Channel-anchored Protein Kinase CK2 and Protein Phosphatase 1 Reciprocally Regulate KCNQ2-containing M-channels via Phosphorylation of Calmodulin

M-type potassium channels, encoded by the KCNQ family genes (KCNQ2–5), require calmodulin as an essential co-factor. Calmodulin bound to the KCNQ2 subunit regulates channel trafficking and stabilizes channel activity. We demonstrate that phosphorylation of calmodulin by protein kinase CK2 (casein kinase 2) rapidly and reversibly modulated KCNQ2 current. CK2-mediated phosphorylation of calmodulin strengthened its binding to KCNQ2 channel, caused resistance to phosphatidylinositol 4,5-bisphosphate depletion, and increased KCNQ2 current amplitude. Accordingly, application of CK2-selective inhibitors suppressed KCNQ2 current. This suppression was prevented by co-expression of CK2 phosphomimetic calmodulin mutants or pretreatment with a protein phosphatase inhibitor, calyculin A. We also demonstrated that functional CK2 and protein phosphatase 1 (PP1) were selectively tethered to the KCNQ2 subunit. We identified a functional KVXF consensus site for PP1 binding in the N-terminal tail of KCNQ2 subunit: mutation of this site augmented current density. CK2 inhibitor treatment suppressed M-current in rat superior cervical ganglion neurons, an effect negated by overexpression of phosphomimetic calmodulin or pretreatment with calyculin A Furthermore, CK2 inhibition diminished the medium after hyperpolarization by suppressing the M-current. These findings suggest that CK2-mediated phosphorylation of calmodulin regulates the M-current, which is tonically regulated by CK2 and PP1 anchored to the KCNQ2 channel complex.     

Casein kinase II contributes to the synergistic effects of BMP7 and BDNF on Smad 1/5/8 phosphorylation in septal neurons under hypoglycemic stress

The combination of bone morphogenetic protein 7 (BMP7) and neurotrophins (e.g. brain-derived neurotrophic factor, BDNF) protects septal neurons during hypoglycemic stress. We investigated the signaling mechanisms underlying this synergistic protection. BMP7 (5 nM) increased phosphorylation and nuclear translocation of BMP-responsive Smads 1/5/8 within 30 min in cultures of rat embryonic septal neurons. BDNF (100 ng/mL) enhanced the BMP7-induced increase in phospho-Smad levels in both nucleus and cytoplasm; this effect was more pronounced after a hypoglycemic stress. BDNF increased both Akt and Erk phosphorylation, but pharmacological blockade of these kinase pathways (with wortmannin and U0126, respectively) did not reduce the Smad phosphorylation produced by the BMP7 + BDNF combination. Inhibitors of casein kinase II (CK2) activity reduced the (BMP7 + BDNF)-induced Smad phosphorylation, and this trophic factor combination increased CK2 activity in hypoglycemic cultures. These findings suggest that BDNF can increase BMP-dependent Smad phosphorylation via a mechanism requiring CK2.     

Phosphorylation by Casein Kinase 2 Facilitates Psh1 Protein-assisted Degradation of Cse4 Protein

Cse4 is the centromeric histone H3 variant in budding yeast. Psh1 is an E3 ubiquitin ligase that controls Cse4 levels through proteolysis. Here we report that Psh1 is phosphorylated by the Cka2 subunit of casein kinase 2 (CK2) to promote its E3 activity for Cse4. Deletion of CKA2 significantly stabilized Cse4. Consistent with phosphorylation promoting the activity of Psh1, Cse4 was stabilized in a Psh1 phosphodepleted mutant strain in which the major phosphorylation sites were changed to alanines. Phosphorylation of Psh1 did not control Psh1-Cse4 or Psh1-Ubc3(E2) interactions. Although Cse4 was highly stabilized in a cka2Δ strain, mislocalization of Cse4 was mild, suggesting that Cse4 misincorporation was prevented by the intact Psh1-Cse4 association. Supporting this idea, Psh1 was also stabilized in a cka2Δ strain. Collectively our data suggest that phosphorylation is crucial in Psh1-assisted control of Cse4 levels and that the Psh1-Cse4 association itself functions to prevent Cse4 misincorporation.     

