PTP-S2, a nuclear tyrosine phosphatase, is phosphorylated and excluded from condensed chromosomes during mitosis

PTP-S2 is a tyrosine specific protein phosphatase that binds to DNA and is localized to the nucleus in association with chromatin. It plays a role in the regulation of cell proliferation. Here we show that the subcellular distribution of this protein changes during cell division. While PTP-S2 was localized exclusively to the nucleus in interphase cells, during metaphase and anaphase it was distributed throughout the cytoplasm and excluded from condensed chromosomes. At telophase PTP-S2 began to associate with chromosomes and at cytokinesis it was associated with chromatin in the newly formed nucleus. It was hyperphosphorylated and showed retarded mobility in cells arrested in metaphase. In vitro experiments showed that it was phosphorylated by CK2 resulting in mobility shift. Using a deletion mutant we found that CK2 phosphorylated PTP-S2 in the C-terminal non-catalytic domain. A heparin sensitive kinase from mitotic cell extracts phosphorylated PTP-S2 resulting in mobility shift. These results are consistent with the suggestion that during metaphase PTP-S2 is phosphorylated (possibly by CK2 or a CK2-like enzyme), resulting in its dissociation from chromatin.     

Identification of human and mouse HIRA-interacting protein-5 (HIRIP5), two mammalian representatives in a family of phylogenetically conserved proteins with a role in the biogenesis of Fe/S proteins

The human HIRA protein is encoded from a region of chromosome 22q that is critical for the DiGeorge syndrome and the velocardiofacial syndrome. We have previously reported that it directly interacts with core histones, with a novel histone-binding protein, HIRIP3, and during mouse embryogenesis, with the developmentally regulated homeodomain protein Pax3, suggesting a promoter-targeted function at the chromatin level. We here report on HIRA-interacting protein 5 (HIRIP5), a small acidic protein that interacted with HIRA in a double-hybrid screen performed in yeast and in in vitro protein interaction experiments. HIRIP5 has highly conserved homologs in both prokaryotes and eukaryotes, including the NFU1 gene product which has been implicated in iron metabolism in mitochondria of the yeast Saccharomyces cerevisiae. By radioactive in situ hybridization, the HIRIP5 gene was mapped to the 2p13-p15 chromosomal region, separate from any region previously associated with DiGeorge syndrome.     

Phosphorylation by protein kinase CK2 changes the DNA binding properties of the human chromatin protein DEK

We have examined the posttranslational modification of the human chromatin protein DEK and found that DEK is phosphorylated by the protein kinase CK2 in vitro and in vivo. Phosphorylation sites were mapped by quadrupole ion trap mass spectrometry and found to be clustered in the C-terminal region of the DEK protein. Phosphorylation fluctuates during the cell cycle with a moderate peak during G(1) phase. Filter binding assays, as well as Southwestern analysis, demonstrate that phosphorylation weakens the binding of DEK to DNA. In vivo, however, phosphorylated DEK remains on chromatin. We present evidence that phosphorylated DEK is tethered to chromatin throughout the cell cycle by the un- or underphosphorylated form of DEK.     

Phosphorylation of maize and Arabidopsis HMGB proteins by protein kinase CK2alpha

In plants, a variety of chromatin-associated high mobility group (HMG) proteins belonging to the HMGB family have been identified. We have examined the phosphorylation of the HMGB proteins from the monocotyledonous plant maize and the dicotyledonous plant Arabidopsis by protein kinase CK2alpha. Maize CK2alpha phosphorylates the maize HMGB1 and HMGB2/3 proteins and the Arabidopsis HMGB1, HMGB2/3, and HMGB4 proteins. Maize HMGB4 and HMGB5 and Arabidopsis HMGB5 are not phosphorylated by CK2alpha. Depending on the HMGB protein up to five amino acid residues are phosphorylated in the course of the phosphorylation reaction. The HMGB1 proteins from both plants are markedly more slowly phosphorylated by CK2alpha than the other HMGB substrate proteins, indicating that certain HMGB proteins are clearly preferred substrates for CK2alpha. The rate of the phosphorylation reaction appears to be related to the ease of interaction between CK2alpha and the HMGB proteins, as indicated by chemical cross-linking experiments. MALDI/TOF mass spectrometry analyses demonstrate that the HMGB1 and HMGB2/3 proteins occur in various phosphorylation states in immature maize kernels. Thus, HMGB1 exists as monophosphorylated, double-phosphorylated, triple-phosphorylated, and tetraphosphorylated protein in kernel tissue, and the tetraphosphorylated form is the most abundant version. The observed in vivo phosphorylation states indicate that protein kinase(s) other than CK2alpha contribute(s) to the modification of the plant HMGB proteins. The fact that the HMGB proteins are phosphorylated to various extents reveals that the existence of differentially modified forms increases the number of distinct HMGB protein variants in plant chromatin that may be adapted to certain functions.     

