Benzimidazole nucleoside analogues as inhibitors of plant (maize seedling) casein kinases.

Halogeno benzimidazole and benzimidazole nucleoside analogues have been screened for inhibitory activity vs. purified plant (maize seedling) casein kinases I, IIA and IIB, and the results compared with those previously reported for some of the compounds as inhibitors of the corresponding mammalian CK-1 and CK-2 (Meggio et al. (1990) Eur. J. Biochem. 187, 89-94). One new analogue, the riboside of 5,7-dibromobenzimidazole, which is sterically constrained to the anti conformation about the glycosidic bond, and is a good inhibitor, exhibited appreciable (5-7-fold) discrimination between the type I and type II enzymes. An increase in the number of halogen substituents on the benzene ring of benzimidazole from two to three led to marked enhancement of inhibitory activity, particularly against the type II enzymes, with a decrease in Ki from 24 to 4 microM. The 2-aza analogue of 5,6-dichlorobenzimidazole, i.e. 5,6-dichlorobenzotriazole, as the free base, even more effectively discriminated between the two types of plant casein kinases, with Ki approximately 100 microM for CK-I, and Ki approximately 9 microM for CK-IIA and CK-IIB. Inhibition in all instances was competitive with respect to ATP (for CK-I), and ATP and GTP (for CK-IIA and CK-IIB). The results are compared with those for halogenated isoquinolinesulfonamide inhibitors reported by Chijiwa et al. (J. Biol. Chem. (1989) 264, 4924-4927), leading to proposals for the synthesis of potentially more effective and more discriminating inhibitors. Attention is drawn to the significant role of the halogen substituents in the mechanism(s) of action of the structurally related benzimidazole, benzotriazole and naphthalene and isoquinoline, inhibitors of protein kinases.

Cloning and sequencing of the casein kinase 2 alpha subunit from Zea mays.

The nucleotide sequence of the cDNA coding for the alpha subunit of casein kinase 2 of Zea mays has been determined. The cDNA clone contains an open reading frame of 996 nucleotides encoding a polypeptide comprising 332 amino acids. The primary amino acid sequence exhibits 75% identity to the alpha subunit and 71% identity to the alpha' subunit of human casein kinase 2.

Specificity determinants of maize casein kinase-IIB are related to but distinct from those of rat liver casein kinase-2.

The site specificity of maize seedling casein kinase-IIB, a type-2 casein kinase exhibiting an unusually low Mr, has been studied with the aid of model acidic peptide substrates for rat liver casein kinase-2. Like the animal enzyme, casein kinase-IIB also readily phosphorylates peptides SEEEEE, SEAEEE and SEEEAE, but not SEEAEE. Maize seedling casein kinase-IIB, however, is almost inactive toward peptides SAEEEE, SAEEEEE and SAAEEEEE which are good substrates for liver casein kinase-2. This indicates that casein kinase-IIB requires acidic residues not only at position +3, similar to rat liver casein kinase-2, but also at position +1, where the animal enzyme tolerates a neutral residue. This and other differences outlined in this report support the view that protein kinases of the same type from different sources may have significant differences in their substrate specificity.

Expression and characterization of a recombinant maize CK-2 alpha subunit.