Regulation of Ceramide Synthase by Casein Kinase 2-dependent Phosphorylation in Saccharomyces cerevisiae

Complex sphingolipids are important components of eukaryotic cell membranes and, together with their biosynthetic precursors, including sphingoid long chain bases and ceramides, have important signaling functions crucial for cell growth and survival. Ceramides are produced at the endoplasmic reticulum (ER) membrane by a multicomponent enzyme complex termed ceramide synthase (CerS). In budding yeast, this complex is composed of two catalytic subunits, Lac1 and Lag1, as well as an essential regulatory subunit, Lip1. Proper formation of ceramides by CerS has been shown previously to require the Cka2 subunit of casein kinase 2 (CK2), a ubiquitous enzyme with multiple cellular functions, but the precise mechanism involved has remained unidentified. Here we present evidence that Lac1 and Lag1 are direct targets for CK2 and that phosphorylation at conserved positions within the C-terminal cytoplasmic domain of each protein is required for optimal CerS activity. Our data suggest that phosphorylation of Lac1 and Lag1 is important for proper localization and distribution of CerS within the ER membrane and that phosphorylation of these sites is functionally linked to the COP I-dependent C-terminal dilysine ER retrieval pathway. Together, our data identify CK2 as an important regulator of sphingolipid metabolism, and additionally, because both ceramides and CK2 have been implicated in the regulation of cancer, our findings may lead to an enhanced understanding of their relationship in health and disease.     

Casein Kinase 2 β-Subunit Is a Regulator of Bone Morphogenetic Protein 2 Signaling

Bone morphogenetic proteins (BMPs) play a crucial role during embryonic development and regulate processes as diverse as neurogenesis, skeletal formation, and hematopoesis. They signal through a hetero-oligomer complex of BMP receptors. Binding of the ligand to the receptors activates several pathways, including Smad and p38. BMP signaling is controlled in the extracellular space, the plasma membrane, and the intracellular space; however, the mechanism of receptor signaling at the plasma membrane and proteins that regulate this process still need to be identified. The experiments presented here identify the protein kinase casein kinase II (CK2) as a BMP receptor type Ia (BRIa) interacting protein. Fluorescence resonance energy transfer revealed that this interaction occurs at the plasma membrane. BMP2 stimulation of C2C12 cells leads to the release of CK2 from BRIa. Blocking this interaction with specific peptides that inhibit the binding sites for CK2 on BRIa demonstrated a redistribution of BRIa on the plasma membrane. Signaling was initiated once CK2 was released from BRIa, leading to the mineralization of C2C12 cells. These data suggest that CK2 is a negative regulator of BMP signaling and osteoblast differentiation.     

Phosphorylation of RanGAP1 stabilizes its interaction with Ran and RanBP1

Ran is a nuclear Ras-like GTPase that is required for various nuclear events including the bi-directional transport of proteins and ribonucleoproteins through the nuclear pore complex, spindle formation, and reassembly of the nuclear envelope. One of the key regulators of Ran is RanGAP1, a Ran specific GTPase activating protein. The question of whether a mechanism exists for controlling nucleocytoplasmic transport through the regulation of RanGAP1 activity continues to be debated. Here we show that RanGAP1 is phosphorylated in vivo and in vitro. Serine-358 (358S) was identified as the major phosphorylation site, by MALDI-TOF-MS spectrometry. Site directed mutagenesis at this position abolished the phosphorylation. Experiments using purified recombinant kinase and specific inhibitors such as DRB and apigenin strongly suggest that casein kinase II (CK2) is the responsible kinase. Although the phosphorylation of 358S of RanGAP1 did not significantly alter its GAP activity, the phosphorylated wild type RanGAP1, but not a mutant harboring a mutation at the phosphorylation site 358S, efficiently formed a stable ternary complex with Ran and RanBP1 in vivo, suggesting that the 358S phosphorylation of RanGAP1 affects the Ran system.     

A Hinge in the Distal End of the PACSIN 2 F-BAR Domain May Contribute to Membrane-Curvature Sensing

The protein kinase C and casein kinase 2 substrates in neurons (PACSINs) represent a subfamily of membrane-binding proteins characterized by an amino-terminal Bin-Amphiphysin-Rvs (F-BAR) domain. PACSINs link membrane trafficking with actin dynamics and regulate the localization of distinct cargo molecules. The F-BAR domain forms a dimer essential for lipid binding. We have obtained crystals of authentic murine PACSIN 2 that contain an ordered F-BAR domain, indicating that additional domains are flexibly connected to F-BAR. The structure shares similarity to other BAR domains and exhibits special features unique to PACSINs. These include the uneven distribution of charged residues on the concave molecular surface and a so-called wedge loop that is driven into the membrane upon binding of PACSIN. The murine PACSIN 2 F-BAR domain requires dimerization for sensing of curved membranes, and the present structure also provides a mechanism for higher-order oligomer formation. Importantly, comparison of murine with human and Drosophila PACSIN 2 F-BAR domains reveals stark differences in the orientation of distal helical segments leading to a wider crescent shape of murine PACSIN 2. We define hinge residues for these movements that may help PACSINs sense and concomitantly reinforce membrane curvature.     