Identification and characterization of a novel testis-specific gene CKT2, which encodes a substrate for protein kinase CK2

Protein kinase CK2 is a serine/threonine kinase known to phosphorylate numerous substrates. CK2 is implicated in several physiologic and pathologic processes, particularly in cancer biology. CK2 is comprised of several subunits, including CK2α, CK2α′ and CK2β. Inactivation of CK2α′ leads to chromatin degeneration of germ cells, resulting in male sterility. To identify additional targets of CK2α′ in testes and to determine the role of CK2α′ in germ cell nuclear integrity, GST pull-down and protein–protein interaction assays were conducted. A novel testis-specific gene, CKT2 (CK2 Target protein 2), was found whose product interacts with and is phosphorylated by CK2 in vitro and in vivo. CKT2 is a 30.2 kDa protein with one coiled-coil domain and six putative phosphorylation sites. High expression of CKT2 correlated with chromatin condensation of spermatids in murine testes. Findings reported herein demonstrate that CKT2 is a target protein of native CK2α′ in testes and suggest that CKT2 plays a role in chromatin regulation of male germ cells.     

Mitotic phosphorylation of DNA topoisomerase II alpha by protein kinase CK2 creates the MPM-2 phosphoepitope on Ser-1469

DNA topoisomerase II alpha is required for chromatin condensation during prophase. This process is temporally linked with the appearance of mitosis-specific phosphorylation sites on topoisomerase IIalpha including one recognized by the MPM-2 monoclonal antibody. We now report that the ability of mitotic extracts to create the MPM-2 epitope on human topoisomerase II alpha is abolished by immunodepletion of protein kinase CK2. Furthermore, the MPM-2 phosphoepitope on topoisomerase II alpha can be generated by purified CK2. Phosphorylation of C-truncated topoisomerase II alpha mutant proteins conclusively shows, that the MPM-2 epitope is present in the last 163 amino acids. Use of peptides containing all conserved CK2 consensus sites in this region indicates that only the peptide containing Arg-1466 to Ala-1485 is able to compete with topoisomerase II alpha for binding of the MPM-2 antibody. Replacement of Ser-1469 with Ala abolishes the ability of the phosphorylated peptide to bind to the MPM-2 antibody while a peptide containing phosphorylated Ser-1469 binds tightly. Surprisingly, the MPM-2 phosphoepitope influences neither the catalytic activity of topoisomerase II alpha nor its ability to form molecular complexes with CK2 in vitro. In conclusion, we have identified protein kinase CK2 as a new MPM-2 kinase able to phosphorylate an important mitotic protein, topoisomerase II alpha, on Ser-1469.     

Chemical dissection of the APC Repeat 3 multistep phosphorylation by the concerted action of protein kinases CK1 and GSK3