CKIIB, one of the CK-2 like enzymes which have been isolated from maize, has been shown to be a monomeric enzyme that cross-reacts with anti CK-2 alpha specific antibodies suggesting a possible relationship between the two proteins (Dobrowolska et al. (1992) Eur. J. Biochem. 204, 299-303). In order to support the immunological data also by biochemical and biophysical experiments the availability of a recombinant CK-2 alpha from maize was a prerequisite. A maize cDNA clone of maize CK-2 alpha was expressed in the bacterial strain BL21 (DE3). The recombinant protein was purified to homogeneity; its molecular mass on one-dimensional SDS PAGE was estimated to be 36.5 kDa. The calculated molecular mass according to the amino acid composition is 39,228 Da (332 amino acids). The recombinant maize CK-2 alpha (rmCK-2 alpha) exhibited mostly the same properties as the recombinant human CK-2 alpha (rhCK-2 alpha). In several respects it behaved differently from CKIIB, thus supporting the notion that either CKIIB is encoded by another gene or it undergoes extensive posttranscriptional and/or posttranslational alterations. Three observations in particular disprove any close relatedness between CKIIB and rmCK-2 alpha, namely: (a) the phosphorylation of calmodulin by CKIIB is dramatically stimulated by polylysine, whereas polylysine inhibits rather than stimulating the phosphorylation of calmodulin by rmCK-2 alpha (and by rhCK-2 alpha). (b) Addition of rhCK-2 beta has no significant influence on the stimulation of the calmodulin phosphorylation by CKIIB whereas in the case of rmCK-2 alpha and rhCK-2 alpha addition of rhCK-2 beta is required for optimal stimulation by polylysine. (c) CKIIB does not self-assemble with rhCK-2 beta to form a high molecular mass complex as it is demonstrated for rmCK-2 alpha.

Substrate recognition by casein kinase-II: the role of histidine-160.

Casein kinase-II (CK-II) belongs to the protein kinases recognizing serine/threonine in proximity to acidic residues in protein substrates. Crystallography and mutagenesis studies on the cAMP-dependent protein kinase (PKA) disclosed that glutamic acid-170 (E170), is important for interaction of substrates with the enzyme. At a position corresponding to E170 in PKA most Ser/Thr kinases have an aspartic or glutamic acid, while CK-II has a histidine residue (H160). In order to examine the relevance of this substitution for CK-II substrate specificity, a mutant of the catalytic alpha subunit (H160D), in which H160 was changed to aspartic acid, was made. Our results show that H160 is not primarily involved in canonical substrate recognition, but does interact with an acidic residue located at position -2 with respect to the target Ser/Thr.

The physical association of casein kinase 2 with nucleolin.

CK2 (formerly called casein kinase 2) is a ubiquitous messenger-independent serine/threonine protein kinase implicated in cell growth and proliferation. To investigate the regulation and functions of this enzyme, experiments were carried out to search for CK2-interacting proteins. The methods employed included an overlay technique, co-purification, co-immunoprecipitation, and the use of glutathione S-transferase (GST) CK2 fusion proteins. By the CK2 overlay technique, one protein of 110 kDa was found to bind to CK2 with very high affinity. The binding was inhibited by CK2 effectors such as heparin, polyarginine, and histone H1, but was not affected by the CK2 substrate, casein. Protein p110 was also detected by co-immunoprecipitation using anti-CK2 antiserum, suggesting an in vivo association of this protein with CK2. Co-purification of p110 with CK2 from Sf-9 cells that overexpressed CK2 was also observed through sequential chromatographic steps. Using GST fusion proteins of CK2, the CK2-p110 interaction was investigated further and was found to occur primarily through CK2 alpha or alpha' subunits, but not the beta subunit. Protein p110 was purified from 3T3 L1 mouse fibroblast cell lines using a GST-CK2 affinity resin. Amino acid sequence analysis of peptides obtained from the protein indicated that it is the nuclear protein, nucleolin. Furthermore, p110 was recognized by anti-nucleolin antiserum. At present, the physiological significance of the strong interaction between CK2 and nucleolin, an excellent substrate for the enzyme, is not clear. However, this association may be important for regulating rDNA transcription.

Regulation of casein kinase II by growth factors: a reevaluation.

Many of the effects of growth factors or hormones are mediated through the activation of protein kinase cascades. In this regard, it is well established that the activity of several protein kinases can be dramatically increased when cells are treated with a variety of stimuli. Since 1987, there have been several reports demonstrating that the activity of casein kinase II (CKII) can be acutely increased by hormones or growth factors. However, these are a number of discrepancies regarding the activation of CKII. In this study, we have examined CKII activities in extracts prepared from cells following treatment with stimuli that had been previously shown to elicit dramatic increases in CKII activity. Human WI.38 diploid lung fibroblasts were stimulated with serum or a variety of other stimuli including insulin, platelet-derived growth factor, fibroblast growth factor, epidermal growth factor, or phorbol myristate acetate. Human A431 epidermal carcinoma cells were similarly treated with epidermal growth factor. No reproducible increases in CKII activity were observed in response to any of these treatments. By comparison, a dramatic increase in kinase activity towards a synthetic peptide based on phosphorylation sites within the ribosomal S6 protein was consistently measured. Our observations indicate that CKII is not regulated in a similar manner by growth factors as are the protein kinases of the MAP kinase cascade, e.g., MAP kinase itself or ribosomal protein S6 kinase.

