK2/PKC were not inhibited. Our data strongly support that PKC promotes leucine deprivation-induced IGFBP-1 hyperphosphorylation via CK2 activation, mechanistically linking decreased amino acid availability and reduced fetal growth.

Regulation of protein kinase CK2 catalytic activity by protein kinase C and phospholipase D2.

We have previously demonstrated that phospholipase D2 (PLD2) overexpression antagonizes protein kinase CK2 (CK2) inhibition-mediated cellular senescence. In the current paper, we show the molecular mechanism of CK2 activation by PLD2 and protein kinase C (PKC). Elevated expression and chemical activation of PLD2 increased the catalytic activity of CK2 and PKC in human colon cancer HCT116 and embryonic kidney HEK293cells, whereas utilization of PLD2 chemical inhibitors and siRNA suppressed this activity. Inhibition of PKC in these cells suppressed PLD2-induced CK2 activation, suggesting that PLD2 enhances CK2 activity through PKC. Overexpression of the PKC isoforms: PKCα, PKCβ, and PKCζ, but not PKCγ, stimulated CK2 activity in the cells. Importantly, purified conventional PKC (cPKC) and atypical PKC (aPKC) enzymes enhanced CK2 activity and phosphorylated serines 194 and 277 within CK2α as well as serine 148 within CK2β. Furthermore, PKC-mediated phosphorylation of these serines on CK2α was crucial for the stimulation of CK2 activity. Taken together, the present results suggest that PLD2 can stimulate CK2 activity through PKC-mediated CK2α phosphorylation.

PP13 - The protein kinase 2 inhibitor tetrabromobenzotriazole protects against renal ischemia reperfusion injury

Protein kinase 2 (CK2), activated by growth factor receptors, has a profound influence on cell proliferation and survival. Recently, CK2 activation was reported to enhance reactive oxygen species production and activate the nuclear factor κB (NF-κB) pathway. Because oxidative stress and inflammation are critical events for tissue destruction during ischemia reperfusion (I/R), we sought to determine whether CK2 was important in the renal response to I/R. Mice underwent 25 min of renal ischemia and were then reperfused. We confirmed an increased expression of CK2α during the reperfusion period, while expression of CK2β remained consistent. We administered tetrabromobenzotriazole (TBBt), a selective CK2α inhibitor before inducing I/R injury. Mice subjected to I/R injury showed typical patterns of acute kidney injury; blood urea nitrogen and serum creatinine levels, tubular necrosis and apoptosis, inflammatory cell infiltration and proinflammatory cytokine production, and oxidative stress were markedly increased when compared to sham mice. However, pretreatment with TBBt abolished these changes and improved renal function and architecture. Suppression of nuclear factor-κB (NF-κB) and mitogen activated protein kinase (MAPK) pathways might have caused the renoprotective effects of TBBt. Taken together, these results suggest that CK2α mediates proapoptotic and proinflammatory signaling, thus the CK2α inhibitor may be used to prevent renal I/R injuries observed in clinical settings.

Mass Spectrometry-Based Analysis of the Phospho-Proteome for Cardiac Dyssynchrony and Resynchronization Therapy