A crucial event in machinery controlled by Wnt signaling is the association of beta-catenin with the adenomatous polyposis coli (APC) protein, which is essential for the degradation of beta-catenin and requires the multiple phosphorylation of APC at six serines (1501, 1503, 1504, 1505, 1507, and 1510) within its repeat three (R3) region. Such a phosphorylation is believed to occur by the concerted action of two protein kinases, CK1 and GSK3, but its mechanistic aspects are a matter of conjecture. Here, by combining the usage of variably phosphorylated peptides reproducing the APC R3 region and Edman degradation assisted localization of residues phosphorylated by individual kinases, we show that the process is initiated by CK1, able to phosphorylate S1510 and S1505, both specified by non-canonical determinants. Phosphorylation of S1505 primes subsequent phosphorylation of S1501 by GSK3. In turn, phospho-S1501 triggers the hierarchical phosphorylation of S1504 and S1507 by CK1. Once phosphorylated, S1507 primes the phosphorylation of both S1510 and S1503 by CK1 and GSK3, respectively, thus completing all six phosphorylation steps. Our data also rule out the intervention of CK2 despite the presence of a potential CK2 phosphoacceptor site, S1510LDE, in the R3 repeat. S1510 is entirely unaffected by CK2, while it is readily phosphorylated even in the unprimed peptide by CK1delta but not by CK1gamma. This discloses a novel motif significantly different from non-canonical sequences phosphorylated by CK1 in other proteins, which appears to be specifically recognized by the delta isoform of CK1.     

Localization of X-ray cross complementing gene 1 protein in the nuclear matrix is controlled by casein kinase II-dependent phosphorylation in response to oxidative damage

Base excision repair/single strand break repair (BER/SSBR) of damaged DNA is a highly efficient process. X-ray cross complementing protein 1 (XRCC1) functions as a key scaffold protein for BER/SSBR factors. Recent work has shown that XRCC1 forms dense foci at sites of DNA damage in a manner dependent on casein kinase II (CK2) phosphorylation. To investigate the mechanism underlying foci formation, we analyzed the subnuclear localization and phosphorylation status of XRCC1 during the repair process by biochemical fractionation of HeLa cellular proteins. The localization was also verified by in situ extraction of the fixed cells. In unchallenged cells, XRCC1 was primarily found in the chromatin fraction in a highly phosphorylated form; in addition, a minor population (10–15%) existed in the nuclear matrix (NM) with no or marginal phosphorylation.After hydrogen peroxide treatment, hyperphosphorylated XRCC1 appeared in the NM and accordingly, those in the chromatin fraction decreased. Foci formation and changes in XRCC1 distribution could be abolished by the knockdown of CK2, the expression of a non-phosphorylatable version of XRCC1, or the inhibition of poly-ADP ribosylation at the damage sites. Other BER factors, like DNA polymerase β, were also found to accumulate in the NM after hydrogen peroxide-induced DNA damage, although its association with the NM seemed relatively weak. Our results suggest that the constitutive phosphorylation of XRCC1 in the chromatin and its DNA damage-induced recruitment to the NM are critical for foci formation, and that the core reactions of BER/SSBR may occur in the NM.     

Phosphorylation of apolipoprotein-E at an atypical protein kinase CK2 PSD/E site in vitro

Apolipoprotein-E (apoE) plays an important role in neuronal lipid transport and is thought to stabilize microtubules by preventing tau hyperphosphorylation. ApoE is also associated with insoluble amyloid detected in Alzheimer disease brain lesions. The apoE C-terminal shares several physicochemical features with alpha-synuclein, another neuronal apolipoprotein-like protein. Alpha-synuclein is phosphorylated by protein kinase CK2 (CK2) at an atypical PSD/E motif in vivo and in vitro. We identified a similar PSD/E motif in apoE and therefore investigated its potential phosphorylation by CK2 in vitro. When a [(32)P]-labeling approach was used, CK2 readily phosphorylated purified human apoE as well as recombinant forms of human apoE3 and apoE4. Using liquid chromatography mass spectrometry techniques, we mapped the major apoE CK2 phosphorylation site to Ser296 within the apoE PSD/E motif. We also found that apoE potently activated CK2 as demonstrated by increased CK2beta subunit autophosphorylation and by increased phosphorylation of tau when the latter was added to the kinase reaction mixtures. Other proteins such as apolipoprotein A-I and albumin did not effectively activate CK2. The phosphorylation of apoE by CK2 as well as the activation of CK2 by apoE may be relevant in vivo where apoE, CK2, and tau are co-localized with additional CK2 targets on neuronal microtubules.     

Properties of single-stranded DNA-binding proteins (SSB-proteins) from chromatin and nonchromatin fractions of Ehrlich ascites tumour: phosphorylation enhances the affinity of SSB-proteins for single-stranded DNA.

