The activity of CK2 in the extracts of COS-7 cells transfected with wild type and mutant subunits of protein kinase CK2.

Protein kinase CK2 is ubiquitous in eukaryotes and is known to phosphorylate many protein substrates. The enzyme is normally a heterotetramer composed of catalytic (alpha and alpha') and regulatory (beta) subunits. The physiological regulation of the enzyme is still unknown but one of the factors that may play an important role in this regulation is the ratio of the catalytic and regulatory subunits present in cells. The possible existence of 'free' CK2 subunits, not forming part of the holoenzyme, may be relevant to the physiological function of the enzyme in substrate selection or in the interaction of the subunits with other partners. The objective of this work was to study in COS-7 cells the effects of transient expression of CK2 subunits and mutants of the catalytic subunit on the CK2 phosphorylating activity of the extracts of these cells. Using pCEFL vectors that introduce hemagglutinin (HA) or a heptapeptide (AU5) tags in the expressed proteins, COS-7 cells were transfected with alpha and beta subunits of Xenopus CK2, with the alpha' subunit of D. rerio, and with Xl CK2alphaA156, which although inactive can bind tightly to CK2beta, and with Xl CK2alphaE75E76, which is resistant to heparin and polyanion inhibition. The efficiency of transient transfection was of 10-20% of treated cells. Expression of CK2alpha or CK2alphaE75E76 in COS-7 cells caused an increase of 5-7-fold of the CK2 activity in the soluble cell extracts. If these catalytic subunits were cotransfected with CK2beta, the activity increased further to 15-20-fold of the controls. Transfection of CK2beta alone also increase the activity of the extracts about 2-fold. Transfection with the inactive CK2alphaA156 yielded extracts with CK2 activities not significantly different from those transfected with the empty vectors. However, co-transfection of CK2alpha or CK2alphaE75E76 with CK2alphaA156 caused a 60-70% decrease in the CK2 activity as compared to those of cells transfected with only the active CK2alpha subunits. These results can be interpreted as meaning that CK2alphaA156 is a dominant negative mutant that can compete with the other catalytic subunits for the CK2beta subunit. Addition of recombinant CK2beta to the assay system of extracts of cells transfected with catalytic subunits causes a very significant increase in their CK2 activity, demonstrating that CK2beta subunit is limiting in the extracts and that an excess of free CK2alpha has been produced in the transfected cells. Transfection of cells with CK2alphaE75E76 results in a CK2 activity of extracts that is 90% resistant to heparin demonstrating that a very large proportion of the CK2 activity is derived from the expression of the exogenous mutant. In both the in vivo and in vitro systems, the sensitivity of CK2alphaE75E76 to heparin increases considerably when it forms part of the holoenzyme CK2alpha2beta2.

Structural interpretation of site-directed mutagenesis and specificity of the catalytic subunit of protein kinase CK2 using comparative modelling.

The catalytic subunit of protein kinase casein kinase 2 (CK2alpha), which has specificity for both ATP and GTP, shows significant amino acid sequence similarity to the cyclin-dependent kinase 2 (CDK2). We constructed site-directed mutants of CK2alpha and used a three-dimensional model to investigate the basis for the dual specificity. Introduction of Phe and Gly at positions 50 and 51, in order to restore the pattern of the glycine-rich motif, did not seriously affect the specificity for ATP or GTP. We show that the dual specificity probably originates from the loop situated around the position His115 to Asp120 (HVNNTD). The insertion of a residue in this loop in CK2 alpha subunits, compared with CDK2 and other kinases, might orient the backbone to interact with the base A and G; this insertion is conserved in all known CK2alpha. The mutant deltaN118, the design of which was based on the modelling, showed reduced affinity for GTP as predicted from the model. Other mutants were intended to probe the integrity of the catalytic loop, alter the polarity of a buried residue and explore the importance of the carboxy terminus. Introduction of Arg to replace Asn189, which is mapped on the activation loop, results in a mutant with decreased k(cat), possibly as a result of disruption of the interaction between this residue and basic residues in the vicinity. Truncation at position 331 eliminates the last 60 residues of the alpha subunit and this mutant has a reduced catalytic efficiency compared with the wild-type. Catalytic efficiency is restored in the truncation mutant by the replacement of a potentially buried Glu at position 252 by Lys, probably owing to a higher stability resulting from the formation of a salt bridge between Lys252 and Asp208.

Interactions of protein kinase CK2 subunits.

Several approaches have been used to study the interactions of the subunits of protein kinase CK2. The inactive mutant of CK2alpha that has Asp 156 mutated to Ala (CK2alphaA156) is able to bind the CK2beta subunit and to compete effectively in this binding with wild-type subunits alpha and alpha'. The interaction between CK2alphaA156 and CK2beta was also demonstrated by transfection of epitope-tagged cDNA constructs into COS-7 cells. Immunoprecipitation of epitope-tagged CK2alphaA156 coprecipitated the beta subunit and vice-versa. The assay of the CK2 activity of the extracts obtained from cells transiently transfected with these different subunits yielded some surprising results: The CK2 specific phosphorylating activity of these cells transfected with the inactive CK2alphaA156 was considerably higher than the control cells transfected with vectors alone. Assays of the immunoprecipitated CK2alphaA156 expressed in these cells, however, demonstrated that the mutant was indeed inactive. It can be concluded that transfection of the inactive CK2alphaA156 affects the endogenous activity of CK2. Transfection experiments with CK2alpha and beta subunits and CK2alphaA156 were also used to confirm the interaction of CK2 with the general CDK inhibitor p21WAF1/CIP1 co-transfected into these cells. Finally a search in the SwissProt databank for proteins with properties similar to those derived from the amino acid composition of CK2beta indicated that CK2beta is related to protein phosphatase 2A and to other phosphatases as well as to a subunit of some ion-transport ATPases.

