Casein kinase 2 phosphorylates and induces the SALL2 tumor suppressor degradation in colon cancer cells.

Spalt-like proteins are Zinc finger transcription factors from Caenorhabditis elegans to vertebrates, with critical roles in development. In vertebrates, four paralogues have been identified (SALL1-4), and SALL2 is the family's most dissimilar member. SALL2 is required during brain and eye development. It is downregulated in cancer and acts as a tumor suppressor, promoting cell cycle arrest and cell death. Despite its critical functions, information about SALL2 regulation is scarce. Public data indicate that SALL2 is ubiquitinated and phosphorylated in several residues along the protein, but the mechanisms, biological consequences, and enzymes responsible for these modifications remain unknown. Bioinformatic analyses identified several putative phosphorylation sites for Casein Kinase II (CK2) located within a highly conserved C-terminal PEST degradation motif of SALL2. CK2 is a serine/threonine kinase that promotes cell proliferation and survival and is often hyperactivated in cancer. We demonstrated that CK2 phosphorylates SALL2 residues S763, T778, S802, and S806 and promotes SALL2 degradation by the proteasome. Accordingly, pharmacological inhibition of CK2 with Silmitasertib (CX-4945) restored endogenous SALL2 protein levels in SALL2-deficient breast MDA-MB-231, lung H1299, and colon SW480 cancer cells. Silmitasertib induced a methuosis-like phenotype and cell death in SW480 cells. However, the phenotype was significantly attenuated in CRISPr/Cas9-mediated SALL2 knockout SW480 cells. Similarly, Sall2-deficient tumor organoids were more resistant to Silmitasertib-induced cell death, confirming that SALL2 sensitizes cancer cells to CK2 inhibition. We identified a novel CK2-dependent mechanism for SALL2 regulation and provided new insights into the interplay between these two proteins and their role in cell survival and proliferation.

Protein kinase CK2 in health and disease: From birth to death: the role of protein kinase CK2 in the regulation of cell proliferation and survival.

Protein kinase CK2 is a serine/threonine kinase with a multitude of protein substrates. The enzyme is ubiquitously expressed in mammalian cells, where it functions in a variety of cellular processes, including cell cycle progression, apoptosis, transcription, and viral infection. While the importance of CK2 in the mammalian life cycle is undisputed, the regulatory mechanisms coordinating its numerous functions remain elusive. In this review, we focus on the various roles of CK2 in the mammalian cell, with particular attention on its functions through the stages of the cell cycle and during the decision to undergo cell death. We highlight how these roles are controlled in part through direct transcriptional regulation by CK2, and how the constitutive activity of CK2 can be hijacked in the case of viral infection. Finally, we discuss possible ways in which these functions are integrated to allow the cell to respond appropriately in the presence of multiple signals.

Functionally important residues in the peptidyl-prolyl isomerase Pin1 revealed by unigenic evolution.

Pin1 is a phosphorylation-dependent member of the parvulin family of peptidyl-prolyl isomerases exhibiting functional conservation between yeast and man. To perform an unbiased analysis of the regions of Pin1 essential for its functions, we generated libraries of randomly mutated forms of the human Pin1 cDNA and identified functional Pin1 alleles by their ability to complement the Pin1 homolog Ess1 in Saccharomyces cerevisiae. We isolated an extensive collection of functional mutant Pin1 clones harboring a total of 356 amino acid substitutions. Surprisingly, many residues previously thought to be critical in Pin1 were found to be altered in this collection of functional mutants. In fact, only 17 residues were completely conserved in these mutants and in Pin1 sequences from other eukaryotic organisms, with only two of these conserved residues located within the WW domain of Pin1. Examination of invariant residues provided new insights regarding a phosphate-binding loop that distinguishes a phosphorylation-dependent peptidyl-prolyl isomerase such as Pin1 from other parvulins. In addition, these studies led to an investigation of residues involved in catalysis including C113 that was previously implicated as the catalytic nucleophile. We demonstrate that substitution of C113 with D does not compromise Pin1 function in vivo nor does this substitution abolish catalytic activity in purified recombinant Pin1. These findings are consistent with the prospect that the function of residue 113 may not be that of a nucleophile, thus raising questions about the model of nucleophilic catalysis. Accordingly, an alternative catalytic mechanism for Pin1 is postulated.

