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PubMed1999

The multifunctional herpes simplex virus IE63 protein interacts with heterogeneous ribonucleoprotein K and with casein kinase 2.

Herpes simplex virus type 1 (HSV-1), the prototype alpha-herpesvirus, causes several prominent diseases. The HSV-1 immediate early (IE) protein IE63 (ICP27) is the only regulatory gene with a homologue in every mammalian and avian herpesvirus sequenced so far. IE63 is a multifunctional protein affecting transcriptional and post-transcriptional processes, and it can shuttle from the nucleus to the cytoplasm. To identify interacting cellular proteins, a HeLa cDNA library was screened in the yeast two-hybrid system using IE63 as bait. Several interacting proteins were identified including heterogeneous nuclear ribonucleoprotein K (hnRNP K), a multifunctional protein like IE63, and the beta subunit of casein kinase 2 (CK2), a protein kinase, and interacting regions were mapped. Confirmation of interactions was provided by fusion protein binding assays, co-immunoprecipitation from infected cells, and CK2 activity assays. hnRNP K co-immunoprecipitated from infected cells with anti-IE63 serum was a more rapidly migrating subfraction than hnRNP K immunoprecipitated by anti-hnRNP K serum. Using anti-IE63 serum, both IE63 and hnRNP K were phosphorylated in vitro by CK2, while in immunoprecipitates using anti-hnRNP K serum, IE63 but not hnRNP K was phosphorylated by CK2. These data provide important new insights into how this key viral regulatory protein exerts its functions.

Animals↗
PubMed1999

Expression of the casein kinase 2 subunits in Chinese hamster ovary and 3T3 L1 cells provides information on the role of the enzyme in cell proliferation and the cell cycle.

In order to investigate the in vivo functions of protein kinase CK2 (CK2), the expression of Myc-tagged versions of the subunits, Myc-CK2alpha and Myc-CK2beta, was carried out in Chinese hamster ovary cells (CHO cells) and in 3T3 L1 fibroblasts. Cell proliferation in these cells was examined. CHO cells that transiently overexpressed the Myc-CK2beta subunit exhibited a severe growth defect, as shown by a much lower value of [(3)H]thymidine incorporation than the vector controls, and a rounded shrunken morphology. In contrast, cells overexpressing Myc-tagged CK2alpha showed a slightly but consistently higher value of [(3)H]thymidine incorporation than the controls. The defect in cell growth and changes in morphology caused by Myc-CK2beta overexpression were partially rescued by coexpression of Myc-tagged CK2alpha. In parallel to the studies in CHO cells, the stable transfection of Myc-CK2alpha and Myc-CK2beta subunits was achieved in 3T3 L1 fibroblast cells. Similarly, the ectopic expression of Myc-CK2beta, but not Myc-CK2alpha, caused a growth defect. By measuring [(3)H]thymidine incorporation, it was found that expression of Myc-CK2beta prolonged the G(1) phase and inhibited up-regulation of cyclin D1 expression during G(1). In addition, a lower mitotic index and lower mitotic cyclin-dependent kinase activities were detected in Myc-CK2beta-expressing cells. Detailed analysis of stable cells that were synchronously released into the cell cycle revealed that the expression of Myc-CK2beta inhibited cells entering into mitosis and prevented the activation of mitotic cyclin-dependent kinases. Taken together, results from both transient and stable expression of CK2 subunits strongly suggest that CK2 may be involved in the control of cell growth and progression of the cell cycle.

3T3 Cells↗
PubMed2000

Phosphorylation of the beta-amyloid precursor protein at the cell surface by ectocasein kinases 1 and 2.