To assess the possible functional role of single-strand DNA-binding (SSB) proteins in eucaryotic cell, a comparative study was made of SSB-proteins isolated from chromatin and the nonchromatin fractions of Ehrlich ascites tumour (EAT) cells. No appreciable differences between the two groups could be found either in SDS-gel electrophoretic patterns or in the ssDNA-binding capacity and stimulation of DNA replication in permeable EAT cells. However, the chromatin SSB-proteins incorporated 1.4-times more labelled phosphate in vivo; phosphate assays in the isolated chromatin and nonchromatin SSB-proteins yielded ca. 3 and 2 moles Pi/mole protein, respectively. Both preparations could be further phosphorylated in vitro with Ca-phospholipid-dependent protein kinase and the catalytic subunit of cAMP-dependent protein kinase, but the non-chromatin proteins were phosphorylated to a greater degree. In parallel with phosphorylation, the SSB-proteins displayed stronger binding to ssDNA cellulose. Phosphorylation may thus be a means of regulating the functions of SSB-proteins, in particular their interaction with chromatin.     

Phosphorylated synaphin/complexin found in the brain exhibits enhanced SNARE complex binding

The cytosolic protein synaphin/complexin critically regulates fast neurotransmitter release at the synapse by binding to SNARE complex. However, the exact mechanism of its action remains unclear, and very little is known about how it is physiologically regulated. Here we show that synaphins (Syps) 1 and 2 can be phosphorylated in vitro by protein kinase CK2 (CK2). The only phosphorylation site by CK2 was serine-115 (Ser-115) of Syps 1 and 2. Syps 1 and 2 exhibited higher affinities to native and recombinant SNARE complexes when phosphorylated at Ser-115. We found Ser-115-phosphorylated Syp 1 (pS115-Syp 1) in the cytosolic fraction of the rat brain using polyclonal antibody specific to pS115-Syps 1 and 2. These results suggest that the activity of Syp is regulated by CK2 phosphorylation of its Ser-115 in vivo. The phosphorylation may provide a new route for modulating fast neurotransmitter release.     

CK2 phosphorylation of SAG at Thr10 regulates SAG stability,but not its E3 ligase activity

Sensitive to Apoptosis Gene (SAG), a RING component of SCF E3 ubiquitin ligase, was shown to be phosphorylated by protein kinase CK2 at the Thr10 residue. It is, however, unknown whether this phosphorylation is stress-responsive or whether the phosphorylation changes its E3 ubiquitin ligase activity. To address these, we made a specific antibody against the phosphor-SAG^Thr10. Transient transfection experiment showed that SAG was phosphorylated at Thr10 which can be significantly inhibited by TBB, a relatively specific inhibitor of protein kinase CK2. To determine whether this SAG phosphorylation is stress-responsive, we defined a chemical-hypoxia condition in which SAG and CK2 were both induced. Under this condition, we failed to detect SAG phosphorylation at Thr10, which was readily detected, however, in the presence of MG132, a proteasome inhibitor, suggesting that the phosphorylated SAG has undergone a rapid degradation. To further define this, we made two SAG mutants, SAG-T10A which abolishes the SAG phosphorylation and SAG-T10E, which mimics the constitutive SAG phosphorylation. The half-life study revealed that indeed, SAG-T10E has a much shorter protein half-life (2 h), as compared to wild-type SAG (10 h). Again, rapid degradation of SAG-T10E in cells can be blocked by MG132. Thus, it appears that CK2-induced SAG phosphorylation at Thr10 regulates its stability through a proteasome-dependent pathway. Immunocytochemistry study showed that SAG as well as its phosphorylation mutants, was mainly localized in nucleus and lightly in cytoplasm. Hypoxia condition did not change their sub-cellular localization. Finally, an in vitro ubiqutination assay showed that SAG mutation at Thr10 did not change its E3 ligase activity when complexed with cullin-1. These studies suggested that CK2 might regulate SAG-SCF E3 ligase activity through modulating SAG’s stability, rather than its enzymatic activity directly.     