In patients suffering from heart failure, conduction abnormalities that create cardiac dyssynchrony worsen morbidity and mortality. However, a pacemaker-based treatment termed Cardiac Resynchronization Therapy (CRT) can significantly restore function and mortality. Our previous work implicated post-translational signaling cascades in modulating this recovery. To further understand the mechanism of recovery, we aimed to catalogue, as completely as possible, the phospho-proteome in canine models of normal cardiac function (Con), cardiac dyssynchrony (Dys), and CRT. The models were generated by implanting pacemakers and pacing 6-weeks dyssynchronously (Dys), 3-weeks dyssynchronously and 3-weeks CRT (CRT), or not at all (Con). Each cardiac tissue sample (n=4 per group) was split into three parts: myofilament-enriched, myofilament-depleted, and phospho-tyrosine enriched. Each part was phospho-enriched and run on an Orbitrap Elite. By one-way ANOVA and Holm-Sidak post-hoc test, there were 104 sites Ser/Thr sites and 85 Tyr sites that were changed in at least one group. These sites were assigned one of three categories based on the pattern of changes: “Dyssynchrony, Not Fixed by CRT”, “Fixed by CRT”, or “CRT Only/Exacerbated by CRT”. Next we predicted the kinase for each phosphorylation site (Group-based Predication System 2.1). Many tyrosine kinases were identified, although no kinase showed specificity to any category. However, the Ser/Thr kinase CK2 was predicted to target a majority of the “Fixed By CRT” category, and was highly specific for this group, as CK2 was not predicted to target sites in any of the other categories. Thus, CRT appears to act as a broad CK2 activator. In the future, these sites need to be validated as CK2 targets. Moreover, the effect of CK2 activation on cardiomyocyte function, sarcomere shortening and calcium transient, will be explored, considering these are already implicated in recovery with CRT.

Liver mTOR Controls IGF-I Bioavailability by Regulation of Protein Kinase CK2 and IGFBP-1 Phosphorylation in Fetal Growth Restriction

Fetal growth restriction (FGR) increases the risk for perinatal complications and predisposes the infant to diabetes and cardiovascular disease later in life. No treatment for FGR is available, and the underlying pathophysiology remains poorly understood. Increased IGFBP-1 phosphorylation has been implicated as an important mechanism by which fetal growth is reduced. However, to what extent circulating IGFBP-1 is phosphorylated in FGR is unknown, and the molecular mechanisms linking FGR to IGFBP-1 phosphorylation have not been established. We used umbilical cord plasma of appropriate for gestational age (AGA) and growth-restricted human fetuses and determined IGFBP-1 and IGF-I concentrations (ELISA) and site-specific IGFBP-1 phosphorylation (Western blotting using IGFBP-1 phospho-site specific antibodies). In addition, we used a baboon model of FGR produced by 30% maternal nutrient restriction and determined mammalian target of rapamycin (mTOR)C1 activity, CK2 expression/activity, IGFBP-1 expression and phosphorylation, and IGF-I levels in baboon fetal liver by Western blot, enzymatic assay, and ELISA. HepG2 cells and primary fetal baboon hepatocytes were used to explore mechanistic links between mTORC1 signaling and IGFBP-1 phosphorylation. IGFBP-1 was hyperphosphorylated at Ser101, Ser119, and Ser169 in umbilical plasma of human FGR fetuses. IGFBP-1 was also hyperphosphorylated at Ser101, Ser119, and Ser169 in the liver of growth-restricted baboon fetus. mTOR signaling was markedly inhibited, whereas expression and activity of CK2 was increased in growth-restricted baboon fetal liver in vivo. Using HepG2 cells and primary fetal baboon hepatocytes, we established a mechanistic link between mTOR inhibition, CK2 activation, IGFBP-1 hyperphosphorylation, and decreased IGF-I-induced IGF-I receptor autophosphorylation. We provide clear evidence for IGFBP-1 hyperphosphorylation in FGR and identified an mTOR and CK2-mediated mechanism for regulation of IGF-I bioavailability. Our findings are consistent with the model that inhibition of mTOR in the fetal liver, resulting in increased CK2 activity and IGFBP-1 hyperphosphorylation, constitutes a novel mechanistic link between nutrient deprivation and restricted fetal growth.

