Phosphorylation of fibrinogen by casein kinase 1

Casein kinase 1 phosphorylated human fibrinogen, in a reaction that did not use GTP as phosphoryl donor and was neither stimulated by cyclic AMP or Ca2+, nor inhibited by the cyclic AMP-dependent protein kinase inhibitor protein. Maximal incorporation averaged 4 mol of phosphate per mol of fibrinogen, most of it in the largest CNBr-fragment of the alpha-chain. Phosphoamino acid analysis revealed that phosphorylation occurred only at seryl residues. The phosphorylation of fibrinogen by casein kinase 1 was reverted by alkaline phosphatase.     

Phosphorylation of casein by cyclic AMP-dependent protein kinase.

The catalytic subunit of rabbit muscle cyclic AMP-dependent protein kinase (EC 2.7.1.37; ATP:protein transferase) has been tested on a variety of caseins. The B variant of β-casein was phosphorylated at a much greater rate than other β-caseins, α_s1-caseins, and κ-caseins. Whole casein homozygous for β-casein B was phosphorylated at 2.5 times the rate of commercial whole casein. Gel electrophoresis experiments indicate that β-casein is the predominant component phosphorylated in commerical casein. It is therefore suggested that phosphorylation of whole casein depends on its content of the specific genetic variant, β-casein B.     

Phosphorylation of maize RAB-17 protein by casein kinase 2

The maize gene RAB-17, which is responsive to abscisic acid, encodes a basic glycine-rich protein containing, in the middle part of its sequence, a cluster of 8 serine residues followed by a putative casein kinase 2-type substrate consensus sequence. This protein was found to be highly phosphorylated in vivo. Here, we show that RAB-17 protein is a real substrate for casein kinase 2. RAB-17 protein is phosphorylated in vitro by casein kinase 2 isolated from rat liver cytosol and from maize embryos. A maximum of 4 mol of phosphate were incorporated per mol of RAB-17 protein following incubation with casein kinase 2. Phosphopeptide mapping experiments show that the peptide phosphorylated by casein kinase 2 in vitro is identical to that derived from the protein phosphorylated in vivo. Purification by high performance liquid chromatography and partial sequencing of the phosphopeptide indicate that it corresponds to the region of the protein (residues 56-89) containing the cluster of serine residues. Our results indicate that RAB-17 is phosphorylated by casein kinase 2 or a kinase with a similar specificity and that phosphorylation takes place in the serine cluster region of the protein both in vitro and in vivo.     

Phosphorylation of casein kinase II

Casein kinase II from rabbit reticulocytes is a tetramer with an alpha,alpha' beta 2 or alpha 2 beta 2 structure; the alpha subunits contain the catalytic activity, and the beta subunits are regulatory in nature [Traugh, J.A., Lin, W. J., Takada-Axelrod, F., & Tuazon, P. T. (1990) Adv. Second Messenger Phosphoprotein Res. 24, 224-229]. When casein kinase II is isolated from rabbit reticulocytes by a rapid two-step purification of the enzyme, both the alpha and beta subunits are phosphorylated to a significant extent. In vitro, purified casein kinase II undergoes autophosphorylation on the beta subunit. In the presence of polylysine and polyarginine, phosphorylation of the beta subunits is inhibited, and the alpha subunits (alpha and alpha') become autophosphorylated. The effectiveness of polylysine coincides with the molecular weight. With basic proteins, including a number of histones and protamine, autophosphorylation of both subunits is observed. With histones, autophosphorylation of each subunit can be greater than that observed with the autophosphorylated enzyme alone or with a basic polypeptide. Thus, the potential exists for modulatory proteins to alter the autophosphorylation state of casein kinase II. Taken together, the data suggest that phosphorylation of the alpha subunit of casein kinase II in vivo may be due to an unidentified protein kinase or due to autophosphorylation. In the latter instance, casein kinase II could be transiently associated with specific intracellular compounds, such as basic proteins, with a resultant stimulation of autophosphorylation.     

Phosphorylation of tryptophanyl-tRNA-synthetase by casein kinase type II

Incubation of tryptophanyl-tRNA synthetase from bovine pancrease with [gamma-32P]ATP of [gamma-32P]GTP and casein kinase II from rabbit liver leads to the incorporation of labeled phosphate into serine residues of synthetase polypeptide. The maximal level of 32P incorporation into synthetase polypeptide (Mr = 60 kDa) 0.15 moles of 32P per 1 mole of polypeptide was observed. Electrophoretic analysis according to O'Farrell showed that kinase phosphorylates exclusively the most acidic polypeptides (pI 4.9) of the synthetase preparation. Pretreatment of synthetase with animal acidic and alkaline phosphatases had no influence on the level of 32P incorporation in synthetase during subsequent incubation in the presence of casein kinase II.     

