Localization of phosphorylated CK2alpha to the mitotic spindle requires the peptidyl-prolyl isomerase Pin1.

CK2 is a serine/threonine kinase with many substrates, largely unknown modes of regulation and essential roles in mitotic progression. CK2α, a catalytic subunit of CK2, is phosphorylated in mitosis, and here we examine the effect of phosphorylation on CK2α localization. Using phosphospecific antibodies, we show that CK2α localizes to the mitotic spindle in a phosphorylation-dependent manner. Mitotic spindle localization requires the unique C-terminus of CK2α, and involves a novel regulatory mechanism in which phosphorylation of CK2α facilitates binding to the peptidyl-prolyl isomerase Pin1, which is required for CK2α mitotic spindle localization. This could explain how the constitutive activity of CK2α might be targeted towards mitotic substrates. Furthermore, because Pin1 has many important spindle substrates, this might represent a general mechanism for localization of mitotic signalling proteins.

Interaction between CK2α and CK2ß, the subunits of protein kinase CK2: thermodynamic contributions of key residues on the CK2α surface.

The protein Ser/Thr kinase CK2 (former name: casein kinase II) exists predominantly as a heterotetrameric holoenzyme composed of two catalytic subunits (CK2α) bound to a dimer of noncatalytic subunits (CK2ß). We undertook a study to further understand how these subunits interact to form the tetramer. To this end, we used recombinant, C-terminal truncated forms of human CK2 subunits that are able to form the holoenzyme. We analyzed the interaction thermodynamics between the binding of CK2α and CK2ß as well as the impact of changes in temperature, pH, and the ionization enthalpy of the buffer using isothermal titration calorimetry (ITC). With structure-guided alanine scanning mutagenesis we truncated individual side chains in the hydrophobic amino acid cluster located within the CK2α interface to identify experimentally the amino acids that dominate affinity. The ITC results indicate that Leu41 or Phe54 single mutations were most disruptive to binding of CK2ß. Additionally, these CK2α mutants retained their kinase activity. Furthermore, the substitution of Leu41 in combination with Phe54 showed that the individual mutations were not additive, suggesting that the cooperative action of both residues played a role. Interestingly, the replacement of Ile69, which has a central position in the interaction surface of CK2α, only had modest effects. The differences between Leu41, Phe54, and Ile69 in interaction relevance correlate with solvent accessibility changes during the transition from unbound to CK2ß-bound CK2α. Identifying residues on CK2α that play a key role in CK2α/CK2ß interactions is important for the future generation of small molecule drug design.

Regulation of cell proliferation and survival: convergence of protein kinases and caspases.

Intricate networks of protein kinases are intimately involved in the regulation of cellular events related to cell proliferation and survival. In addition to protein kinases, cells also contain networks of proteases including aspartic-acid directed caspases organized in cascades that play a major role in the regulation of cell survival through their involvement in the initiation and execution phases of apoptosis. Perturbations in regulatory protein kinase and caspase networks induce alterations in cell survival and frequently accompany transformation and tumorigenesis. Furthermore, recent studies have documented that caspases or their substrates are subject to phosphorylation in cells illustrating a potential convergence of protein kinase and caspase signaling pathways. Interestingly, a number of caspase substrates are protected from cleavage when they are phosphorylated at sites that are adjacent to caspase cleavage sites. While it is theoretically possible that many distinct protein kinases could protect proteins from caspase-mediated cleavage, protein kinase CK2 is of particular interest because acidic amino acids, including aspartic acid residues that are recognized by caspases, are its dominant specificity determinants.

Protein kinase CK2 catalyzes tyrosine phosphorylation in mammalian cells.

