MPM-2 antibody-reactive phosphorylations can be created in detergent-extracted cells by kinetochore-bound and soluble kinases

1997 ◽  
Vol 110 (17) ◽  
pp. 2013-2025 ◽  
Author(s):  
L. Renzi ◽  
M.S. Gersch ◽  
M.S. Campbell ◽  
L. Wu ◽  
S.A. Osmani ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Hela Cell ◽  
Map Kinase ◽  
Kinase Inhibitors ◽  
Casein Kinase Ii ◽  
Cell Extract ◽  
Casein Kinase ◽  
Mitotic Chromosomes ◽  
Phosphatase Inhibitors ◽  
Hela Cell Extract

The MPM-2 antibody labels mitosis-specific and cell cycle-regulated phosphoproteins. The major phosphoproteins of mitotic chromosomes recognized by the MPM-2 antibody are DNA topoisomerase II (topoII) alpha and beta. In immunofluorescence studies of PtK1 cytoskeletons, prepared by detergent lysis in the presence of potent phosphatase inhibitors, the MPM-2 antibody labels phosphoproteins found at kinetochores, chromosome arms, midbody and spindle poles of mitotic cells. In cells extracted without phosphatase inhibitors, labeling of the MPM-2 antibodies at kinetochores is greatly diminished. However, in cytoskeletons this epitope can be regenerated through the action of kinases stably bound at the kinetochore. Various kinase inhibitors were tested in order to characterize the endogenous kinase responsible for these phosphorylations. We found that the MPM-2 epitope will not rephosphorylate in the presence of the broad specificity kinase inhibitors K-252a, staurosporine and 2-aminopurine. Several other inhibitors had no effect on the rephosphorylation indicating that the endogenous MPM-2 kinase at kinetochores is not p34cdc2, casein kinase II, MAP kinase, protein kinase A or protein kinase C. The addition of N-ethylmaleimide inactivated the endogenous kinetochore kinase; this allowed testing of several purified kinases in the kinetochore rephosphorylation assay. Active p34cdc2-cyclin B, casein kinase II and MAP kinase could not generate the MPM-2 phosphoepitope. However, bacterially expressed NIMA from Aspergillus and ultracentrifuged mitotic HeLa cell extract were able to catalyze the rephosphorylation of the MPM-2 epitope at kinetochores. Furthermore, fractionation of mitotic HeLa cell extract showed that kinases that create the MPM-2 epitope at kinetochores and chromosome arms are distinct. Our results suggest that multiple kinases (either soluble or kinetochore-bound), including a homolog of mammalian NIMA, can create the MPM-2 phosphoepitope. The kinetochore-bound kinase that catalyzes the formation of the MPM-2 phosphoepitope may play an important role in key events such as mitotic kinetochore assembly and sister chromatid separation at anaphase.

Involvement of Ras/MAP Kinase in the Regulation of Ca2+ Channels in Adult Bullfrog Sympathetic Neurons by Nerve Growth Factor

1998 ◽  
Vol 80 (3) ◽  
pp. 1352-1361 ◽  
Author(s):  
Saobo Lei ◽  
William F. Dryden ◽  
Peter A. Smith
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Protein Kinase ◽  
Map Kinase ◽  
Kinase Inhibitors ◽  
Sympathetic Neurons ◽  
Mitogen Activated Protein Kinase ◽  
Nerve Growth ◽  
Channel Current ◽  
Mitogen Activated Protein