Inducible expression of the regulatory protein kinase CK2beta subunit: incorporation into complexes with catalytic CK2 subunits and re-examination of the effects of CK2beta on cell proliferation.

The regulatory subunit of protein kinase CK2, designated CK2beta, exists both free in cells and in complexes with the CK2 catalytic subunits. Growing evidence suggests that CK2beta has functions dependent and independent of the CK2 catalytic subunits. There have been indications that CK2beta has functions associated with DNA damage responses and in the control of cell proliferation. For example, transient and stable constitutive overexpression of CK2beta in mammalian cells was previously shown to perturb cell cycle progression and to attenuate proliferation. To systematically investigate the molecular mechanisms responsible for these effects of CK2beta on cell proliferation, we generated human osteosarcoma U2OS cell lines with tetracycline-regulated expression of CK2beta. Increased expression of CK2beta results in increases in total cellular CK2 activity, but no changes in cell cycle profiles or proliferation. Furthermore, following exposure to ultraviolet radiation, p53 induction was identical regardless of the levels of CK2beta in cells. Mouse 3T3-L1 cells stably transfected with CK2beta also showed no alterations in cell proliferation. The differences between these results and those previously reported emphasize the complex nature of CK2beta and its cellular functions. Furthermore, these results indicate that increased expression of CK2beta is not by itself sufficient to effect alterations in cell proliferation.

Assembly of protein kinase CK2: investigation of complex formation between catalytic and regulatory subunits using a zinc-finger-deficient mutant of CK2beta.

Protein kinase CK2 is a tetrameric enzyme comprised of two regulatory subunits (CK2beta) and two catalytic subunits (CK2alpha and/or CK2alpha'). The crystal structure of dimeric CK2beta demonstrated that a zinc finger mediates CK2beta dimerization, therefore we constructed a mutant in which cysteine residues 109 and 114 were mutated to serine. Our objectives were to examine the effects of disrupting the zinc finger of the regulatory CK2beta subunit on CK2 tetramer assembly. Examination of this zinc-finger-deficient mutant of CK2beta using a yeast two-hybrid assay demonstrates that the mutant fails to form CK2beta homodimers. In order to extend these studies, we co-transfected COS-7 cells with epitope-tagged constructs and performed co-immunoprecipitation assays. The results from these studies demonstrate that the mutant fails to form CK2beta homodimers and fails to interact with catalytic CK2 subunits. Furthermore, we demonstrate that the mutant CK2beta is not appreciably phosphorylated in cells. Using in vitro binding assays, we demonstrated that the mutant CK2beta protein fails to interact with glutathione S-transferase-CK2alpha'. Finally, we demonstrate that the mutant is translated at an equivalent rate to wild-type CK2beta, but is degraded much more rapidly. Overall, our results are consistent with the model that beta-beta dimerization precedes incorporation of catalytic subunits into tetrameric CK2 complexes, and that beta-beta dimerization is a prerequisite for the stable incorporation of catalytic subunits into CK2 complexes.

Tyrosine kinase-mediated serine phosphorylation of adenylyl cyclase.