Phosphorylation by casein kinase 2 regulates Nap1 localization and function

In Saccharomyces cerevisiae, the evolutionarily conserved nucleocytoplasmic shuttling protein Nap1 is a cofactor for the import of histones H2A and H2B, a chromatin assembly factor and a mitotic factor involved in regulation of bud formation. To understand the mechanism by which Nap1 function is regulated, Nap1-interacting factors were isolated and identified by mass spectrometry. We identified several kinases among these proteins, including casein kinase 2 (CK2), and a new bud neck-associated protein, Nba1. Consistent with our identification of the Nap1-interacting kinases, we showed that Nap1 is phosphorylated in vivo at 11 sites and that Nap1 is phosphorylated by CK2 at three substrate serines. Phosphorylation of these serines was not necessary for normal bud formation, but mutation of these serines to either alanine or aspartic acid resulted in cell cycle changes, including a prolonged S phase, suggesting that reversible phosphorylation by CK2 is important for cell cycle regulation. Nap1 can shuttle between the nucleus and cytoplasm, and we also showed that CK2 phosphorylation promotes the import of Nap1 into the nucleus. In conclusion, our data show that Nap1 phosphorylation by CK2 appears to regulate Nap1 localization and is required for normal progression through S phase.     

Phosphorylation in vitro of human fibrinogen with casein kinase TS and characterization of phosphorylated sites

Human fibrinogen was phosphorylated by casein kinase TS. The [32P]phosphate incorporated varied between 0.5 and 1 mol of phosphate per mole of fibrinogen. The phosphate was localized to Ser523 and Ser590 and serine and threonine residues between amino acids 259 and 268 in the A alpha-chain. In addition, Thr416 and Ser420 were phosphorylated in the gamma'-chain, which is a variant of the gamma-chain, constituting 7-10% of the gamma-chain population. The functional significance of casein kinase TS-induced phosphorylation of fibrinogen remains unknown; however, a slight but consistent increase of the turbidity in a gelation assay was observed for phosphorylated compared to unphosphorylated fibrinogen.     

Phosphorylation of protein kinase C by casein kinase-1

Because phosphorylation of protein kinase C (PKC) may provide a mechanism for regulation of this enzyme, we have examined the ability of two other kinases to phosphorylate PKC. Our results show that casein kinase 1 (CK-1), but not casein kinase 2 (CK-2), can phosphorylate PKC in the absence of Ca2+ and phospholipids. The 32P incorporation into PKC in the presence of Ca2+ and phospholipids is also enhanced by CK-1.     

Phosphorylation of the insulin receptor by a casein kinase I-like enzyme.

A serine protein kinase that phosphorylates the beta-subunit of the insulin receptor has been partially purified 5,000-fold from HeLa cell membranes. The enzyme has been purified by ion-exchange and hydroxylapatite chromatography and sucrose gradient centrifugation; it has an apparent molecular weight of 36,000-43,000 daltons. It exhibits the following properties: (a) it catalyzes the phosphorylation of the autophosphorylated insulin receptor more efficiently than the nonautophosphorylated insulin receptor, (b) it decreases insulin receptor phosphorylation of tubulin but has no effect on insulin receptor phosphorylation of microtubule-associated proteins or reduced and carboxyamidomethylated lysozyme. The enzyme also phosphorylates casein and ribosomal protein S6 and shares many properties with casein kinase I: (a) similar molecular weight, (b) utilization of ATP but not GTP as phosphoryl donor, and (c) sensitivity to inhibition by heparin. Based on several criteria the receptor serine kinase is neither protein kinase C nor the cAMP-dependent protein kinase.     