The beta-amyloid precursor protein (betaAPP) is one of the rare proteins known to be phosphorylated within its ectodomain. We have shown previously that betaAPP can be phosphorylated within secretory vesicles and at the cell surface (Walter, J., Capell, A., Hung, A. Y. , Langen, H., Schnölzer, M., Thinakaran, G., Sisodia, S. S., Selkoe, D. J., and Haass, C. (1997) J. Biol. Chem. 272, 1896-1903). We have now specifically characterized the phosphorylation of cell surface-located betaAPP and identified two ectoprotein kinases that phosphorylate betaAPP at the outer face of the plasma membrane. By using selective protein kinase inhibitors and by investigating the usage of ATP and GTP as cosubstrates, we demonstrate that membrane-bound betaAPP as well as secreted forms of betaAPP can be phosphorylated by casein kinase (CK) 1- and CK2-like ectoprotein kinases. The ectodomain of betaAPP was also phosphorylated by purified CK1 and CK2 in vitro, but not by protein kinases A and C. Phosphorylation of betaAPP by ectoprotein kinases and by purified CK1 and CK2 occurred within an acidic domain in the N-terminal half of the protein. Heparin strongly inhibited the phosphorylation of cell-surface betaAPP by ecto-CK1 and ecto-CK2, indicating a regulatory role of this extracellular matrix component in betaAPP phosphorylation.

Amyloid beta-Protein Precursor↗
PubMed2002

Regulation of phosducin-like protein by casein kinase 2 and N-terminal splicing.

Phosducin-like protein (PhLP) is a member of the phosducin family of G-protein betagamma-regulators and exists in two splice variants. The long isoform PhLP(L) and the short isoform PhLP(S) differ by the presence or absence of an 83-amino acid N terminus. In isolated biochemical assay systems, PhLP(L) is the more potent Gbetagamma-inhibitor, whereas the functional role of PhLP(S) is still unclear. We now report that in intact HEK 293 cells, PhLP(S) inhibited Gbetagamma-induced inositol phosphate generation with approximately 20-fold greater potency than PhLP(L). Radiolabeling of transfected HEK 293 cells with [(32)P] revealed that PhLP(L) is constitutively phosphorylated, whereas PhLP(S) is not. Because PhLP(L) has several consensus sites for the constitutively active kinase casein kinase 2 (CK2) in its N terminus, we tested the phosphorylation of the recombinant proteins by either HEK cell cytosol in the presence or absence of kinase inhibitors or by purified CK2. PhLP(L) was a good CK2 substrate, whereas PhLP(S) and phosducin were not. Progressive truncation and serine/threonine to alanine mutations of the PhLP(L) N terminus identified a serine/threonine cluster (Ser-18/Thr-19/Ser-20) within a small N-terminal region of PhLP(L) (amino acids 5-28) as the site in which PhLP(L) function was modified in HEK 293 cells. In native tissue, PhLP(L) also seems to be regulated by phosphorylation because phosphorylated and non-phosphorylated forms of PhLP(L) were detected in mouse brain and adrenal gland. Moreover, the alternatively spliced isoform PhLP(S) was also found in adrenal tissue. Therefore, the physiological control of G-protein regulation by PhLP seems to involve phosphorylation by CK2 and alternative splicing of the regulator.

Alternative Splicing↗
PubMed2002

Casein kinase 2 interacts with cyclin-dependent kinase 11 (CDK11) in vivo and phosphorylates both the RNA polymerase II carboxyl-terminal domain and CDK11 in vitro.

The PITSLRE protein kinases, hereafter referred to as cyclin-dependent kinase 11 (CDK11) due to their association with cyclin L, are part of large molecular weight protein complexes that contain RNA polymerase II (RNAP II) as well as numerous transcription and RNA processing factors. Data presented here demonstrate that the influence of CDK11(p110) on transcription and splicing does not involve phosphorylation of the RNAP II carboxyl-terminal domain by CDK11(p110). We have isolated a DRB- and heparin-sensitive protein kinase activity that co-purifies with CDK11(p110) after ion exchange and affinity purification chromatography. This protein kinase was identified as casein kinase 2 (CK2) by immunoblot and mass spectrometry analyses. In addition to the RNAP II carboxyl-terminal domain, CK2 phosphorylates the CDK11(p110) amino-terminal domain. These data suggest that CDK11(p110) isoforms participate in signaling pathways that include CK2 and that its function may help to coordinate the regulation of RNA transcription and processing events. Future experiments will determine how phosphorylation of CDK11(p110) by CK2 specifically affects RNA transcription and/or processing events.