Receptor tyrosine kinase (RTK) activation is associated with modulation of heptahelical receptor-stimulated adenylyl cyclase responses. The mechanisms underlying the RTK-mediated enhancement of adenylyl cyclase function remain unclear. In the present studies, we show that the tyrosine kinase-dependent enhancement of adenylyl cyclase isoform VI function parallels an enhancement in serine phosphorylation of the enzyme. This effect was mediated by both RTK activation, with IGF-1, and by tyrosine phosphatase inhibition, with sodium orthovanadate. This enhancement of adenylyl cyclase function was not attenuated by inhibitors of ERK, PKC, PKA, or PI3 kinase activity but was blunted by inhibition of endogenous p74(raf-1)() activity. To characterize the molecular site of this effect we identified multiple candidate serine residues in and adjacent to the adenylyl cyclase VI C1b catalytic region and performed serine-to-alanine site-directed mutagenesis using adenylyl cyclase VI as a template. Mutation of serine residues 603 and 608 or serine residues 744, 746, 750, and 754 attenuated both the tyrosine kinase-mediated enhancement of enzyme phosphorylation as well as the sensitization of function. Together, these data define a novel tyrosine kinase-mediated mechanism leading to serine phosphorylation of adenylyl cyclase isoform VI and the sensitization of adenylyl cyclase responsiveness.

Functional specialization of CK2 isoforms and characterization of isoform-specific binding partners.

In mammals, protein kinase CK2 has two isozymic forms of its catalytic subunit, designated CK2alpha and CK2alpha'. CK2alpha and CK2alpha' exhibit extensive similarity within their catalytic domains but have completely unrelated C-terminal sequences. To systematically examine the cellular functions of each CK2 isoform in mammalian cells, we have generated human osteosarcoma U2-OS cell lines with the expression of active or inactive versions of each CK2 isoform under the control of an inducible promoter. Examination of these cell lines provides evidence for functional specialization of CK2 isoforms at the cellular level in mammals with indications that CK2alpha' is involved in the control of proliferation and/or cell survival. To understand the molecular basis for functional differences between CK2alpha and CK2alpha', we have undertaken studies to identify proteins that interact specifically with each isoform of CK2 and could contribute to the regulation of their independent functions. A novel pleckstrin-homology domain containing protein, designated CK2-interacting protein 1 (i.e. CKIP-1) was isolated using the yeast two hybrid system as a protein that interacts with CK2alpha but not CK2alpha'. When expressed in cells as a fusion with green fluorescent protein, CKIP-1 localizes to the cell membrane and to the nucleus. In this study, we present evidence from deletion analysis of CKIP-1 suggesting that a C-terminal region containing a putative leucine zipper has a role in regulating its nuclear localization. Collectively, our data supports a model whereby CKIP-1 is a non-enzymatic regulator of CK2alpha that regulates the cellular functions of CK2alpha by targeting or anchoring CK2alpha to specific cellular localization or by functioning as an adapter to integrate CK2alpha-mediated signaling events with components of other signal transduction pathways.

Identification and characterization of CKIP-1, a novel pleckstrin homology domain-containing protein that interacts with protein kinase CK2.

The catalytic subunits of protein kinase CK2, CK2alpha and CK2alpha', are closely related to each other but exhibit functional specialization. To test the hypothesis that specific functions of CK2alpha and CK2alpha' are mediated by specific interaction partners, we used the yeast two-hybrid system to identify CK2alpha- or CK2alpha'-binding proteins. We report the identification and characterization of a novel CK2-interacting protein, designated CKIP-1, that interacts with CK2alpha, but not CK2alpha', in the yeast two-hybrid system. CKIP-1 also interacts with CK2alpha in vitro and is co-immunoprecipitated from cell extracts with epitope-tagged CK2alpha and an enhanced green fluorescent protein fusion protein encoding CKIP-1 (i.e. EGFP-CKIP-1) when they are co-expressed. CK2 activity is detected in anti-CKIP-1 immunoprecipitates performed with extracts from non-transfected cells indicating that CKIP-1 and CK2 interact under physiological conditions. The CKIP-1 cDNA is broadly expressed and encodes a protein with a predicted molecular weight of 46,000. EGFP-CKIP-1 is localized within the nucleus and at the plasma membrane. The plasma membrane localization is dependent on the presence of an amino-terminal pleckstrin homology domain. We postulate that CKIP-1 is a non-enzymatic regulator of one isoform of CK2 (i.e. CK2alpha) with a potential role in targeting CK2alpha to a particular cellular location.

The regulatory beta subunit of protein kinase CK2 mediates formation of tetrameric CK2 complexes.