PD-1 Increases PTEN Phosphatase Activity While Decreasing PTEN Protein Stability by Inhibiting Casein Kinase 2

Programmed death 1 (PD-1) is a potent inhibitor of T cell responses. PD-1 abrogates activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, but the mechanism remains unclear. We determined that during T cell receptor (TCR)/CD3- and CD28-mediated stimulation, PTEN is phosphorylated by casein kinase 2 (CK2) in the Ser380-Thr382-Thr383 cluster within the C-terminal regulatory domain, which stabilizes PTEN, resulting in increased protein abundance but suppressed PTEN phosphatase activity. PD-1 inhibited the stabilizing phosphorylation of the Ser380-Thr382-Thr383 cluster within the C-terminal domain of PTEN, thereby resulting in ubiquitin-dependent degradation and diminished abundance of PTEN protein but increased PTEN phosphatase activity. These effects on PTEN were secondary to PD-1-mediated inhibition of CK2 and were recapitulated by pharmacologic inhibition of CK2 during TCR/CD3- and CD28-mediated stimulation without PD-1. Furthermore, PD-1-mediated diminished abundance of PTEN was reversed by inhibition of ubiquitin-dependent proteasomal degradation. Our results identify CK2 as a new target of PD-1 and reveal an unexpected mechanism by which PD-1 decreases PTEN protein expression while increasing PTEN activity, thereby inhibiting the PI3K/Akt signaling axis.     

Cyclin-dependent Kinase 5 (Cdk5)-dependent Phosphorylation of p70 Ribosomal S6 Kinase 1 (S6K) Is Required for Dendritic Spine Morphogenesis

The maturation and maintenance of dendritic spines depends on neuronal activity and protein synthesis. One potential mechanism involves mammalian target of rapamycin, which promotes protein synthesis through phosphorylation of eIF4E-binding protein and p70 ribosomal S6 kinase 1 (S6K). Upon extracellular stimulation, mammalian target of rapamycin phosphorylates S6K at Thr-389. S6K also undergoes phosphorylation at other sites, including four serine residues in the autoinhibitory domain. Despite extensive biochemical studies, the importance of phosphorylation in the autoinhibitory domain in S6K function remains unresolved, and its role has not been explored in the cellular context. Here we demonstrated that S6K in neuron was phosphorylated at Ser-411 within the autoinhibitory domain by cyclin-dependent kinase 5. Ser-411 phosphorylation was regulated by neuronal activity and brain-derived neurotrophic factor (BDNF). Knockdown of S6K in hippocampal neurons by RNAi led to loss of dendritic spines, an effect that mimics neuronal activity blockade by tetrodotoxin. Notably, coexpression of wild type S6K, but not the phospho-deficient S411A mutant, could rescue the spine defects. These findings reveal the importance of cyclin-dependent kinase 5-mediated phosphorylation of S6K at Ser-411 in spine morphogenesis driven by BDNF and neuronal activity.     

Effect of Ethanol on Nuclear Casein Kinase II Activity in Brain

Casein kinase II (CK II) plays an important role in serine/threonine dependent protein phosphorylation. In brain it is associated with long term potentiation besides its involvement in DNA, RNA and protein metabolism. Ethanol has been shown to induce cognitive impairment and affects DNA, RNA and protein metabolism at various steps. Since CK II is central in all these events, which are specifically affected by ethanol, the role of nuclear CK II is investigated in the present study. Total nuclear casein kinase activity was unaffected while heparin sensitive nuclear casein kinase II activity showed a 30% decrease in the brain from chronic alcohol fed rats. Cytosolic CK II activity was also unaffected. Immunological detection by western analysis using CK II antibodies showed no alteration in the quantity of enzyme. The decrease in nuclear casein kinase II might be responsible for ethanol induced cognitive impairment in the brain.     