Lei, Saobo, William F. Dryden, and Peter A. Smith. Involvement of Ras/MAP kinase in the regulation of Ca2+ channels in adult bullfrog sympathetic neurons by nerve growth factor. J. Neurophysiol. 80: 1352–1361, 1998. The cellular mechanisms that underlie nerve growth factor (NGF) induced increase in Ca2+-channel current in adult bullfrog sympathetic B-neurons were examined by whole cell recording techniques. Cells were maintained at low density in neuron-enriched, defined-medium, serum-free tissue culture for 6 days in the presence or absence of NGF (200 ng/ml). The increase in Ba2+ current ( I Ba) density induced by NGF was attenuated by the RNA synthesis inhibitor cordycepin (20 μM), by the DNA transcription inhibitor actinomycin D (0.01 μg/ml), by inhibitors of Ras isoprenylation (perillic acid 0.1–1.0 mM or α-hydroxyfarnesylphosphonic acid 10–100 μM), by tyrosine kinase inhibitors genistein (20 μM) or lavendustin A (1 μM), and by PD98059 (10–100 μM), an inhibitor of mitogen-activated protein kinase kinase. Inhibitors of the phosphatidylinositol 3-kinase (PI3K) pathway (wortmannin, 100 nM, or LY29400, 100 μM) were ineffective as were inhibitors of phospholipase Cγ (U73122 or neomycin, both 100 μM). The effect of NGF persisted in Ca2+-free medium that contained 1.8 mM Mg2+ and 2 mM ethylene glycol-bis(β-aminoethyl ether)- N, N, N′, N′-tetraacetic acid. It was mimicked by a Trk antibody that was capable of inducing neurite outgrowth in explant cultures of bullfrog sympathetic ganglion. Antibodies raised against the low-affinity p75 neurotrophin receptor were ineffective in blocking the effect of NGF on I Ba. These results suggest that NGF-induced increase in Ca2+ channel current in adult sympathetic neurons results, at least in part, from new channel synthesis after Trk activation of Ras and mitogen activated protein kinase by a mechanism that is independent of extracellular Ca2+.

scholarly journals Inhibition of Wnt/β-Catenin Signaling by p38 MAP Kinase Inhibitors Is Explained by Cross-Reactivity with Casein Kinase Iδ/ɛ

2011 ◽  
Vol 18 (4) ◽  
pp. 485-494 ◽  
Author(s):  
Folkert Verkaar ◽  
Antoon A. van der Doelen ◽  
Jos F.M. Smits ◽  
W. Matthijs Blankesteijn ◽  
Guido J.R. Zaman
Keyword(s):  
Map Kinase ◽  
Kinase Inhibitors ◽  
Cross Reactivity ◽  
P38 Map Kinase ◽  
Casein Kinase ◽  
P38 Map Kinase Inhibitors

Inhibition of protein kinase C- and casein kinase II-mediated phosphorylation of GAP-43 by S100β

1994 ◽  
Vol 25 (3-4) ◽  
pp. 297-304 ◽  
Author(s):  
Li-Hsien Lin ◽  
Linda J. Van Eldik ◽  
Neil Osheroff ◽  
Jeanette J. Norden
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Kinase C

Identification of Glycyrrhizin-Binding Protein Kinase as Casein Kinase II and Characterization of Its Associated Phosphate Acceptors in Mouse Liver

1996 ◽  
Vol 227 (1) ◽  
pp. 102-109 ◽  
Author(s):  
Shigeyoshi Harada ◽  
Atsushi Karino ◽  
Yoshihito Shimoyama ◽  
Fazel Shamsa ◽  
Kenzo Ohtsuki
Keyword(s):  
Protein Kinase ◽  
Mouse Liver ◽  
Binding Protein ◽  
Casein Kinase Ii ◽  
Casein Kinase

Transcription factor binding is limited by the 5'-flanking regions of a Drosophila tRNAHis gene and a tRNAHis pseudogene

1984 ◽  
Vol 4 (12) ◽  
pp. 2714-2722
Author(s):  
L Cooley ◽  
J Schaack ◽  
D J Burke ◽  
B Thomas ◽  
D Söll
Keyword(s):  
Complex Formation ◽  
Hela Cell ◽  
Cell Extract ◽  
Deletion Analysis ◽  
Stable Complex ◽  
Flanking Sequence ◽  
Factor Binding ◽  
Real Gene ◽  
Hela Cell Extract ◽  
Flanking Regions