Protein kinase CK2 and JNK control Nrf2 activation in response to skin contact sensitizers in THP1 cells

Protein kinase CK2 and JNK control Nrf2 activation in response to skin contact sensitizers in THP1 cells

In vitro mechanism for downregulation of ER ‐α expression by epigallocatechin gallate in ER +/ PR + human breast cancer cells

Exposure of the breast to estrogens and other sex hormones is an important cancer risk factor and estrogen receptor downregulators are attracting significant clinical interest. Epigallocatechin gallate (EGCG), a polyphenolic compound found in green tea, has gained considerable attention for its antitumor properties. Here we aimed to investigate the molecular mechanisms through which EGCG regulates ER-α expression in ER+ PR+ breast cancer cells. Western blotting analysis, real-time PCR, and transient transfections of deletion fragments of the ER-α gene promoter show that EGCG downregulates ER-α protein, mRNA, and gene promoter activity with a concomitant reduction of ER-α genomic and nongenomic signal. These events occur through p38(MAPK) /CK2 activation, causing the release from Hsp90 of progesterone receptor B (PR-B) and its consequent nuclear translocation as evidenced by immunofluorescence studies. EMSA, and ChIP assay reveal that, upon EGCG treatment, PR-B is recruited at the half-PRE site on ER-α promoter. This is concomitant with the formation of a corepressor complex containing NCoR and HDAC1 while RNA polymerase II is displaced. The events are crucially mediated by PR-B isoform, since they are abrogated with PR-B siRNA. Our data provide evidence for a mechanism by which EGCG downregulates ER-α and explains the inhibitory action of EGCG on the proliferation of ER+ PR+ cancer cells tested. We suggest that the EGCG/PR-B signaling should be further exploited for clinical approach.

Identification of a novel function of the clathrin-coated structure at the plasma membrane in facilitating GM-CSF receptor-mediated activation of JAK2

It is well known that ligand binding to the high-affinity GM-CSF receptor (GMR) activates JAK2. However, how and where this event occurs in a cellular environment remains unclear. Here, we demonstrate that clathrin- but not lipid raft-mediated endocytosis is crucial for GMR signaling. Knockdown expression of clathrin heavy chain or intersectin 2 (ITSN2) attenuated GMR-mediated activation of JAK2, whereas inhibiting clathrin-coated pits or plagues to bud off the membrane by the dominant-negative mutant of dynamin enhanced such event. Moreover, unlike the wild-type receptor, an ITSN2-non-binding mutant of GMR defective in targeting to clathrin-coated pits or plagues [collectively referred to as clathrin-coated structures (CCSs) here] failed to activate JAK2 at such locations. Additional experiments demonstrate that ligand treatment not only enhanced JAK2/GMR association at CCSs, but also induced a conformational change of JAK2 which is required for JAK2 to be activated by CCS-localized CK2. Interestingly, ligand-independent activation of the oncogenic mutant of JAK2 (JAK2V617F) also requires the targeting of this mutant to CCSs. But JAK2V617F seems to be constitutively in an open conformation for CK2 activation. Together, this study reveals a novel functional role of CCSs in GMR signaling and the oncogenesis of JAK2V617F.

Protein kinase CK2 mediates peroxynitrite-induced heme oxygenase-1 expression in articular chondrocytes

Heme oxygenase-1 (HO-1) is induced as an adaptive mechanism against oxidative stress in chondrocytes, which play an important role in the maintenance and degradation of cartilage. In the present study, we examined the role of protein kinase casein kinase (CK2) on peroxynitrite-induced expression of HO-1 in primary articular chondrocytes. 3-Μorpholinosydnonimine hydrochloride (SIN-1) has been shown to mediate cell death by activating apoptosis-related molecules in cells. In this study, we used a low concentration of SIN-1 that did not induce apoptosis to elucidate the mechanism by which SIN-1 upregulates HO-1 expression. In chondrocytes, SIN-1 induced HO-1 expression with spontaneous downregulation in a different manner than with high concentrations of SIN-1. Importantly, SIN-1 treatment of chondrocytes increased CK2 activation. Additionally, inhibition of CK2 with 4,5,6,7-tetrabromobenzotriazole (TBB) or siRNA did not induce HO-1 expression and reduced NF-E2-related factor2 (Nrf2) accumulation in chondrocytes. Therefore, we examined whether CK2 directly regulates Nrf2, which is a transcription factor that regulates the expression of HO-1. Indeed, TBB treatment inhibited phosphorylation and nuclear translocation of Nrf2 in SIN-1-treated cells. Moreover, using an immunoprecipitation assay, we confirmed that SIN-1 treatment enhanced the interaction between CK2 and Nrf2. Taken together, our findings suggest that peroxynitrite activates Nrf2 via CK2 signaling, leading to the upregulation of HO-1 in primary chondrocytes.

