Regulation of Erythroid Krüppel-like Factor (EKLF) Transcriptional Activity by Phosphorylation of a Protein Kinase Casein Kinase II Site within Its Interaction Domain

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.

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

Journal of Cell Science ◽

10.1242/jcs.110.17.2013 ◽

1997 ◽

Vol 110 (17) ◽

pp. 2013-2025 ◽

Cited By ~ 3

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.

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A role for casein kinase II phosphorylation in the regulation of IRF-1 transcriptional activity

A Molecular and Cellular View of Protein Kinase CK2 ◽

10.1007/978-1-4419-8624-5_21 ◽

1999 ◽

pp. 169-180

Author(s):

Rongtuan Lin ◽

John Hiscott

Keyword(s):

Transcriptional Activity ◽

Casein Kinase Ii ◽

Casein Kinase

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Protein phosphatases active on acetyl-CoA carboxylase phosphorylated by casein kinase I, casein kinase II and the cAMP-dependent protein kinase

Biochemical and Biophysical Research Communications ◽

10.1016/0006-291x(85)91733-4 ◽

1985 ◽

Vol 130 (3) ◽

pp. 1132-1138 ◽

Cited By ~ 10

Author(s):

Lee A. Witters ◽

Geoffrey W. Bacon

Keyword(s):

Protein Kinase ◽

Protein Phosphatases ◽

Casein Kinase Ii ◽

Casein Kinase ◽

Acetyl Coa Carboxylase ◽

Casein Kinase I ◽

Dependent Protein Kinase ◽

Acetyl Coa ◽

Camp Dependent Protein Kinase ◽

Dependent Protein

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

Molecular and Cellular Biology ◽

10.1128/mcb.16.3.892 ◽

1996 ◽

Vol 16 (3) ◽

pp. 892-898 ◽

Cited By ~ 34

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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Identifying and characterizing casein kinase II in human platelets

Blood ◽

10.1182/blood.v83.12.3517.3517 ◽

1994 ◽

Vol 83 (12) ◽

pp. 3517-3523 ◽

Cited By ~ 12

Author(s):

CH Hoyt ◽

CJ Oh ◽

JB Beekman ◽

DW Litchfield ◽

KM Lerea

Keyword(s):

Protein Kinase ◽

Protein Kinases ◽

Casein Kinase Ii ◽

Human Platelets ◽

Casein Kinase ◽

Immunogold Electron Microscopy ◽

Beta Subunits ◽

Specific Substrate ◽

Substrate Peptide ◽

Independent Protein

Abstract We have recently shown that inhibition of protein phosphatases in platelets causes increases in protein phosphorylations with a concomitant inhibition of platelet responses. The burst in protein phosphorylation appears to be catalyzed by messenger-independent protein kinases. The aim of the present study was to characterize the presence of broad families of protein kinases found in platelets. Lysates of control and thrombin-stimulated platelets were prepared, and proteins were separated on MONO Q fast protein liquid chromatography. In addition to the presence of histone protein kinase and tyrosine kinase activities, human platelets contain casein kinase II (CKII) activity as assessed by phosphorylation of a specific substrate peptide. Western blot analysis and immunogold electron microscopy studies further showed the presence of alpha-, alpha'-, and beta- subunits of CKII. The enzyme appears to be distributed throughout the cytosol and not secreted after thrombin treatment. Immunoprecipitation studies suggest that at least some of the holoenzymes exist as an alpha alpha' beta 2 complex. Although no activation of the enzyme was detected after thrombin treatment, our results show that CKII is a major messenger-independent protein kinase in platelets.

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DNA repair protein O6-alkylguanine-DNA alkyltransferase is phosphorylated by two distinct and novel protein kinases in human brain tumour cells

Biochemical Journal ◽

10.1042/bj3510393 ◽

2000 ◽

Vol 351 (2) ◽

pp. 393-402 ◽

Cited By ~ 12

Author(s):

Srinivas R. S. MULLAPUDI ◽

Francis ALI-OSMAN ◽

Jiang SHOU ◽

Kalkunte S. SRIVENUGOPAL

Keyword(s):

Dna Repair ◽

Protein Kinase ◽

Tyrosine Kinase ◽

Protein Kinases ◽

Casein Kinase Ii ◽

Casein Kinase ◽

Biochemical Modulation ◽

Cell Extracts ◽

Dna Alkyltransferase

We showed recently that human O6-alkylguanine-DNA alkyltransferase (AGT), an important target for improving cancer chemotherapy, is a phosphoprotein and that phosphorylation inhibits its activity [Srivenugopal, Mullapudi, Shou, Hazra and Ali-Osman (2000) Cancer Res. 60, 282–287]. In the present study we characterized the cellular kinases that phosphorylate AGT in the human medulloblastoma cell line HBT228. Crude cell extracts used Mg2+ more efficiently than Mn2+ for phosphorylating human recombinant AGT (rAGT) protein. Both [γ-32P]ATP and [γ-32P]GTP served as phosphate donors, with the former being twice as efficient. Specific components known to activate protein kinase A, protein kinase C and calmodulin-dependent kinases did not stimulate the phosphorylation of rAGT. Phosphoaminoacid analysis after reaction in vitro with ATP or GTP showed that AGT was modified at the same amino acids (serine, threonine and tyrosine) as in intact HBT228 cells. Although some of these properties pointed to casein kinase II as a candidate enzyme, known inhibitors and activators of casein kinase II did not affect rAGT phosphorylation. Fractionation of the cell extracts on poly(Glu/Tyr)-Sepharose resulted in the adsorption of an AGT kinase that modified the tyrosine residues and the exclusion of a fraction that phosphorylated AGT on serine and threonine residues. In-gel kinase assays after SDS/PAGE and non-denaturing PAGE revealed the presence of two AGT kinases of 75 and 130kDa in HBT228 cells. The partly purified tyrosine kinase, identified as the 130kDa enzyme by the same assays, was strongly inhibited by tyrphostin 25 but not by genestein. The tyrosine kinase used ATP or GTP to phosphorylate the AGT protein; this reaction inhibited the DNA repair activity of AGT. Evidence that the kinases might physically associate with AGT in cells was also provided. These results demonstrate that two novel cellular protein kinases, a tyrosine kinase and a serine/threonine kinase, both capable of using GTP as a donor, phosphorylate the AGT protein and affect its function. The new kinases might serve as potential targets for strengthening the biochemical modulation of AGT in human tumours.

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