Protein kinase CK2 exhibits oncogenic activity in mice and is over-expressed in a number of tumors or leukemic cells. On the basis of its amino acid sequence and a wealth of experimental information, CK2 has traditionally been classified as a protein serine/threonine kinase. In contrast to this traditional view of CK2, recent evidence has shown that CK2 can also phosphorylate tyrosine residues under some circumstances in vitro and in yeast. In this study, we provide definitive evidence demonstrating that CK2 also exhibits tyrosine kinase activity in mammalian cells. Tyrosine phosphorylation of CK2 in cells and in CK2 immunoprecipitates is dependent on CK2 activity and is inhibited by the CK2 selective inhibitor 4,5,6,7-tetrabromobenzotriazole. Examination of phosphotyrosine profiles in cells reveals a number of proteins, including CK2 itself, which exhibit increased tyrosine phosphorylation when CK2 levels are increased. Peptide arrays to evaluate the specificity determinants for tyrosine phosphorylation by CK2 reveal that its specificity for tyrosine phosphorylation is distinct from its specificity for serine/threonine phosphorylation. Of particular note is the requirement for an aspartic acid immediately C-terminal to the phosphorylatable tyrosine residue. Collectively, these data provide conclusive evidence that CK2 catalyzes the phosphorylation of tyrosine residues in mammalian cells, a finding that adds a new level of complexity to the challenge of elucidating its cellular functions. Furthermore, these results raise the possibility that increased CK2 levels that frequently accompany transformation may contribute to the increased tyrosine phosphorylation that occurs in transformed cells.

Subtype-specific regulation of equilibrative nucleoside transporters by protein kinase CK2.

Two subtypes of equilibrative transporters, es (equilibrative inhibitor-sensitive) and ei (equilibrative inhibitor-insensitive), are responsible for the majority of nucleoside flux across mammalian cell membranes. Sequence analyses of the representative genes, ENT1 {equilibrative nucleoside transporter 1; also known as SLC29A1 [solute carrier family 29 (nucleoside transporters), member 1]} and ENT2 (SLC29A2), suggest that protein kinase CK2-mediated phosphorylation may be involved in the regulation of es- and ei-mediated nucleoside transport. We used human osteosarcoma cells transfected with catalytically active or inactive alpha' and alpha subunits of CK2 to assess the effects of CK2 manipulation on nucleoside transport activity. Expression of inactive CK2alpha' (decreased CK2alpha' activity) increased the number of binding sites (approximately 1.5-fold) for the es-specific probe [3H]NBMPR ([3H]nitrobenzylthioinosine), and increased (approximately 1.8-fold) the V(max) for 2-chloro[3H]adenosine of the NBMPR-sensitive (es) nucleoside transporter. There was a concomitant decrease in the V(max) of the NBMPR-resistant (ei-mediated) uptake of 2-chloro[3H]adenosine. This inhibition of CK2alpha' activity had no effect, however, on either the K(D) of [3H]NBMPR binding or the K(m) of 2-chloro[3H]adenosine uptake. Quantitative PCR showed a transient decrease in the expression of both hENT1 (human ENT1) and hENT2 mRNAs within 4-12 h of induction of the inactive CK2alpha' subunit, but both transcripts had returned to control levels by 24 h. These data suggest that inhibition of CK2alpha' reduced ei activity by attenuation of hENT2 transcription, while the increase in es/hENT1 activity was mediated by post-translational action of CK2. The observed modification in es activity was probably due to a CK2alpha'-mediated change in the phosphorylation state of the ENT1 protein, or an interacting protein, effecting an increase in the plasma membrane lifetime of the transport proteins.

Phosphorylation regulates the stability of the regulatory CK2beta subunit.

Protein kinase CK2 is a protein serine/threonine kinase that exhibits elevated expression in a number of cancers and displays oncogenic activity in mice. The regulatory CK2beta subunit has a central role in assembly of functional tetrameric CK2 complexes where it participates in modulation of catalytic activity and substrate specificity. Since overexpression of CK2beta results in elevated levels of CK2 activity, we investigated the molecular mechanisms that control its degradation since perturbations in these pathways could contribute to elevated CK2 in cancer. In this study, we demonstrate that CK2beta is degraded by a proteasome-dependent pathway and that it is ubiquitinated. We have also investigated the role of phosphorylation and a putative destruction box in regulating its stability in cells. Importantly, replacement of three serine residues within the autophosphorylation site of CK2beta with glutamic acid residues resulted in a significant decrease in its degradation indicating that autophosphorylation is involved in regulating its stability. Notably, although the autophosphorylation site of CK2beta is remarkably conserved between species, this is the first functional role ascribed to this site. Furthermore, based on these results, we speculate that alterations in the phosphorylation or dephosphorylation of the regulatory CK2beta subunit could underlie the elevated expression of CK2 that is observed in cancer cells.