Casein Kinase II↗
PubMed2002

p53 serine 392 phosphorylation increases after UV through induction of the assembly of the CK2.hSPT16.SSRP1 complex.

Previously, we purified a UV-responsive p53 serine 392 kinase from F9 and HeLa cells and found that its activity is attributed to a high molecular weight protein complex containing the protein kinase CK2, along with the chromatin-associated factors hSPT16 and SSRP1. Here we determine that these proteins interact in vitro and in cells via non-overlapping domains and provide evidence consistent with the idea that hSPT16 and SSRP1 change the conformation of CK2 upon binding such that it specifically targets p53 over other substrates. Also, UV irradiation apparently induces the association of the complex, thereby increasing the specificity of CK2 for p53 at the expense of other cellular CK2 substrates and leading to an overall increase in p53 serine 392 phosphorylation.

Animals↗
PubMed2006

The interleukin-1beta gene is transcribed from a poised promoter architecture in monocytes.

Cytokine transcription is usually regulated by transcription factor binding and chromatin remodeling following an inducing signal. By contrast, these data showed the interleukin (IL)-1beta promoter assembles into a "poised" structure, as evidenced by nuclease accessibility and loss of core histones immediately surrounding the transcription start site. Strikingly, these properties do not change upon transcriptional activation by lipopolysaccharide. Furthermore, association of two key transcriptional activators, PU.1 and C/EBPbeta, is robust pre- and post-stimulation indicating the IL-1beta promoter is packaged into a nontranscribed but poised promoter architecture in cells capable of rapidly inducing IL-1beta. Monocyte stimulation causes recruitment of a third factor, IRF-4, to the IL-1beta enhancer. PU.1 phosphorylation at a CK2 kinase consensus element is required for this recruitment. We showed that CK2 phosphorylates PU.1, CK2 inhibitors abrogate IL-1beta induction, and CK2 inducibly associates with the IL-1beta enhancer. Taken together, these data indicate a novel two-step mechanism for IL-1beta transcription: 1) formation of a poised chromatin architecture, and 2) phosphorylation of an enhancer-bound factor that recruits other activators. We propose that this poised structure may generally characterize rapidly activated genes.

Casein Kinase II↗
PubMed2003

Pea DNA topoisomerase I is phosphorylated and stimulated by casein kinase 2 and protein kinase C.

DNA topoisomerase I catalyzes the relaxation of superhelical DNA tension and is vital for DNA metabolism; therefore, it is essential for growth and development of plants. Here, we have studied the phosphorylation-dependent regulation of topoisomerase I from pea (Pisum sativum). The purified enzyme did not show autophosphorylation but was phosphorylated in an Mg(2+)-dependent manner by endogenous protein kinases present in pea nuclear extracts. This phosphorylation was abolished with calf intestinal alkaline phosphatase and lambda phosphatase. It was also phosphorylated by exogenous casein kinase 2 (CK2), protein kinase C (PKC; from animal sources), and an endogenous pea protein, which was purified using a novel phorbol myristate acetate affinity chromatography method. All of these phosphorylations were inhibited by heparin (inhibitor of CK2) and calphostin (inhibitor of PKC), suggesting that pea topoisomerase I is a bona fide substrate for these kinases. Spermine and spermidine had no effect on the CK2-mediated phosphorylation, suggesting that it is polyamine independent. Phospho-amino acid analysis showed that only serine residues were phosphorylated, which was further confirmed using antiphosphoserine antibody. The topoisomerase I activity increased after phosphorylation with exogenous CK2 and PKC. This study shows that these kinases may contribute to the physiological regulation of DNA topoisomerase I activity and overall DNA metabolism in plants.

Casein Kinase II↗
PubMed2005

Signaling via the angiotensin-converting enzyme results in the phosphorylation of the nonmuscle myosin heavy chain IIA.