Protein kinase CK2 is a tetrameric enzyme composed of two catalytic (alpha and/or alpha') subunits and two regulatory (beta) subunits. Because CK2beta is synthesized in excess of CK2alpha, we hypothesized that formation of CK2beta homodimers precedes the incorporation of the catalytic subunits of CK2 into complexes. To test this hypothesis, we cotransfected cells with two epitope-tagged variants of CK2beta. The results of these cotransfection studies demonstrate that interactions between two CK2beta subunits take place in the absence of CK2alpha. Together with results from previous biosynthetic labeling studies, these results suggest that formation of CK2beta homodimers occurs before incorporation of catalytic subunits of CK2 into CK2 complexes. We also cotransfected Cos-7 cells with a deletion fragment of CK2beta (i.e. Myc-beta1-166) together with full-length hemagglutinin (HA)-tagged CK2beta and/or CK2alpha'. Although complexes between Myc-beta1-166 and HA-beta were readily detected, we obtained no evidence of direct interactions between Myc-beta1-166 and HA-CK2alpha'. These results suggest that residues within the N-terminal 166 amino acids of CK2beta are sufficient for interactions between CK2beta subunits, whereas the C-terminal domain of CK2beta is required for complex formation with the catalytic subunits of CK2. Finally, we observed that expression of full-length HA-beta promotes phosphorylation of Myc-beta1-166 by HA-CK2alpha'.

Inducible expression of protein kinase CK2 in mammalian cells. Evidence for functional specialization of CK2 isoforms.

Protein kinase CK2 (formerly casein kinase II) exhibits elevated expression in a variety of cancers, induces lymphocyte transformation in transgenic mice, and collaborates with Ha-Ras in fibroblast transformation. To systematically examine the cellular functions of CK2, human osteosarcoma U2-OS cells constitutively expressing a tetracycline-regulated transactivator were stably transfected with a bidirectional plasmid encoding either catalytic isoform of CK2 (i.e. CK2alpha or CK2alpha') together with the regulatory CK2beta subunit in order to increase the cellular levels of either CK2 isoform. To interfere with either CK2 isoform, cells were also transfected with kinase-inactive CK2alpha or CK2alpha' (i. e. GK2alpha (K68M) or CK2alpha'(K69M)) together with CK2beta. In these cells, removal of tetracycline from the growth medium stimulated coordinate expression of catalytic and regulatory CK2 subunits. Increased expression of active forms of CK2alpha or CK2alpha' resulted in modest decreases in cell proliferation, suggesting that optimal levels of CK2 are required for optimal proliferation. By comparison, the effects of induced expression of kinase-inactive CK2alpha differed significantly from the effects of induced expression of kinase-inactive CK2alpha'. Of particular interest is the dramatic attenuation of proliferation that is observed following induction of CK2alpha'(K69M), but not following induction of CK2alpha(K68M). These results provide evidence for functional specialization of CK2 isoforms in mammalian cells. Moreover, cell lines exhibiting regulatable expression of CK2 will facilitate efforts to systematically elucidate its cellular functions.

Expression and regulation of protein kinase CK2 during the cell cycle.