Protein Disulfide Isomerase Regulates Sickle Erythrocyte Volume Via ET-1 Dependent Casein Kinase II Mechanism

Abstract 2114Disordered K+ efflux and osmotically induced water loss leads to red blood cell (RBC) dehydration and plays a role in the pathophysiology of Sickle Cell Disease. We previously reported that activation of endothelin-1 (ET-1) receptors in sickle erythrocyte was partially responsible for dense sickle cell formation. However, the mechanism by which ET-1 regulates RBC volume remains unclear. Serine/threonine kinases have been shown to regulate K+ transport in RBC. Casein Kinase II (CK2), a serine/threonine kinase, phosphorylates acidic proteins, regulates calmodulin activity and cytoskeletal proteins and is present in RBC. CK2 activity is blocked by apigenin, emodin, heparin, and ornithine decarboxylase. Previous reports have shown a role for flavonoids such as apigenin as substrates for erythrocyte plasma membrane oxidoreductases. We recently observed a role for Protein Disulfide Isomerase (PDI) in regulating cellular hydration and K+ efflux in human RBC. PDI catalyzes disulfide interchange reactions in the plasma membrane, mediates redox modifications and is up-regulated under hypoxic conditions. However the relationship between CK2 and PDI in the setting of cellular hydration status is un-explored. Our results indicate that erythrocyte membrane CK2 activity increases when sickle cells are incubated with 500 nM ET-1 for 30 min (2.8 ± 0.1 to 4.9 ± 0.01 nmol/min/mL * 106 cell) an event that is blunted by pre-incubation with the ET-1 B receptor blocker, BQ788 (2.5 ± 0.1 nmol/min/mL * 106 cell, n=3, p<0.04) and 20 μM apigenin (2.7 ± 0.4 nmol/min/mL * 106 cell, n=3, p<0.04). We examined the role of CK2 activation on cellular dehydration. We incubated sickle erythrocytes for 3 hours in deoxygenation-oxygenation cycles in the presence or absence of 20μM apigenin or 2μM 4,5,6,7-tetrabromobenzotriazole (TBB), a specific CK2 inhibitor, and measured the changes in erythrocyte density by phthalate oil density analysis. We observed that inhibition of CK2 led to reduced deoxygenation-stimulated cellular dehydration in sickle erythrocytes by apigenin (D50= 1.106 to 1.100 g/mL) or TBB (D50 =1.097 g/mL). We then studied the role of CK2 inhibitors on PDI activity by Insulin Turbidity Assay and observed that apigenin and TBB led to significant reductions in PDI activity in vitro (64% and 42% respectively). We also studied the effects of the flavonoids: naringenin, naringin, apigenin and rutin on PDI activity and observed reductions in PDI activity that were greater with apigenin>rutin>TBB>naringin>naringenin (n=2, P<0.05). Furthermore, we observed that K+ flux via Gardos channel activation is correlated with PDI activity in vitro in sickle erythrocytes. Taken together our results implicate CK2 and PDI as intermediate regulators of ET-1 stimulated cellular volume systems in red blood cells. Supported by NIH R01-HL09632 to AR. Disclosures:No relevant conflicts of interest to declare.