Identification of proteins that associate with protein kinase CK2.

In order to aid in an understanding of the cellular functions of protein kinase CK2, a search for interacting proteins was carried out using a 32P-labeled CK2 overlay method. Several proteins were found to associate with CK2 by this assay; among them, one protein of 110 kDa appeared to be the most prominent one. The possible association of CK2 with p110 was suggested by experiments involving the co-immunoprecipitation using anti-CK2 antibodies. Further analysis using GST-CK2 fusion proteins demonstrated that the CK2-p110 interaction occurred through the CK2alpha/alpha' subunits. To identify p110, it was purified using a GST-CK2 affinity column, and internal amino acid sequencing was then performed. p110 was found to be nucleolin, a nucleolar protein that may be important for rRNA synthesis; a possible role of CK2 in the control of this process is suggested. Using the same CK2 overlay technique, another interacting protein, insulin receptor substrate 1 (IRS-1), was also identified. By applying a modified overlay method using individual 35S-labeled CK2 subunits, obtained by in vitro translation in rabbit reticulate lysates, it was determined that CK2 associates with IRS-1 through its alpha/alpha' subunits; i.e. in keeping with the fact that IRS-1 is a known substrate for CK2. However, further work is needed to examine the association of CK2 with IRS-1 in vivo in order to fully understand the significance of the interaction.

Purification and characterization of maize seedling casein kinase IIB, a monomeric enzyme immunologically related to the alpha subunit of animal casein kinase-2.

Casein kinase IIB (CKIIB), a protein kinase related to animal casein kinase-2 (CK2), has been purified to homogeneity. It appears to be a monomeric enzyme, composed by an individual 39 kDa subunit, homologous to the alpha/alpha' subunits of animal CK2 and devoid of the autophosphorylatable 25-kDa alpha subunit of animal CK2, which display an heterotetrameric alpha 2 beta 2/alpha alpha' beta 2 structure. Such a conclusion is supported by the following lines of evidence: (1) CKIIB displays an apparent 39,000 Mr by gel filtration on Ultrogel AcA 34 and it gives rise to a single prominent protein band of similar Mr (38,000) upon SDS/PAGE; (2) upon incubation of the enzyme with [32P]ATP, no radiolabeled bands are detectable which might be attributable to either canonical or atypical beta subunits; (3) the 39-kDa band immunoreacts with antisera that recognize the alpha subunit of rat and chicken CK2; (4) conversely, no component immunologically related with the beta subunit could be detected in CKIIB by Western-blot analyses with antisera that recognize animal beta subunits; (5) the recombinant beta subunit of human CK2 is readily phosphorylated by CKIIB, the reaction being prevented, rather than stimulated, by polylysine, a behaviour typical of animal CK2 autophosphorylation. While the responsiveness of CKIIB to either heparin inhibition or polylysine stimulation are reminiscent of those of animal CK2, its peptide substrate specificity is significantly different and its thermolability is increased. Altogether these data would indicate that maize seedling CKIIB represents a naturally occurring monomeric form of CK2 devoid of non-catalytic subunits. Its properties, compared to those of animal CK2, suggest that the beta subunits of animal CK2 may be responsible for structural modifications conferring an altered specificity and an increased stability to the catalytic subunit.

Model studies on iron(III) ion affinity chromatography. II. Interaction of immobilized iron(III) ions with phosphorylated amino acids, peptides and proteins.