There are indications from genetic, biochemical and cell biological studies that protein kinase CK2 (formerly casein kinase II) has a variety of functions at different stages in the cell cycle. To further characterize CK2 and its potential roles during cell cycle progression, one of the objectives of this study was to systematically examine the expression of all three subunits of CK2 at different stages in the cell cycle. To achieve this objective, we examined levels of CK2alpha, CK2alpha' and CK2beta on immunoblots as well as CK2 activity in samples prepared from: (i) elutriated populations of MANCA (Burkitt lymphoma) cells, (ii) serum-stimulated GL30-92/R (primary human fibroblasts) cells and (iii) drug-arrested chicken bursal lymphoma BK3A cells. On immunoblots, we observed a significant and co-ordinate increase in the expression of CK2alpha and CK2alpha' following serum stimulation of quiescent human fibroblasts. By comparison, no major fluctuations in CK2 activity were detected during any other stages during the cell cycle. Furthermore, we did not observe any dramatic differences between the relative levels of CK2alpha to CK2alpha' during different stages in the cell cycle. However, we observed a significant increase in the amount of CK2beta relative to CK2alpha in cells arrested with nocodazole. We also examined the activity of CK2 in extracts or in immunoprecipitates prepared from drug-arrested cells. Of particular interest is the observation that the activity of CK2 is not changed in nocodazole-arrested cells. Since CK2 is maximally phosphorylated in these cells, this result suggests that the phosphorylation of CK2 by p34cdc2 does not affect the catalytic activity of CK2. However, the activity of CK2 was increased by incubation with p34cdc2 in vitro. Since this activation was independent of ATP we speculate that p34cdc2 may have an associated factor that stimulates CK2 activity. Collectively, the observations that relative levels of CK2beta increase in mitotic cells, that CK2alpha and CK2beta are phosphorylated in mitotic cells and that p34cdc2 affects CK2 activity in vitro suggest that CK2 does have regulatory functions associated with cell division.

Ribonucleotide reductase R2 protein is phosphorylated at serine-20 by P34cdc2 kinase.

Ribonucleotide reductase is a rate-limiting enzyme in DNA synthesis and is composed of two different proteins, R1 and R2. The R2 protein appears to be rate-limiting for enzyme activity in proliferating cells, and it is phosphorylated by p34cdc2 and CDK2, mediators of cell cycle transition events. A sequence in the R2 protein at serine-20 matches a consensus sequence for p34cdc2 and CDK2 kinases. We tested the hypothesis that the serine-20 residue was the major p34cdc2 kinase site of phosphorylation. Three peptides were synthesized (from Asp-13 to Ala-28) that contained either the wild type amino acid sequence (Asp-Gln-Gln-Gln-Leu-Gln-Leu-Ser-Pro-Leu-Lys-Arg-Leu-Thr-Leu-Ala, serine peptide) or a mutation, in which the serine residue was replaced with an alanine residue (alanine peptide) or a threonine residue (threonine peptide). Only the serine peptide and threonine peptide were phosphorylated by p34cdc2 kinase. In two-dimensional phosphopeptide mapping experiments of serine peptide and Asp-N endoproteinase digested R2 protein, peptide co-migration patterns suggested that the synthetic phosphopeptide containing serine-20 was identical to the major Asp-N digested R2 phosphopeptide. To further test the hypothesis that serine-20 is the primary phosphorylated residue on R2 protein, three recombinant R2 proteins (R2-Thr, R2-Asp and R2-Ala) were generated by site-directed mutagenesis, in which the serine-20 residue was replaced with threonine, aspartic acid or alanine residues. Wild type R2 and threonine-substituted R2 proteins (R2-Thr) were phosphorylated by p34cdc2 kinase, whereas under the same experimental conditions, R2-Asp and R2-Ala phosphorylation was not detected. Furthermore, the phosphorylated amino acid residue in the R2-Thr protein was determined to be phosphothreonine. Therefore, by replacing a serine-20 residue with a threonine, the phosphorylated amino acid in R2 protein was changed to a phosphothreonine. In total, these results firmly establish that a major p34cdc2 phosphorylation site on the ribonucleotide reductase R2 protein occurs near the N-terminal end at serine-20, which is found within the sequence Ser-Pro-Leu-Lys-Arg-Leu. Comparison of ribonucleotide reductase activities between wild type and mutated forms of the R2 proteins suggested that mutation at serine-20 did not significantly affect enzyme activity.

Tra1p is a component of the yeast Ada.Spt transcriptional regulatory complexes.