Characterizing the convergence of protein kinase CK2 and caspase-3 reveals isoform-specific phosphorylation of caspase-3 by CK2α': implications for pathological roles of CK2 in promoting cancer cell survival.

Protein kinase CK2 has emerged as a promising candidate for the treatment of a number of cancers. This enzyme is comprised of two catalytic subunits (CK2 and/or CK2α') that form complexes with homodimers of regulatory CK2ß subunits. While catalytic and regulatory CK2 subunits are generally expressed at similar levels to form tetrameric complexes, asymmetric expression of CK2 subunits has been associated with various forms of cancer and the enhanced survival of cancer cells. To elucidate mechanisms responsible for regulation of cancer cell survival by CK2, we recently employed computational and experimental strategies that revealed widespread overlap between sites for CK2 phosphorylation and caspase cleavage. Among candidates with overlapping CK2 and caspase cleavage sites was caspase-3 that is phosphorylated by CK2 to prevent its activation by upstream caspases. To elucidate the precise relationship between CK2 and caspase-3, we modulated expression of individual CK2 subunits and demonstrated that CK2α' exhibits a striking preference for caspase-3 phosphorylation in cells as compared to CK2α and that CK2ß exhibits the capacity to abolish caspase-3 phosphorylation. Since caspase-3 represents the first CK2 substrate selectively phosphorylated by CK2α' in cells, our work highlights divergent functions of the different forms of CK2. Given the involvement of CK2 in a diverse series of biological events and its association with various cancers, this work has important implications for identifying pathological roles of distinct forms of CK2 that could instruct efforts to selectively target individual CK2 subunits for therapy.

The islet size to oxygen consumption ratio reliably predicts reversal of diabetes posttransplant.

ß-Cell replacement therapy by either whole-organ pancreas or islets of Langerhans transplantation can restore carbohydrate control to diabetic patients and reduces complications associated with the disease. One of the variables inherent in islet transplantation is the isolation of functional islets from donor pancreata. Islet isolations fail to consistently produce good-quality functional islets. A rapid pretransplant assay to determine posttransplant function of islets would be an invaluable tool. We have tested the novel hypothesis that modified oxygen consumption rates (OCR), standardized to DNA quantity (nmol/min-mg DNA), would serve as a pretransplant assessment of the metabolic potency of the islets postisolation. This study compares the ability of current in vitro assays to predict in vivo restoration of normoglycemia in a diabetic nude mouse posttransplantation of adult pig islets. There is known to be a diversity of islet sizes within each preparation. This parameter has not heretofore been effectively considered a critical factor in islet engraftment. Our results suggest a surprising finding that islet size influences the probability of restoring carbohydrate control. Based on this observation, we thus developed a novel predictor of islet graft function that combines the effects of both islet OCR and size. When OCR was divided by the islet index (size), a highly significant predictor of graft function was established (p = 0.0002, n = 75). Furthermore, when OCR/islet index values exceeded 70.0 nmol/min-mg DNA/islet index, an effective threshold of diabetes reversal was observed. This assay can be performed with as few as 1,000 islet equivalents (IEQ) and conducted in less than 60 min. Our data suggest that, using this novel method to assess islet cell function prior to transplantation, OCR/islet index thresholds provide a valuable tool in identifying which islet preparations are most likely to restore glycemic control posttransplant.

A novel technique for the transplantation of pancreatic islets within a vascularized device into the greater omentum to achieve insulin independence.

BACKGROUND: The greater omentum with its vascularization and blood flow has been considered as a location for islet transplantation; however, there is a need to provide a controlled and protected site for the islets within the omentum that would be applicable to donor islets and future stem cell technologies. Here we describe the use of a novel device implanted within the omentum with a subcutaneous delivery port that offers an environment for donor islets. METHODS: A prototype cell pouch device was wrapped in the greater omentum and an islet implantation port was exposed subcutaneously in diabetic Lewis rats. After tissue growth throughout the device, islet isografts were implanted and long-term glucose control was evaluated. RESULTS: By using this technique, 7 of 10 diabetic rat recipients showed long-term normal blood glucose levels after minimal islet dose transplants. Histologic assessment revealed collagen formation and vascularization within the device. CONCLUSIONS: The implanted device assessed using this technique provides a safe and efficacious environment for the support of pancreatic islets contained within a removable device as a cell therapy in a highly vascularized setting.