CD5 through CK2 activation regulates B-1a B-cell survival, proliferation, and T-independent type 2 antibody response. (104.15)

Abstract CD5 expression is the characteristic feature of B-1a B-cells and B10 regulatory B-cells; however, the physiological role of this receptor on the development, persistence, and/or function of these B-cell populations remains undefined. To begin addressing this question we generated a CD5 mutant knock-in mouse (CD5ΔCK2BD) in which the serine threonine kinase, CK2, binding domain in the CD5-cytoplasmic tail was deleted. CD5 plays a critical role in regulating T-cell survival following antigen receptor engagement and this activity is dependent on CK2 activation. In the CD5ΔCK2BD mouse, the ability to activate CK2 is abrogated. B-1a B-cells in the CD5ΔCK2BD mouse turnover at a rate two-fold greater than that in WT mice. This increased turnover rate compensated for the decreased B-cell survival to maintain a near normal B-1a B-cell number in the peritoneal cavity. However, total serum natural IgM and IgA was significantly lower than that in WT mice. CD5ΔCK2BD B-1a B-cells proliferated poorly in response to anti-IgM stimulation but not LPS stimulation. The CD5ΔCK2BD mouse also exhibited an impaired anti-phosphatidylcholine (PC) response following immunization with heat killed S. pneumoniae, a prototypic B-1a B-cell response. Immune responses to T-independent type 1 antigen were not altered in the CD5ΔCK2BD mouse. These data provide the first evidence that CD5 has a direct role in regulating B-1a B-cell physiology.

CD5 modulates regulatory T cell function, proliferation, and survival. (168.12)

Abstract We previously showed that CD5-/- mice develop less severe experimental autoimmune encephalomyelitis (EAE). This is unexpected since CD5-/- T cells are hyperresponsive to myelin peptide immunization. The decreased severity is partially due to loss of CD5-dependent survival signals that are mediated by the activation of the serine/threonine kinase CK2. EAE in CD5 mutant knock-in mice (CD5ΔCK2BD), in which the ability to bind/activate CK2 is abrogated, was also less severe and recapitulates the findings in CD5-/- mice. CD5-/- Treg cells are hypersuppressive in in vitro suppression assays and therefore may also contribute to lower EAE severity in the CD5-/- mouse. This led us to address if CD5 regulates Treg cell function through activation of CK2. In contrast to the hypersuppressive activity of Treg cells from CD5-/- mice, Treg cells from CD5ΔCK2BD mice were less efficient in their ability to suppress responder CD4+ T cells than WT Treg cells. The proliferation of CD5-/- Tregs in response to stimulation with anti-CD3 in the presence of APCs and IL-2 was greater than WT and CD5ΔCK2BD. From these results we infer that the CD5-dependent CK2 activation pathway is a positive regulator of Treg suppressive activity. In contrast, the inhibitory (ITIM) domain is a negative regulator of Treg biology and proliferation. The combined signaling pathways from these two domains of CD5 balance Treg function and maintain homeostasis.

Functional Polymorphism of the CK2α Intronless Gene Plays Oncogenic Roles in Lung Cancer

Protein kinase CK2 is frequently up-regulated in human cancers, although the mechanism of CK2 activation in cancer remains unknown. In this study, we investigated the role of the CK2α intronless gene (CSNK2A1P, a presumed CK2α pseudogene) in the pathogenesis of human cancers. We found evidence of amplification and over-expression of the CSNK2A1P gene in non- small cell lung cancer and leukemia cell lines and 25% of the lung cancer tissues studied. The mRNA expression levels correlated with the copy numbers of the CSNK2A1P gene. We also identified a novel polymorphic variant (398T/C, I133T) of the CSNK2A1P gene and showed that the 398T allele is selectively amplified over the 398C allele in 101 non-small cell lung cancer tissue samples compared to those in 48 normal controls (p = 0.013<0.05). We show for the first time CSNK2A1P protein expression in transfected human embryonic kidney 293T and mouse embryonic fibroblast NIH-3T3 cell lines. Both alleles are transforming in these cell lines, and the 398T allele appears to be more transforming than the 398C allele. Moreover, the 398T allele degrades PML tumor suppressor protein more efficiently than the 398C allele and shows a relatively stronger binding to PML. Knockdown of the CSNK2A1P gene expression with specific siRNA increased the PML protein level in lung cancer cells. We report, for the first time, that the CSNK2A1P gene is a functional proto-oncogene in human cancers and its functional polymorphism appears to degrade PML differentially in cancer cells. These results are consistent with an important role for the 398T allele of the CSNK2A1P in human lung cancer susceptibility.