The yeast Ada and TBP class of Spt proteins interact in multiple complexes that are required for transcriptional regulation. We have identified Tra1p as a component of these complexes through tandem mass spectrometry analysis of proteins that associate with Ngg1p/Ada3p. TRA1 is an essential gene and encodes a 3744-amino acid protein that is a member of a group of proteins including the catalytic subunit of DNA-dependent protein kinase, ATM and TRRAP, with carboxyl-terminal regions related to phosphatidylinositol 3-kinases. The interaction between Tra1p and Ada/Spt components was verified by the reciprocal coimmunoprecipitation of Ada2p and Tra1p from whole cell extracts in one or more complexes containing Spt7p. Tra1p cofractionated with Ngg1p and Spt7p through consecutive chromatography on Mono Q, DNA-cellulose, and Superose 6 columns. Binding of Tra1p to DNA-cellulose required Ada components. The association of Tra1p with two Ada.Spt complexes was suggested by its cofractionation with Ngg1p and Spt7p in two peaks on the Mono Q column. In the absence of Ada2p, the elution profile of Tra1p shifted to a distinct peak. Despite the similarity of Tra1p to a group of putative protein kinases, we have not detected protein kinase activity within immunoprecipitates of Tra1p or the Ada.Spt complexes.

Regulation of casein kinase 2 by direct interaction with cell surface receptor CD5.

The transmembrane protein CD5, expressed on all T cells and the B1 subset of B cells, modulates antigen receptor-mediated activation. We used the yeast two-hybrid system to identify proteins that interact with its cytoplasmic domain and play a role in CD5 proximal signaling events. We found that the beta subunit of the serine/threonine kinase casein kinase 2 (CK2) interacts specifically with the cytoplasmic domain of CD5. Co-immunoprecipitation experiments showed activation-independent association of CK2 with CD5 in human and murine B and T cell lines and murine splenocytes. The interaction of CK2 holoenzyme with CD5 is mediated by the amino terminus of the regulatory subunit beta. CK2 binds and phosphorylates CD5 at the CK2 motifs flanked by Ser459 and Ser461. Cross-linking of CD5 leads to the activation of CD5-associated CK2 in a murine B-lymphoma cell line and a human T-leukemia cell line and is independent of net recruitment of CK2 to CD5. In contrast, CK2 is not activated following cross-linking of the B cell receptor complex or the T cell receptor complex. This direct regulation of CK2 by a cell surface receptor provides a novel pathway for control of cell activation that could play a significant role in regulation of CD5-dependent antigen receptor activation in T and B cells.

Cooperative interaction between the DNA-binding domains of PU.1 and IRF4.

The two lymphoid-specific transcription factors PU.1 and IRF4 form a cooperative ternary complex at immunoglobulin enhancer elements such as the lambdaB and kappaE3' sites. We report here that the synergy of this interaction can be reconstituted in part with the DNA-binding domains of the two proteins. The minimal DNA binding-domain of IRF4 was mapped to residues 20 to 137, corresponding to the conserved DNA-binding region of other interferon regulatory factors (IRFs). This domain can bind weakly to a synthetic murine lambdaB element, while IRF4 constructs that contain residues 1 to 19 require the presence of PU.1 for DNA-binding at similar concentrations. Fluorescence polarization of fluorescein-labelled DNA was used to show that the presence of residues 1 to 19 decreases the binding affinity of IRF4 N-terminal constructs from two- to fivefold. However, all constructs bound better to the lambdaB element in the presence of the DNA-binding domain of PU.1. This cooperative interaction was not dependent on phosphorylation of the PEST domain of PU.1, but was dependent on the proper spacing of the binding sites for PU.1 and IRF4. These data suggest that at least part of the cooperative interaction between full-length PU.1 and IRF4 involves the DNA-binding domains of the two proteins. NMR spectroscopy of 15N-labelled PU.1 and IRF4 constructs indicates that the PEST domain of PU.1 and residues 1 to 19 of IRF4 may be unstructured in the isolated proteins.

DNA binding by cut homeodomain proteins is down-modulated by casein kinase II.