The chromatographic behaviour of phosphoamino acids, phosphopeptides and phosphoproteins and their non-phosphorylated counterparts was studied on Fe(III)-Chelating Sepharose and Fe(III)-Chelating Superose. The phosphorylated compounds, in contrast to their non-phosphorylated or dephosphorylated counterparts, adsorb to immobilized iron(III) ions at pH 5.5 and can be desorbed by an increase in pH. Phosphoamino acids were eluted at pH 6.5-6.7, whereas monophosphopeptides and phosphoprotamine eluted in the pH range 6.9-7.5. Molecules possessing clusters(s) of carboxylic groups are weakly retained (gamma-carboxyglutamic acid, Ala-Ser-Glu5) or bound (polyglutamic acid, beta-casein) to the immobilized iron(III) ions at pH 5.5. Dephosphorylated beta-casein was desorbed at pH 7.0, whereas for elution of native (non-dephosphorylated) beta-casein, phosphate buffer of pH 7.7 was required. The homopolymer of polyglutamic acid was desorbed in the pH range 6.0-6.3, whereas copolymers of glutamic acid and tyrosine require pH 7.0-7.3 or even phosphate buffer at pH 7.7 for elution.

Specific interaction between casein kinase 2 and the nucleolar protein Nopp140.

Casein kinase 2 (CK2) is a multifunctional second messenger-independent protein serine/threonine kinase that phosphorylates many different proteins. To understand the function and regulation of this enzyme, biochemical methods were used to search for CK2-interacting proteins. Using immobilized glutathione S-transferase fusion proteins of CK2, the nucleolar protein Nopp140 was identified as a CK2-associated protein. It was found that Nopp140 binds primarily to the CK2 regulatory subunit, beta. The possible in vivo association of Nopp140 with CK2 was also suggested from a coimmunoprecipitation experiment in which Nopp140 was detected in immunoprecipitates of CK2 prepared from cell extracts. Further studies using an overlay technique with radiolabeled CK2 as a probe revealed a direct CK2-Nopp140 interaction. Using deletion mutants of CK2beta subunits, the binding region of the CK2beta subunit to Nopp140 has been mapped. It was found that the NH2-terminal 20 amino acids of CK2beta are involved. Since Nopp140 has been identified as a nuclear localization sequence-binding protein and has been shown to shuttle between the cytoplasm and the nucleus, the finding of a CK2-Nopp140 interaction could shed light on our understanding of the function and regulation of CK2 and Nopp140.

Heparin decreases activator protein-1 binding to DNA in part by posttranslational modification of Jun B.

Heparin is a potent inhibitor of the proliferation and migration of vascular smooth muscle cells. This agent selectively inhibits the transcription of tissue-type plasminogen activator and interstitial collagenase, probably by decreasing the binding of activator protein-1 (AP-1) to phorbol ester-responsive elements in the promoters of these genes. Decreased AP-1 binding is not due to a direct inhibition by heparin, since heparinase digestion of nuclear extracts prepared from heparin-treated smooth muscle cells does not restore AP-1 binding activity. Treatment of cells with heparin suppresses the expression of Jun B, one of the components of AP-1. The major effect of heparin is at the level of posttranslational modification of Jun B. Results from pulse-chase labeling experiments show that the newly synthesized Jun B is rapidly converted to a higher-molecular-weight form and that conversion is suppressed by heparin. Evidence is presented suggesting that the heparin-inhibited event is phosphorylation of Jun B.

Osmotic stress induces rapid activation of a salicylic acid-induced protein kinase and a homolog of protein kinase ASK1 in tobacco cells.

In tobacco cells, osmotic stress induced the rapid activation of two protein kinases that phosphorylate myelin basic protein. Immunological studies demonstrated that the 48-kD kinase is the salicylic acid-induced protein kinase (SIPK), a member of the mitogen-activated protein kinase family. SIPK was activated 5 to 10 min after the cells were exposed to osmotic stresses, and its activity persisted for approximately 30 min. In contrast, the 42-kD kinase was activated within 1 min after osmotic stress, and its activity was maintained for approximately 2 hr. Moreover, in addition to myelin basic protein, the 42-kD kinase phosphorylated casein and two transcription factors, c-Jun and ATF-2. This latter enzyme was inactivated by a serine/threonine-specific phosphatase but, unlike SIPK, was not affected by a tyrosine-specific phosphatase. After the 42-kD kinase was purified to apparent homogeneity, tryptic peptide analysis indicated that it is a homolog of Arabidopsis serine/threonine kinase1 (ASK1).