The phosphorylation of the short C-terminal cytoplasmic domain of the somatic angiotensin-converting enzyme (ACE) is involved in the regulation of enzyme shedding. We determined whether the phosphorylation of the cytoplasmic domain of ACE (ACEct) on Ser1270 regulates the cleavage/secretion of the enzyme by affecting its association with other proteins. ACE was associated with beta-actin and the nonmuscle myosin heavy chain IIA (MYH9) in endothelial cells, as determined by coimmunoprecipitation experiments as well as an ACEct affinity column. The ACE-associated MYH9 immunoprecipitated from (32)P-labeled endothelial cells was basally phosphorylated and cell stimulation with ACE inhibitors, or with bradykinin, increased the phosphorylation of MYH9. Casein kinase 2 (CK2) but not protein kinase C phosphorylated MYH9 in vitro, CK2 coprecipitated with MYH9 from endothelial cells and the phosphorylation of MYH9 in intact cells paralleled the phosphorylation of ACE on Ser1270 by CK2. The CK2 inhibitor 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole attenuated the phosphorylation of ACE and MYH9, disrupted their association, and enhanced the cleavage/secretion of ACE from the plasma membrane. Cytochalasin D decreased the interaction between ACE and MYH9 and stimulated ACE shedding. Although MYH9 was still able to associate with residual amounts of a nonphosphorylatable S1270A ACE mutant, no ACE inhibitor-induced increase in MYH9 phosphorylation could be detected in S1270A-expressing cells. These data indicate that the interaction of ACE with MYH9 determines ACE shedding and is modulated by phosphorylation processes. Furthermore, because ACE inhibitors affect the phosphorylation of MYH9, the phosphorylation of this class II myosin might contribute to the phenomenon of ACE signaling in endothelial cells.

Amino Acid Sequence↗
PubMed2004

Phosphorylation of Epstein-Barr virus ZEBRA protein at its casein kinase 2 sites mediates its ability to repress activation of a viral lytic cycle late gene by Rta.

ZEBRA, a member of the bZIP family, serves as a master switch between latent and lytic cycle Epstein-Barr virus (EBV) gene expression. ZEBRA influences the activity of another viral transactivator, Rta, in a gene-specific manner. Some early lytic cycle genes, such as BMRF1, are activated in synergy by ZEBRA and Rta. However, ZEBRA suppresses Rta's ability to activate a late gene, BLRF2. Here we show that this repressive activity is dependent on the phosphorylation state of ZEBRA. We find that two residues of ZEBRA, S167 and S173, that are phosphorylated by casein kinase 2 (CK2) in vitro are also phosphorylated in vivo. Inhibition of ZEBRA phosphorylation at the CK2 substrate motif, either by serine-to-alanine substitutions or by use of a specific inhibitor of CK2, abolished ZEBRA's capacity to repress Rta activation of the BLRF2 gene, but did not alter its ability to initiate the lytic cycle or to synergize with Rta in activation of the BMRF1 early-lytic-cycle gene. These studies illustrate how the phosphorylation state of a transcriptional activator can modulate its behavior as an activator or repressor of gene expression. Phosphorylation of ZEBRA at its CK2 sites is likely to play an essential role in proper temporal control of the EBV lytic life cycle.

Casein Kinase II↗
PubMed2002

Role for Hes1-induced phosphorylation in Groucho-mediated transcriptional repression.