The Drosophila and mammalian Cut homeodomain proteins contain, in addition to the homeodomain, three other DNA binding regions called Cut repeats. Cut-related proteins thus belong to a distinct class of homeodomain proteins with multiple DNA binding domains. Using nuclear extracts from mammalian cells, Cut-specific DNA binding was increased following phosphatase treatment, suggesting that endogenous Cut proteins are phosphorylated in vivo. Sequence analysis of Cut repeats revealed the presence of sequences that match the consensus phosphorylation site for casein kinase II (CKII). Therefore, we investigated whether CKII can modulate the activity of mammalian Cut proteins. In vitro, a purified preparation of CKII efficiently phosphorylated Cut repeats causing an inhibition of DNA binding. In vivo, overexpression of the CKII alpha and beta caused a decrease in DNA binding by Cut. The CKII phosphorylation sites within the murine Cut (mCut) protein were identified by in vitro mutagenesis as residues Ser400, Ser789, and Ser972 within Cut repeat 1, 2, and 3, respectively. Cut homeodomain proteins were previously shown to function as transcriptional repressors. Overexpression of CKII reduced transcriptional repression by mCut, whereas a mutant mCut protein containing alanine substitutions at these sites was not affected. Altogether our results indicate that the transcriptional activity of Cut proteins is modulated by CKII.

Expression and localization of epitope-tagged protein kinase CK2.

Protein kinase CK2, formerly known as casein kinase II, is a ubiquitous protein serine/threonine kinase. The enzyme exists in tetrameric complexes composed of two catalytic (CK2 alpha and/or CK2 alpha') subunits and two subunits (CK2 beta) that appear to have a role in modulating the activity of the catalytic subunits. With the exception of their unrelated carboxy-terminal domains, the two isozymic forms of mammalian CK2 display extensive sequence identity. Furthermore, CK2 alpha and CK2 alpha' exhibit remarkable conservation between species, suggesting that they may have unique functions. In the present study, the cDNAs encoding CK2 alpha and CK2 alpha' were modified by addition of the hemagglutinin tag of the influenza virus at the amino terminus of the respective proteins. The epitope-tagged proteins were transfected into Cos-7 cells and the localization of the expressed proteins determined by indirect immunofluorescence using monoclonal antibodies specific for the epitope tag. The use of transfection favors the formation of homotetrameric complexes (i.e., alpha 2 beta 2, alpha' 2 beta 2) instead of heterotetrameric complexes (i.e., alpha alpha' beta 2) that are present in many cells. Epitope-tagged CK2 alpha and CK2 alpha' displayed kinase activity and the ability to form complexes with CK2 beta. The results of these studies also indicate definitively that CK2 alpha and CK2 alpha' are both localized predominantly within the nucleus. Mutation of conserved lysine residues within the ATP binding domains of CK2 alpha and CK2 alpha' resulted in loss of kinase activity. However, examination of these mutants indicates that kinase activity is not essential for formation of complexes between subunits of CK2 and is not required for nuclear localization of CK2.

Fibroblast growth factor-2 decreases metabolic coupling and stimulates phosphorylation as well as masking of connexin43 epitopes in cardiac myocytes.