CK2, a protein kinase of the next millennium.

Protein kinase CK2 is a ubiquitous eukaryotic protein kinase composed of two catalytic subunits, alpha and/or alpha', and two regulatory subunits, beta. In order to define similarities and dissimilarities between the alpha and alpha' catalytic subunits, which might account for their particular cellular functions, different forms of the enzyme were expressed in Sf9 cells and their properties determined. Both catalytic subunits were expressed separately, and also along with the regulatory beta subunit, in order to obtain free alpha and alpha', as well as alpha2beta2 and alpha'2beta2 forms. Our results confirm that the beta subunit acts to stabilize the alpha and alpha' subunits and also influences the substrate specificity and kinetic properties of the enzyme. Although significant differences between the specificities of holoenzymes alpha2beta2 and alpha'2beta2 as determined using a number of substrates were not detected, autophosphorylation studies on alpha2beta2 and alpha'2beta2 revealed significant differences in this property. The regulatory subunit beta was phosphorylated less rapidly by the alpha' subunit than by the alpha subunit, and the extent of phosphorylation of beta by alpha was also greater than that of beta by alpha'. It was also noted that the thermo-stability and the extent of its activation by NaCl were greater for alpha2beta2 than for alpha'2beta2. These different properties may relate to distinct functions of the two form of CK2.

Calcium-dependent protein kinase from maize seedlings activated by phospholipids.

A calcium- and phospholipid-dependent protein kinase of apparent molecular mass 54 kDa (designated ZmCPKp54) was partially purified from etiolated maize seedlings. Activity of ZmCPKp54 is stimulated by phosphatidylserine and phosphatidylinositol, but is not essentially affected by diolein and phorbol esters. The enzyme cross-reacts with polyclonal antibodies against the calmodulin like-domain of the calcium-dependent protein kinase, but not with antibodies against catalytic or regulatory domains of protein kinase C. ZmCPKp54 is not able to phosphorylate the specific substrates of protein kinase C (MARCKS peptide and protein kinase C substrate peptide derived from pseudosubstrate sequence) and its activity is not inhibited by specific PKC inhibitors (bisindolylmaleimide, protein kinase C pseudosubstrate inhibitory peptide). The substrate specificity and sensitivity to the inhibitors of the maize enzyme resembles calcium-dependent protein kinase. The biochemical and immunological properties indicate that ZmCPKp54 belongs to the calcium-dependent protein kinase family.

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.

Overexpression of mitogen-activated protein kinase kinase (MAPKK) and its mutants in NIH 3T3 cells. Evidence that MAPKK involvement in cellular proliferation is regulated by phosphorylation of serine residues in its kinase subdomains VII and VIII.

Mitogen-activated protein kinase kinase (MAPKK) is a dual specificity protein kinase that exhibits a high degree of specificity toward its downstream target, mitogen-activated protein kinase (MAPK). In this study, we used stable overexpression of MAPKK and its mutants in NIH 3T3 cells to study effects on downstream components of the MAPK signaling cascade and to correlate them to physiological responses. We have mutated the potential regulatory serine residue 222 to alanine (S222A) or to glutamate (S222E) and serines 212 and 218 together to alanine residues (S212A,S218A). Lysine 97 was mutated to alanine (K97A) to provide an inactive enzyme. Overexpression of the wild type MAPKK had no effect on any of the parameters examined. The K97A and S222A mutants served as dominant negatives by suppressing MAPKK, MAPK, and p90rsk activation in vivo. S222E enhanced all of these activities, and S212A,S218A had a small inhibitory effect. A similar trend was observed when cellular proliferation was examined and the different effects were accompanied by altered cellular shape. Taken together, our results demonstrate a direct linkage between the MAPK signaling pathway and the control of cellular proliferation and morphology and also establish that phosphorylation of serine 222 is essential for MAPKK activation together with the phosphorylation of an additional serine(s) (probably serine 218).