Transcriptional corepressors of the Groucho/transducin-like Enhancer of split (Gro/TLE) family regulate a number of developmental pathways in both invertebrates and vertebrates. They form transcription repression complexes with members of several DNA-binding protein families and participate in the regulation of the expression of numerous genes. Despite their pleiotropic roles, little is known about the mechanisms that regulate the functions of Gro/TLE proteins. It is shown here that Gro/TLEs become hyperphosphorylated in response to neural cell differentiation and interaction with the DNA-binding cofactor Hairy/Enhancer of split 1 (Hes1). Hyperphosphorylation of Gro/TLEs is correlated with a tight association with the nuclear compartment through interaction with chromatin, suggesting that hyperphosphorylated Gro/TLEs may mediate transcriptional repression via chromatin remodeling mechanisms. Pharmacological inhibition of protein kinase CK2 reduces the Hes1-induced hyperphosphorylation of Gro/TLEs and causes a decrease in the chromatin association of the latter. Moreover, the transcription repression activity of Gro/TLEs is reduced by protein kinase CK2 inhibition. Consistent with these observations, Gro/TLEs are phosphorylated in vitro by purified protein kinase CK2. Taken together, these results implicate protein kinase CK2 in Gro/TLE functions. They suggest further that this kinase is involved in a hyperphosphorylation mechanism activated by Hes1 that promotes the transcription repression functions of Hes1-Gro/TLE protein complexes.

Animals↗
PubMed2005

The pleckstrin homology domain-containing protein CKIP-1 is involved in regulation of cell morphology and the actin cytoskeleton and interaction with actin capping protein.

CKIP-1 is a pleckstrin homology domain-containing protein that interacts with protein kinase CK2. To elucidate the functions of CKIP-1, we generated human osteosarcoma cell lines with tetracycline-regulated expression of Flag-CKIP-1. Flag-CKIP-1 expression resulted in distinct changes in cellular morphology. Therefore, we examined the actin profile by immunofluorescence, quantitative measurement of phalloidin binding, and immunoblot analysis. These studies demonstrate that Flag-CKIP-1 expression resulted in increases in F-actin staining and protein levels of beta-actin. To elucidate the mechanisms behind the observed phenotype, we utilized tandem affinity purification to isolate CKIP-1 interacting proteins. Mass spectrometry analysis led to the identification of the actin capping protein subunits, CPalpha and CPbeta, as novel CKIP-1 interaction partners. Interactions were confirmed by coimmunoprecipitation and by colocalization. Furthermore, we demonstrate that Ser9 of CPalpha is phosphorylated by protein kinase CK2 in vitro, that CPalpha is phosphorylated in vivo, and that treatment with a CK2-specific inhibitor results in a decrease in CPalpha phosphorylation. Finally, we demonstrate that CKIP-1 and CK2 inhibit the activity of actin capping protein at the barbed ends of actin filaments. Overall, our results are consistent with CKIP-1 playing a role in the regulation of the actin cytoskeleton through its interactions with actin capping protein.

Actin Capping Proteins↗
PubMed2003

5,6-Dichloro-ribifuranosylbenzimidazole- and apigenin-induced sensitization of colon cancer cells to TNF-alpha-mediated apoptosis.

Tumor necrosis factor-alpha (TNF-alpha) is a multifunctional cytokine involved in the expression of many genes integral to the inflammatory response. In addition, it activates both apoptotic and survival pathways, the latter being mediated through the activation of the transcription factor nuclear factor-kappaB (NF-kappaB). Protein kinase CK2, a serine-threonine kinase that is universally upregulated in human malignancies, may be involved at multiple levels in this process. However, its role in mediating a survival response within colon cancer cells remains incompletely understood. Here we report that inhibition of CK2 in HCT-116 and HT-29 cells with the use of two specific CK2 inhibitors, 5,6-dichloro-ribifuranosylbenzimidazole (DRB) and apigenin, effected a synergistic reduction in cell survival when used in conjunction with TNF-alpha. Furthermore, there was a demonstrable synergistic reduction in colony formation in soft agar with the use of the same combinations. Western blot analysis showed that poly-ADP ribose polymerase and procaspase-3 cleavage complemented the fluorescence-activated cell sorter analysis findings of significantly increased subdiploid DNA-containing cell populations using these conditions. Remarkably, these events occurred in the absence of any reduction in the expression of the Bcl-2 family members Bcl-2, Mcl-1, and Bcl-xL or any change in the proapoptotic molecules Bad or Bax. One-hybrid NF-kappaB promoter assays utilizing a Gal4-p65 transactivation domain construct revealed that the TNF-induced transactivation was inhibited by both DRB and apigenin. This was associated with a concomitant reduction in the expression of a recognized anti-apoptotic NF-kappaB target, manganese superoxide dismutase, demonstrated by Q-PCR. Our findings indicate a potentially novel strategy for the treatment of colon cancer, one that targets CK2 simultaneous with TNF-alpha administration.