Casein Kinase 2 (CK2)-mediated Phosphorylation of Hsp90β as a Novel Mechanism of Rifampin-induced MDR1 Expression

The P-glycoprotein (P-gp) encoded by the MDR1 gene is a drug-exporting transporter located in the cellular membrane. P-gp induction is regarded as one of the main mechanisms underlying drug-induced resistance. Although there is great interest in the regulation of P-gp expression, little is known about its underlying regulatory mechanisms. In this study, we demonstrate that casein kinase 2 (CK2)-mediated phosphorylation of heat shock protein 90β (Hsp90β) and subsequent stabilization of PXR is a key mechanism in the regulation of MDR1 expression. Furthermore, we show that CK2 is directly activated by rifampin. Upon exposure to rifampin, CK2 catalyzes the phosphorylation of Hsp90β at the Ser-225/254 residues. Phosphorylated Hsp90β then interacts with PXR, causing a subsequent increase in its stability, leading to the induction of P-gp expression. In addition, inhibition of CK2 and Hsp90β enhances the down-regulation of PXR and P-gp expression. The results of this study may facilitate the development of new strategies to prevent multidrug resistance and provide a plausible mechanism for acquired drug resistance by CK2-mediated regulation of P-gp expression.     

Casein kinase 1-dependent phosphorylation of familial advanced sleep phase syndrome-associated residues controls PERIOD 2 stability

The mammalian circadian clock component PERIOD2 (PER2) plays a critical role in circadian rhythm entrainment. Recently, a missense mutation at a putative phosphorylation site in hPER2, Ser-662, was identified in patients that suffer from familial advanced sleep phase syndrome (FASPS). Patients with FASPS display abnormal sleep-wake patterns characterized by a lifelong pattern of sleep onset in the early evening and offset in the early morning. Although the phosphorylation of PER2 is strongly implied from functional studies, it has not been possible to study the site-specific phosphorylation of PER2 on Ser-662, and the biochemical functions of this residue are unclear. Here, we used phospho-specific antibodies to show that PER2 is phosphorylated on Ser-662 and flanking casein kinase (CK) sites in vivo. The phosphorylation of PER2 was carried out by the combined activities of casein kinase 1δ (CK1 δ) and casein kinase 1ε (CK1ε) and was antagonized by protein phosphatase 1. PER2 phosphorylation was rapidly induced in response to circadian entrainment of mammalian cell lines and occurred in both cytosolic and nuclear compartments. Importantly, we found that the pool of Ser-662-phosphorylated PER2 proteins was more stable than the pool of total PER2 molecules, implying that the FASPS phosphorylation cluster antagonizes PER2 degradation. Consistent with this idea, a Ser-662→Ala mutation that abrogated PER2 phosphorylation significantly reduced its half-life, whereas a phosphomimetic Ser-662→Asp substitution led to an elevation in half-life. Our combined findings provide new insights into PER2 regulation and the biochemical basis of FASPS.     

The Drosophila Splicing Factor PSI Is Phosphorylated by Casein Kinase II and Tousled-Like Kinase

Alternative splicing of pre-mRNA is a highly regulated process that allows cells to change their genetic informational output. These changes are mediated by protein factors that directly bind specific pre-mRNA sequences. Although much is known about how these splicing factors regulate pre-mRNA splicing events, comparatively little is known about the regulation of the splicing factors themselves. Here, we show that the Drosophila splicing factor P element Somatic Inhibitor (PSI) is phosphorylated at at least two different sites by at minimum two different kinases, casein kinase II (CK II) and tousled-like kinase (tlk). These phosphorylation events may be important for regulating protein-protein interactions involving PSI. Additionally, we show that PSI interacts with several proteins in Drosophila S2 tissue culture cells, the majority of which are splicing factors.     

Carbon monoxide neurotransmission activated by CK2 phosphorylation of heme oxygenase-2

Carbon monoxide (CO) is a putative gaseous neurotransmitter that lacks vesicular storage and must be synthesized rapidly following neuronal depolarization. We show that the biosynthetic enzyme for CO, heme oxygenase-2 (HO2), is activated during neuronal stimulation by phosphorylation by CK2 (formerly casein kinase 2). Phorbol ester treatment of hippocampal cultures results in the phosphorylation and activation of HO2 by CK2, implicating protein kinase C (PKC) in CK2 stimulation. Odorant treatment of olfactory receptor neurons augments HO2 phosphorylation and activity as well as cyclic guanosine monophosphate (cGMP) levels, with all of these effects selectively blocked by CK2 inhibitors. Likewise, CO-mediated nonadrenergic, noncholinergic (NANC) relaxation of the internal anal sphincter requires CK2 activity. Our findings provide a molecular mechanism for the rapid neuronal activation of CO biosynthesis, as required for a gaseous neurotransmitter.     