A peptide-based target screen implicates the protein kinase CK2 in the global regulation of caspase signaling.

The convergence of caspase and protein kinase signaling pathways has become increasingly evident, as illustrated by the protection of caspase substrates from cleavage upon undergoing phosphorylation at or near to their caspase recognition motifs. To investigate the global role of phosphorylation in the regulation of caspase signaling, we designed a peptide match program to identify sequences from the human proteome that contained overlapping recognition motifs for caspases and kinases. We identified the protein kinase CK2 as the most prominent kinase with a consensus site for phosphorylation that overlapped with caspase recognition motifs. We then evaluated potential targets of CK2 and caspases by combining peptide array target screens with identification of caspase substrates. We identified numerous shared candidate targets of CK2 and caspases, including procaspase-3, which functions at a level at which both intrinsic and extrinsic apoptotic signals converge. Together, these data support a role for CK2-dependent phosphorylation as a global mechanism for inhibiting caspase signaling pathways.

Protein kinase CK2 is a constitutively active enzyme that promotes cell survival: strategies to identify CK2 substrates and manipulate its activity in mammalian cells.

Protein kinase CK2 is a constitutively active protein serine/threonine kinase that is ubiquitously expressed and essential for the survival of eukaryotic cells. On the basis of its elevated expression in a number of human cancers and its ability to promote tumorigenesis in transgenic mice, CK2 has emerged as a promising candidate for molecular-targeted therapy. Accordingly, there has been considerable interest in identifying the cellular events that are regulated by CK2 and the cellular substrates of CK2 that are responsible for mediating its actions in cells. Large-scale phosphoproteomics studies are revealing extensive lists of candidate CK2 substrates on the basis that these proteins are phosphorylated at sites conforming to the consensus for phosphorylation by CK2. However, efforts to validate the vast majority of these candidates as bona fide physiological CK2 substrates have been hindered by the lack of systematic strategies to identify its direct substrates and manipulate its activity in intact cells. To overcome these limitations, we describe experimental procedures for isolating CK2 from bacteria and from mammalian cells to enable in vitro phosphorylation of candidate substrates. We also outline strategies for manipulating the levels and activity of CK2 in intact cells. Collectively, the methods that are presented in this chapter should enable the identification and characterization of CK2 substrates and CK2-regulated processes both in vitro and in living cells.

Tyrosine phosphorylation of protein kinase CK2 by Src-related tyrosine kinases correlates with increased catalytic activity.

Casein kinase-2 (CK2) is a pleiotropic and constitutively active serine/threonine protein kinase composed of two catalytic (alpha and/or alpha') and two regulatory beta-subunits, whose regulation is still not well understood. In the present study, we show that the catalytic subunits of human CK2, but not the regulatory beta-subunits, are readily phosphorylated by the Src family protein tyrosine kinases Lyn and c-Fgr to a stoichiometry approaching 2 mol phosphotyrosine/mol CK2alpha with a concomitant 3-fold increase in catalytic activity. We also show that endogenous CK2alpha becomes tyrosine-phosphorylated in pervanadate-treated Jurkat cells. Both tyrosine phosphorylation and stimulation of activity are suppressed by the specific Src inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4- d ]pyrimidine. By comparison, mutations giving rise to inactive forms of CK2alpha do not abrogate and, in some cases, stimulate Lyn and c-Fgr-dependent tyrosine phosphorylation of CK2. Several radiolabelled phosphopeptides could be resolved by HPLC, following tryptic digestion of CK2alpha that had been phosphoradiolabelled by incubation with [(32)P]ATP and c-Fgr. The most prominent phosphopeptide co-migrates with a synthetic peptide encompassing the 248-268 sequence, phosphorylated previously by c-Fgr at Tyr(255) in vitro. The identification of Tyr(255) as a phosphorylated residue was also supported by MS sequencing of both the phosphorylated and non-phosphorylated 248-268 tryptic fragments from CK2alpha and by on-target phosphatase treatment. A CK2alpha mutant in which Tyr(255) was replaced by phenylalanine proved less susceptible to phosphorylation and refractory to stimulation by c-Fgr.