Antineoplastic Agents↗
PubMed2004

Antitumor effect of a novel proapoptotic peptide that impairs the phosphorylation by the protein kinase 2 (casein kinase 2).

Protein Kinase (casein kinase 2, CK2) is a serine-threonine kinase that is frequently dysregulated in many human tumors. Therefore we hypothesized that peptides capable of binding to the CK2 acidic domain may exhibit potential anticancer properties. By screening a random cyclic peptide phage display library, we have identified a novel peptide, P15, that abrogated CK2 phosphorylation by blocking the substrate in vitro. To verify its potential antineoplastic effect, P15 was fused to the cell-penetrating peptide derived from the HIV-Tat protein. Interestingly, P15-Tat induced apoptosis as evidenced by rapid caspase activation and cellular cytotoxicity in a variety of tumor cell lines. Furthermore, direct injection of P15-Tat into C57BL6 mice bearing day 7-established solid tumors, resulted in substantial regression of the tumor mass. Our findings describe a new proapoptotic cyclic peptide that blocks the CK2 phosphorylation and exhibits antitumor effect in vivo, indicating that the P15 peptide may potentially be used clinically to treat solid tumors or as an adjuvant for cancer therapy.

Animals↗
PubMed2006

Transforming growth factor-beta-induced expression of the apolipoprotein E gene requires c-Jun N-terminal kinase, p38 kinase, and casein kinase 2.

OBJECTIVE: The cytokine transforming growth factor-beta (TGF-beta) and apolipoprotein E (apoE) play potent antiatherogenic roles. Despite such importance, the mechanisms underlying the regulation of apoE expression by TGF-beta have not been characterized and were therefore investigated. METHODS AND RESULTS: Using THP-1 cell line as a model system, with key findings confirmed in primary cultures, we show that TGF-beta induces the expression of apoE, and this is prevented by pharmacological inhibitors of c-Jun N-terminal kinase (JNK), p38 kinase, and casein kinase 2 (CK2). In support for an important role for these pathways, TGF-beta activates JNK, p38 kinase, and CK2, and dominant-negative (DN) forms of these proteins inhibit the cytokine-induced apoE expression. TGF-beta also increases the phosphorylation and expression of c-Jun, a downstream target for JNK action and a component of activator protein-1 (AP-1), and DN c-Jun inhibits the induction of apoE expression in response to the cytokine. AP-1 DNA binding was also induced by TGF-beta, and the action of p38 kinase, JNK, and CK2 converged on the activation of c-Jun/AP-1. CONCLUSIONS: These studies reveal a novel role for JNK, p38 kinase, CK2, and c-Jun/AP-1 in the TGF-beta-induced expression of apoE.

Apolipoproteins E↗
PubMed2006

Estrogen modulation of MgATPase activity of nonmuscle myosin-II-B filaments.