Identification of a Novel Protein Interaction Motif in the Regulatory Subunit of Casein Kinase 2

Casein kinase 2 (CK2) regulates multiple cellular processes and can promote oncogenesis. Interactions with the CK2β regulatory subunit of the enzyme target its catalytic subunit (CK2α or CK2α′) to specific substrates; however, little is known about the mechanisms by which these interactions occur. We previously showed that by binding CK2β, the Epstein-Barr virus (EBV) EBNA1 protein recruits CK2 to promyelocytic leukemia (PML) nuclear bodies, where increased CK2-mediated phosphorylation of PML proteins triggers their degradation. Here we have identified a KSSR motif near the dimerization interface of CK2β as forming part of a protein interaction pocket that mediates interaction with EBNA1. We show that the EBNA1-CK2β interaction is primed by phosphorylation of EBNA1 on S393 (within a polyserine region). This phosphoserine is critical for EBNA1-induced PML degradation but does not affect EBNA1 functions in EBV replication or segregation. Using comparative proteomics of wild-type (WT) and KSSR mutant CK2β, we identified an uncharacterized cellular protein, C18orf25/ARKL1, that also binds CK2β through the KSSR motif and show that this involves a polyserine sequence resembling the CK2β binding sequence in EBNA1. Therefore, we have identified a new mechanism of CK2 interaction used by viral and cellular proteins.     

PACSIN 2 represses cellular migration through direct association with cyclin D1 but not its alternate splice form cyclin D1b

Cyclin D1 overexpression is a common feature of many human malignancies. Genomic deletion analysis has demonstrated a key role for cyclin D1 in cellular proliferation, angiogenesis and cellular migration. To investigate the mechanisms contributing to cyclin D1 functions, we purified cyclin D1a-associated complexes by affinity chromatography and identified the PACSIN 2 (protein kinase C and casein kinase substrate in neurons 2) protein by mass spectrometry. The PACSIN 2, but not the related PACSIN 1 and 3, directly bound wild-type cyclin D1 (cyclin D1a) at the carboxyl terminus and failed to bind cyclin D1b, the alternative splicing variant of cyclin D1. PACSIN 2 knockdown induced cellular migration and reduced cell spreading in LNCaP cells expressing cyclin D1a. In cyclin D1^−/− mouse embryonic fibroblasts (MEFs), cyclin D1a, but not cyclin D1b, reduced the cell spreading to a polarized morphology. siPACSIN 2 had no effect on cellular migration of cyclin D1^−/− MEFs. Cyclin D1a restored the migratory ability of cyclin D1^−/− MEFs, which was further enhanced by knocking down PACSIN 2 with siRNA. The cyclin D1-associated protein, PACSIN 2, regulates cell spreading and migration, which are dependent on cyclin D1 expression.Key words: PACSIN 2, cyclin D1, polymorphism, cellular migration, cell spreading, cancer     

Casein kinase 2 regulates the mRNA-destabilizing activity of tristetraprolin

Tristetraprolin (TTP) is an AU-rich element-binding protein that regulates mRNA stability. We previously showed that TTP acts as a negative regulator of VEGF gene expression in colon cancer cells. The p38 MAPK pathway is known to suppress the TTP activity. However, until now the signaling pathway to enhance TTP function is not well known. Here, we show that casein kinase 2 (CK2) enhances the TTP function in the regulation of the VEGF expression in colon cancer cells. CK2 increased TTP protein levels and enhanced VEGF mRNA decaying activity of TTP. TTP was not a direct target of CK2. Instead, CK2 increased the phosphorylation of MKP-1, which led to a decrease in the phosphorylation of p38 MAPK. Inhibition of MKP-1 by siRNA attenuated the increase in TTP function and the decrease of p38 phosphorylation induced by CK2α overexpression. TGF-β1 increased the expressions of CK2 and TTP and the TTP function. The siRNA against CK2α or TTP reversed TGF-β1-induced increases in the expression of CK2 and TTP and the TTP function. Our data suggest that CK2 enhances the protein level and activity of TTP via the modulation of the MKP-1-p38 MAPK signaling pathway and that TGF-β1 enhances the activity of CK2.