Cardiac gap junction (GJ) channels, composed of connexins, allow electrical and metabolic couplings between cardiomyocytes, properties important for coordinated action of the heart as well as tissue homeostasis and control of growth and differentiation. Fibroblast growth factor-2 (FGF-2) is an endogenous growth-promoting protein, believed to participate in the short- and long-term responses of the heart to injury. We have examined short-term effects of FGF-2 on cardiac myocyte GJ-mediated metabolic coupling, using cultures of neonatal rat cardiomyocytes. FGF-2 decreased coupling between cardiomyocytes assessed by scrape dye loading as well as microinjection and dye transfer within 30 minutes of administration. Genistein blocked the effects of FGF-2. To determine the mechanism, we next assessed the effect of FGF-2 on expression, distribution, and phosphorylation of connexin43 (Cx43), which is a major cardiomyocyte connexin. FGF-2 did not affect Cx43 mRNA or protein accumulation and synthesis, and it did not change Cx43 localization at sites of intercellular contact as assessed by immunostaining with a polyclonal anti-Cx43 antibody raised against a synthetic peptide containing residues 346 to 363 of Cx43. FGF-2, however, decreased staining intensity at sites of intermyocyte contact when a monoclonal anti-Cx43 antibody was used, suggesting a localized masking of epitope(s) recognized by the monoclonal but not the polyclonal antibody. These epitopes appear to reside within residues 261 to 270 of Cx43, as indicated by full quenching of monoclonal antibody staining with synthetic peptides. In addition, FGF-2 induced a more than twofold increase in Cx43 phosphorylation. Phosphoamino acid analysis indicated increased phosphorylation of Cx43 on serine residues. Although tyrosine phosphorylation of Cx43 was not detected in either treated or control cells, a fraction of Cx43 was immunoprecipitated with anti-phosphotyrosine-specific antibodies in FGF-2-treated myocytes, suggesting interaction (and hence coprecipitation) with phosphotyrosine-containing protein(s). In conclusion, we have identified Cx43 and intercellular communication as targets of FGF-2-triggered and tyrosine phosphorylation-dependent signal transduction in cardiac myocytes. It is suggested that phosphorylation of Cx43 on serine induced by FGF-2 contributes to decreased metabolic coupling between cardiomyocytes.

Granzyme B induces apoptosis and cyclin A-associated cyclin-dependent kinase activity in all stages of the cell cycle.

Granzyme B rapidly induces apoptosis in the presence of the pore-forming protein perforin. We have examined the cell cycle restriction of this apoptosis by separating Jurkat cells into fractions representing different stages of the cell cycle by centrifugal elutriation. Cells were susceptible to apoptosis from G1 through to G2/M, with no significant resistance detected at any stage. Similarly, cells arrested at G1/S or G2/M with either hydroxyurea or nocodazole were slightly more sensitive than asynchronously growing cells. Granzyme B induces Cdc2 kinase activity and requires its induction for apoptosis. Cyclin-dependent kinase (CDK) activity is regulated by phosphorylation and association with cyclins that also control subcellular localization of the CDK/cyclin complexes. Cdc2 associates with both cyclin A, which is synthesized at G1 and S, and cyclin B, which is produced later during S and G2 before G2/M transition. We find that the CDK activity induced by granzyme B is associated primarily with cyclin A in both asynchronous and G1/S-arrested cells, while cyclin B-associated kinase activity is minimal. Because cyclin A is also able to associate with Cdk2, a kinase that is important for G1/S transition, we examined the activation of this CDK during granzyme B-induced apoptosis and find that Cdk2 is induced as rapidly as Cdc2. In conclusion, we have identified a lack of cell cycle restriction of granzyme B-induced apoptosis and the rapid activation of both cyclin A/Cdc2 and cyclin A/Cdk2 kinase activity.

Developmental changes in transcription factors associated with the nuclear matrix of chicken erythrocytes.

The nuclear matrix has roles in organizing nuclear DNA and in controlling transcription. Transcription factors are associated with the nuclear matrix, with the spectra of transcription factors differing from one cell type to another. In this study we identified the transcription factors and enzymes functioning in the regulation of gene expression that were associated with nuclear matrix and nonmatrix nuclear fractions in erythrocytes isolated from chick embryos at different stages of development, anemic and normal adult birds. We found that the primitive erythroid nuclear matrix had the greatest histone deacetylase activity and highest levels of several transcription factors, including GATA-1, CACCC-binding proteins, and NF1. These transcription factors have key roles in erythroid-specific gene expression. The levels of these transcription factors were lower in the nonmatrix and matrix fractions isolated from definitive erythrocytes. For primitive and definitive erythrocytes, the level of CACCC-binding proteins in the nuclear matrix fraction was greater than that of Sp1. The relative levels of these transcription factors were reversed in the nonmatrix fraction. Casein kinase II was not found in erythroid nuclear matrices. The observed erythroid lineage specific alterations in erythroid nuclear matrix transcription factor composition and abundance may be involved in erythroid-specific gene expression.