Diosgenin inhibits macrophage-derived inflammatory mediators through downregulation of CK2, JNK, NF-κB and AP-1 activation

Diosgenin is a precursor of steroid hormones, which can be found in several plant species. Diosgenin has been shown to have a variety of biological activities including anti-inflammatory activity, but through a mechanism that is unclear. Especially, the effect of this agent on macrophage function has not been characterized in detail. In the present study, we examined the effects of diosgenin on the production of inflammatory mediators in lipopolysaccharide (LPS)/interferon γ (IFN-γ)-activated murine macrophage. Macrophages pre-exposed to diosgenin (0.1–10 μΜ) were stimulated with LPS/IFN-γ. Pretreatment with diosgenin resulted in the inhibition of NO production and inducible nitric oxide synthase (iNOS) protein and mRNA expression in a concentration-dependent manner. In addition, diosgenin inhibits production of reactive oxygen species (ROS), interleukin-1 (IL-1), and IL-6, but not that of tumor necrosis factor-α (TNF-α). Inhibition of these inflammatory mediators appears to be at the transcriptional level, since diosgenin decreased LPS/IFN-γ-induced NF-κB and AP-1 activity. Diosgenin blocked CK2 activation and phosphorylation of c-Jun NH 2-terminal kinase (JNK), but not that of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase 1/2 (ERK 1/2). These results indicate that the inhibition of these signaling molecules expression was correlated with the reduced production of inflammatory mediators in macrophages. Taken together the present data suggest that diosgenin reduces the production of inflammatory meditators by inhibiting LPS/IFN-γ-triggered CK2, JNK, NF-κB and AP-1 activation, thereby implicating a mechanism by which diosgenin may exert its immunosuppressive effects.

CD5-dependent CK2 activation pathway regulates T cell activation and threshold for anergy (143.27)

Abstract We have reported that a major biological activity of CD5 is to regulate the survival of activated T cells, a property mediated by the CD5-dependent CK2 activation pathway. In order to address the physiological role of the CD5-dependent CK2 activation pathway in T cell activation, the generation of Th effector cells, and autoimmunity, we generated by gene targeting a mouse (CD5ΔCK2BD) that expressed CD5 lacking the ability to bind and activate CK2. CD4 and CD8 T cells from CD5ΔCK2BD mice exhibited enhanced AICD and hyporeactivity to antigen receptor stimulation compared to T cells from CD5WT and CD5-/- mice. The ability to mobilize calcium and upregulate CD69 were greatly enhanced in CD5-/- T cells but not in CD5WT T cells or CD5ΔCK2BD T cells, indicating that the CD5-dependent CK2 activation pathway did not regulate TCR-proximal signaling pathways. CD5-/- mice developed attenuated experimental autoimmune encephalomyelitis (EAE) when immunized with 150 μg MOG, yet the disease in CD5ΔCK2BD mice was comparable to that seen in CD5WT mice. In contrast, immunization with tolerogenic doses of MOG resulted in attenuated EAE in CD5WT mice but not in CD5-/- and CD5ΔCK2BD mice. The results indicated that loss of the CD5-dependent CK2 pathway made T cells refractory to induction of anergy, a prediction that was recapitulated in in vitro recall assays. Our results reveal a novel role for CD5 in setting thresholds for anergy in T cells. Supported by grants from the NIH and NMSS.