The study tested the hypothesis that estrogen controls epithelial paracellular resistance through modulation of myosin. The objective was to understand how estrogen modulates nonmuscle myosin-II-B (NMM-II-B), the main component of the cortical actomyosin in human epithelial cervical cells. Experiments used human cervical epithelial cells CaSki as a model, and end points were NMM-II-B phosphorylation, filamentation, and MgATPase activity. The results were as follows: 1) treatment with estrogen increased phosphorylation and MgATPase activity and decreased NMM-II-B filamentation; 2) estrogen effects could be blocked by antisense nucleotides for the estrogen receptor-alpha and by ICI-182,780, tamoxifen, and the casein kinase-II (CK2) inhibitor, 5,6-dichloro-1-beta-(D)-ribofuranosylbenzimidazole and attenuated by AG1478 and PD98059 (inhibitors of epithelial growth factor receptor and ERK/MAPK) but not staurosporine [blocker of protein kinase C (PKC)]; 3) treatments with the PKC activator sn-1,2-dioctanoyl diglyceride induced biphasic effect on NMM-II-B MgATPase activity: an increase at 1 nm to 1 microM and a decrease in activity at more than 1 microM; 4) sn-1,2-dioctanoyl diglyceride also decreased NMM-II-B filamentation in a monophasic and saturable dose dependence (EC(50) 1-10 microM); 5) when coincubated directly with purified NMM-II-B filaments, both CK2 and PKC decreased filamentation and increased MgATPase activity; 6) assays done on disassembled NMM-II-B filaments showed MgATPase activity in filaments obtained from estrogen-treated cells but not estrogen-depleted cells; and 7) incubations in vitro with CK2, but not PKC, facilitated MgATPase activity, even in disassembled NMM-II-B filaments. The results suggest that estrogen, in an effect mediated by estrogen receptor-alpha and CK2 and involving the epithelial growth factor receptor and ERK/MAPK cascades, increases NMM-II-B MgATPase activity independent of NMM-II-B filamentation status.

Biological Transport↗
PubMed2006

BID, an interaction partner of protein kinase CK2alpha.

Recombinant murine BID protein was used as an in vitro substrate for the CK2 holoenzyme and the catalytic CK2alpha subunit. The results obtained show that BID can only serve as a substrate for the catalytic CK2alpha subunit. Phosphorylation of BID using the CK2 holoenzyme was only possible in the presence of polylysine, supporting the notion that BID behaves similarly to calmodulin. Co-immunoprecipitation of BID and CK2 subunits revealed that BID is preferentially associated with the CK2alpha subunit. Enzyme kinetic analyses yielded a Km value for BID that is a level of magnitude lower than that measured for casein and the synthetic peptide, suggesting more specific and tight binding of BID to CK2alpha. In contrast are the Vmax values observed, with a significantly higher phosphorylation rate measured for casein and the synthetic peptide than for BID. When BID was phosphorylated by polylysine-stimulated CK2 holoenzyme prior to caspase-8 cleavage, the formation of tC-BID was reduced in comparison to treatment with caspase-8 in the absence of protein kinase. Mass spectrometric analysis of BID phosphorylated by CK2alpha before and after cleavage with caspase-8 showed phosphorylation of residues Thr58 and Ser76.

Amino Acid Sequence↗
PubMed2004

Regulation of the NMDA receptor complex and trafficking by activity-dependent phosphorylation of the NR2B subunit PDZ ligand.

Interactions between NMDA receptors (NMDARs) and the PDZ [postsynaptic density-95 (PSD-95)/Discs large/zona occludens-1] domains of PSD-95/SAP90 (synapse-associated protein with a molecular weight of 90 kDa) family proteins play important roles in the synaptic targeting and signaling of NMDARs. However, little is known about the mechanisms that regulate these PDZ domain-mediated interactions. Here we show that casein kinase II (CK2) phosphorylates the serine residue (Ser1480) within the C-terminal PDZ ligand (IESDV) of the NR2B subunit of NMDAR in vitro and in vivo. Phosphorylation of Ser1480 disrupts the interaction of NR2B with the PDZ domains of PSD-95 and SAP102 and decreases surface NR2B expression in neurons. Interestingly, activity of the NMDAR and Ca2+/calmodulin-dependent protein kinase II regulates CK2 phosphorylation of Ser1480. Furthermore, CK2 colocalizes with NR1 and PSD-95 at synaptic sites. These results indicate that activity-dependent CK2 phosphorylation of the NR2B PDZ ligand regulates the interaction of NMDAR with PSD-95/SAP90 family proteins as well as surface NMDAR expression and may be a critical mechanism for modulating excitatory synaptic function and plasticity.

2-Amino-5-phosphonovalerate↗