In vivo and in vitro phosphorylation of Candida albicans 20S proteasome

In this paper we demonstrate that the Candida albicans 20S proteasome is in vivo phosphorylated and is a good in vitro substrate (S(0.5) 14nM) of homologous protein kinase CK2 (CK2). We identify alpha6/C2, alpha3/C9, and alpha5/Pup2 proteasome subunits as the main in vivo phosphorylated and in vitro CK2-phosphorylatable proteasome components. In vitro phosphorylation by homologous CK2 holoenzyme occurs only in the presence of polylysine, a characteristic that distinguishes the yeast proteasomes from mammalian proteasomes which are phosphorylated by CK2 in the absence of polycations. The major in vivo phosphate acceptor is the alpha3/C9 subunit, being phosphorylated in serine, both in vivo and in vitro. The phosphopeptides generated by endoproteinase Glu-C digestion from in vivo labeled alpha3/C9 subunit, from in vitro phosphorylation by homologous CK2 holoenzyme, and from the recombinant alpha3/C9 subunit phosphorylated by recombinant human CK2-alpha subunit are identical, suggesting that CK2 is likely responsible for in vivo phosphorylation of this subunit. Direct mutational analysis shows that serine 248 is the residue of the alpha3/C9 subunit phosphorylated by CK2. The in vitro stoichiometry of phosphorylation of the alpha6/C2 and alpha3/C9 proteasome subunits by CK2 can be estimated as 0.7-0.8 and 0.4-0.5 mol of phosphate per mole of subunit, respectively. These results are consistent with the relative abundance of the unphosphorylated and phosphorylated isoforms of these subunits present in the purified 20S proteasome preparation. Our demonstration of phosphorylation of C. albicans proteasome suggests that phosphorylation might be a general mechanism of regulation of proteasome activity.     

Protein kinase CK2 differentially phosphorylates maize chromosomal high mobility group B (HMGB) proteins modulating their stability and DNA interactions.

The high mobility group (HMG) proteins of the HMGB family are architectural factors in eukaryotic chromatin, which are involved in the regulation of various DNA-dependent processes. We have examined the post-translational modifications of five HMGB proteins from maize suspension cultured cells, revealing that HMGB1 and HMGB2/3, but not HMGB4 and HMGB5, are phosphorylated by protein kinase CK2. The phosphorylation sites have been mapped to the acidic C-terminal domains by analysis of tryptic peptides derived from HMGB1 and HMGB2/3 using nanospray ion trap mass spectrometry. In native HMGB1, Ser(149) is constitutively phosphorylated, whereas Ser(133) and Ser(136) are differentially phosphorylated. The functional significance of the CK2-mediated phosphorylation of HMGB proteins was analyzed by circular dichroism measurements showing that the phosphorylation increases the thermal stability of the HMGB proteins. Electrophoretic mobility shift assays demonstrate that the phosphorylation reduces the affinity of the HMGB proteins for linear DNA. The specific recognition of DNA minicircles is not affected by the phosphorylation, but a different pattern of protein-DNA complexes is formed. Collectively, these findings show that phosphorylation of residues within the acidic C-terminal domain of the HMGB proteins can modulate protein stability and the DNA binding properties of the HMGB proteins.     

Phosphorylation and regulation of a G protein-coupled receptor by protein kinase CK2

We demonstrate a role for protein kinase casein kinase 2 (CK2) in the phosphorylation and regulation of the M3-muscarinic receptor in transfected cells and cerebellar granule neurons. On agonist occupation, specific subsets of receptor phosphoacceptor sites (which include the SASSDEED motif in the third intracellular loop) are phosphorylated by CK2. Receptor phosphorylation mediated by CK2 specifically regulates receptor coupling to the Jun-kinase pathway. Importantly, other phosphorylation-dependent receptor processes are regulated by kinases distinct from CK2. We conclude that G protein-coupled receptors (GPCRs) can be phosphorylated in an agonist-dependent fashion by protein kinases from a diverse range of kinase families, not just the GPCR kinases, and that receptor phosphorylation by a defined kinase determines a specific signalling outcome. Furthermore, we demonstrate that the M3-muscarinic receptor can be differentially phosphorylated in different cell types, indicating that phosphorylation is a flexible regulatory process where the sites that are phosphorylated, and hence the signalling outcome, are dependent on the cell type in which the receptor is expressed.