Familial intracranial arachnoid cysts.

Three siblings with intracranial arachnoid cysts are described, two males and one female. One of the males has symmetric, bilateral, temporoparietal convexity cysts, and the others have singular, unilateral cysts. Three additional siblings in the family and other known relatives are clinically unaffected. As far as we know, this is the second reported case of familial intracranial arachnoid cysts and the first involving three siblings. The significance of these cysts and a review of the literature are presented.

Dopamine receptors on isolated membranes of rat thymocytes.

Using [3H]DA as the ligand of choice, we have shown that rat thymocytes possess dopamine binding sites. The binding of [3H]DA was specific, saturable and of high affinity. [3H]DA binding was rapid and reversible; [3H]DA was totally displaced by low concentrations of dopamine, norepinephrine, and apomorphine. The binding sites were insensitive to neuroleptic drugs such as sulpiride, bromocriptine, and haloperidol. On the basis of these findings, we have suggested that thymocytes possess a D-3 receptor type.

Adult rat oligodendrocytes grown in vitro upon an extracellular matrix have the ability to proliferate.

The use of extracellular matrix (ECM) as a natural substrate for cell culture has markedly improved the growth and morphological differentiation of isolated adult rat oligodendrocytes. ECM-grown oligodendrocytes exhibited cyclic nucleotide phosphodiesterase (CNP)-activity which increased with time in culture and network formation. As much as 50-70% of the cells incorporated [3H]thymidine as visualized by the high labeling index of galactocerebroside (GalC)-positive cells. Chemical and enzymatic modifications of the ECM suggested that laminin in conjunction with other ECM constituents, plays a role in the induction of proliferation and/or differentiation responses in mature oligodendrocytes.

Human alpha-fetoprotein-rich fraction inhibits galactocerebroside antibody-mediated lysis of oligodendrocytes in vitro.

Polyclonal rabbit antiserum to galactocerebroside (anti-GalC) produces titer-dependent lysis of cultured Percoll-isolated bovine and rat oligodendrocytes. In this study anti-GalC produced complement-dependent lysis of 76% of the bovine cells and 65% of the rat cells maintained for 3 to 6 days in vitro. With the concomitant addition of human umbilical cord serum fractions containing fetal alpha-fetoprotein (AFP), lysis was decreased to 31% and 39%, respectively. Control antisera (anti-complete Freund's adjuvant/albumin) showed a cytotoxicity index of 15% for bovine cells and 23% for rat cells. Neither albumin, nor normal human serum, nor any of several pregnancy-associated hormones reduced the lysis produced by anti-GalC. AFP-rich fraction reduced oligodendrocyte lysis when mixed with anti-GalC or complement, but not when first incubated with the cells. Similar findings were obtained when AFP was assayed in specific oligodendrocyte radioimmunoassays utilizing anti-GalC antibody. Our experiments indicate that AFP activity may result from its binding to anti-GalC antibody; it is possible that the Fc portion of the antibody is involved. These data provide in vitro evidence of a possible immunosuppressive role of AFP in the central nervous system.

Enhanced growth and morphological differentiation of isolated adult rat oligodendrocytes in vitro: use of a naturally produced extracellular matrix.

Attachment, growth and morphological differentiation of isolated adult rat oligodendrocytes cultured on a naturally produced basement membrane-like extracellular matrix (ECM) occurred much faster than on poly-L-lysine (PLL) coated tissue culture dishes. In each individual trial the cells cultured on ECM exhibited, within 3-5 days in culture, a massive outgrowth of long and branched cytoplasmic processes. Outgrowth to such an extent, using PLL or plastic tissue culture dishes, was not observed even after 2 weeks in culture. The successful high plating efficiency, rapid growth and network formation as well as its resemblance to the in vivo environment of cells make this naturally produced substrate a superior substitute for PLL and therefore more attractive for studying the behavior and function of oligodendrocytes in vitro.

Anterior decompression of the spine for metastatic epidural cord compression: a promising avenue of therapy?

Most metastatic epidural tumors arise in a vertebral body and invade the anterior epidural space. Therefore, it is logical to decompress the spine anteriorly and not by traditional laminectomy. Surgical decompression is indicated if relapse occurs after radiotherapy and further radiation cannot be administered, if there is neurological deterioration during radiotherapy, and when histological diagnosis of the primary tumor is lacking. This pilot study consists of eleven consecutive anterior decompressions of the spine performed in nine patients. In seven instances other treatment modalities had been exhausted, and in four patients a tissue diagnosis was lacking. Before operation eight of the patients were nonambulatory, four of them paraplegic. Following decompression all but one patient became ambulatory. At operation the main bulk of the compressing tumor was found anterior or anterolateral to the cord. Spine stabilization was done when stability was a problem. Wound infection in one patient was the only postoperative complication. The encouraging outcome of our management prompts us to suggest that anterior decompression of the spine should be considered more often in metastatic compression of the cord and cauda equina.

Myasthenia gravis following viral infection.

The pathogenesis of myasthenia gravis is autoimmune, the real etiology, however, remains unknown. Virus has been suggested as an etiological agent of the disease. In this study we present 5 myasthenic patients, whose symptoms began a few weeks after a proven viral infection. The possibility of viral infection as etiology of myasthenia gravis is raised, and the mechanisms discussed.