We determined the sequence of a Drosophila tRNA gene cluster containing a tRNAHis gene and a tRNAHis pseudogene in close proximity on the same DNA strand. The pseudogene contains eight consecutive base pairs different from the region of the bona fide gene which codes for the 3' portion of the anticodon stem of tRNAHis. The tRNAHis gene is transcribed efficiently in Drosophila Kc cell extract, whereas the pseudogene is not. The pseudogene is also a much poorer competitor than the real gene in a stable transcription complex formation assay, even though the sequence alteration in the pseudogene does not affect the sequence or spacing of the putative internal transcription control regions. Recombinant clones were constructed in which the 5'-flanking regions are exchanged. The transcription efficiencies and competitive abilities of the recombinant clones resemble those of the genes from which the 5' flank was derived; for example, the tRNAHis pseudogene with the 5'-flanking sequence of the tRNAHis gene is now efficiently transcribed. Deletion analysis of the pseudogene 5' flank failed to uncover an inhibitory element. Deletion analysis of the real gene showed very high dependence on the presence of the wild-type 5'-flanking sequence for factor binding to the internal control regions and stable complex formation. The 5'-flanking sequence of a Drosophila tRNAArg gene active in the Drosophila Kc cell extract does not restore transcriptional activity or stable complex formation. The tRNAHis gene and pseudogene behave atypically in HeLa cell extract. Both genes compete for HeLa transcription factors, but neither of them is efficiently transcribed. Removal of the 5'-flanking sequences of each gene and replacement with various sequences, including the tRNAArg gene 5' flank, does not allow increased transcription in HeLa cell extract.

Nucleoplasmin associates with and is phosphorylated by casein kinase II

1995 ◽  
Vol 108 (2) ◽  
pp. 779-787 ◽  
Author(s):  
I. Vancurova ◽  
T.M. Paine ◽  
W. Lou ◽  
P.L. Paine
Keyword(s):  
Protein Kinase ◽  
Polyclonal Antibody ◽  
Nuclear Transport ◽  
Specific Inhibitor ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Transferase Activity ◽  
Catalytic Subunits ◽  
Kinetics Of

Nucleoplasmin is a phosphorylated nuclear-accumulating protein. We report herein that the kinetics of its cytoplasm-->nucleus transport are affected by its degree of phosphorylation. Therefore, we sought to identify any protein kinase which specifically associates with nucleoplasmin. We discovered that nucleoplasmin co-isolates by two independent methods (immunoabsorption and chromatography) in a complex including a kinase which phosphorylates nucleoplasmin. The co-purifying kinase is casein kinase II-like because: (i) it phosphorylates casein; (ii) its phospho-transferase activity can be competed out by GTP; (iii) it is stimulated by polylysine; and (iv) it is inhibited by heparin. Moreover, a polyclonal antibody to the alpha (38 kDa) and alpha' (36 kDa) catalytic subunits of casein kinase II specifically recognizes 38 and 36 kDa polypeptides in the nucleoplasmin-complex, and a specific inhibitor of casein kinase II inhibits nucleoplasmin's nuclear transport. Additionally, we found that phosphorylation of nucleoplasmin by its associated casein kinase II is strongly inhibited by histones and that, in addition to nucleoplasmin, another protein (p100) in the nucleoplasmin-complex is phosphorylated by casein kinase II.

scholarly journals Casein kinase II is required for efficient transcription by RNA polymerase III.

1996 ◽  
Vol 16 (3) ◽  
pp. 892-898 ◽  
Author(s):  
D J Hockman ◽  
M C Schultz
Keyword(s):  
Protein Kinase ◽  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Rna Polymerases ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Polymerase Iii

Casein kinase II (CKII) is a ubiquitous and highly conserved serine/threonine protein kinase found in the nucleus and cytoplasm of most cells. Using a combined biochemical and genetic approach in the yeast Saccharomyces cerevisiae, we assessed the role of CKII in specific transcription by RNA polymerases I, II, and III. CKII is not required for basal transcription by RNA polymerases I and II but is important for polymerase III transcription. Polymerase III transcription is high in extracts with normal CKII activity but low in extracts from a temperature-sensitive mutant that has decreased CKII activity due to a lesion in the enzyme's catalytic alpha' subunit. Polymerase III transcription of 5S rRNA and tRNA templates in the temperature-sensitive extract is rescued by purified, wild-type CKII. An inhibitor of CKII represses polymerase III transcription in wild-type extract, and this repression is partly overcome by supplementing reaction mixtures with active CKII. Finally, we show that polymerase III transcription in vivo is impaired when CKII is inactivated. Our results demonstrate that CKII, an oncogenic protein kinase previously implicated in cell cycle and growth control, is required for high-level transcription by RNA polymerase III.

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