Phosphorylation of rat liver mitochondrial glycerol-3-phosphate acyltransferase by casein kinase 2

We have previously shown rat liver mitochondrial glycerol-3-phosphate acyltransferase (mtGAT), which catalyzes the first step in de novo glycerolipid biosynthesis, is stimulated by casein kinase 2 (CK2) and that a phosphorylated protein of approximately 85 kDa is present in CK2-treated mitochondria. In this paper, we have identified the (32)P-labeled 85-kDa protein as mtGAT. We have also investigated whether the phosphorylation of mtGAT is because of CK2. Mitochondria were treated with CK2 and [gamma-(32)P]GTP as the phosphate donor. Autoradiography, Western blot, and immunoprecipitation results showed mtGAT was phosphorylated by CK2. Next, we incubated mitochondria with CK2 and either ATP or GTP, in the presence of heparin, a known inhibitor of CK2. Heparin inhibited CK2-induced stimulation of mtGAT activity; this inhibition resulted in decreased (32)P-labeling of mtGAT. Additionally, mitochondria were treated with CK2 and [gamma-(32)P]ATP in the presence of staurosporine (a serine/threonine protein kinase inhibitor), genistein (a tyrosine kinase inhibitor), and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB, a CK2 inhibitor). Only DRB, the CK2 inhibitor, greatly reduced the amount of (32)P-incorporation into mtGAT by CK2. Finally, isolated mitochondrial outer membrane was incubated with cytosol in the presence of [gamma-(32)P]GTP; (32)P-labeled mtGAT was detected. Collectively, these data suggest that CK2 phosphorylates mtGAT. The impact of our results in the regulation of mtGAT and other anabolic processes is discussed.     

Phosphorylation of type II (beta) protein kinase C by casein kinase II.

Rat brain type II (beta) protein kinase C (PKC) was phosphorylated by rat lung casein kinase II (CK-II). Neither type I (gamma) nor type III (alpha) PKC was significantly phosphorylated by CK-II. CK-II incorporated 0.2-0.3 mol of phosphate into 1 mol of type II PKC. This phosphate was located at the single seryl residue (Ser-11) in the V1-variable region of the regulatory domain of the PKC molecule. A glutamic acid cluster was located at the carboxyl-terminal side of Ser-11, showing the consensus sequence for phosphorylation by CK-II. The velocity of this phosphorylation was enhanced by the addition of Ca2+, diolein, and phosphatidylserine, which are all required for the activation of PKC. Phosphorylation of casein or synthetic oligopeptides by CK-II was not affected by Ca2+, diolein, or phosphatidylserine. Available evidence suggests that CK-II phosphorylates preferentially the activated form of type II PKC. It remains unknown, however, whether this reaction has a physiological significance.     

Phosphorylation of casein kinase II by p34cdc2 in vitro and at mitosis.

In human epidermal carcinoma A431 cells, the beta subunit of casein kinase II is phosphorylated at an autophosphorylation site and at serine 209 which can be phosphorylated in vitro by p34cdc2 (Litchfield, D. W., Lozeman, F. J., Cicirelli, M. F., Harrylock, M., Ericsson, L. H., Piening, C. J., and Krebs, E. G. (1991) J. Biol. Chem. 266, 20380-20389). Given the importance of p34cdc2 in the regulation of cell cycle events, we were interested in examining the phosphorylation of casein kinase II during different stages of the cell cycle. In this study it is demonstrated that the extent of phosphorylation of serine 209 in the beta subunit is significantly increased relative to phosphorylation of the autophosphorylation site when chicken bursal lymphoma BK3A cells are arrested at mitosis by nocodazole treatment. This result suggests that serine 209 is a likely physiological target for p34cdc2. In addition, the alpha subunit of casein kinase II also undergoes dramatic phosphorylation with an associated alteration in its electrophoretic mobility when BK3A cells or human Jurkat cells are arrested with nocodazole. Phosphopeptide mapping studies indicate that p34cdc2 can phosphorylate in vitro the same peptides on the alpha subunit that are phosphorylated in cells arrested at mitosis. These phosphorylation sites were localized to serine and threonine residues in the carboxyl-terminal domain of alpha. Taken together, the results of this study indicate that casein kinase II is a probable physiological substrate for p34cdc2 and suggest that its functional properties could be affected in a cell cycle-dependent manner.     

Phosphorylation of casein by mitochondrial protein kinase(s)

Summary Chromatin fractions (DNA, histones and nonhistone chromosomal proteins NHCP) have been isolated from human peripheral B and T lymphocytes using different methods and analyzed in order to identify their lipid content.While DNA and histone fractions do not reveal the presence of lipids, a 2% of phospholipids is present in the NHCP fraction. The phospholipids associated with NHCP present a constant relative ratio among sphingomyelin, phosphatidyl-choline and phosphatidyl-ethanolamine both in B and T lymphocytes, whichever are the extraction procedures employed.These findings are related to the possible derepressive role of phospholipids on DNA-dependent RNA synthesis.     

Phosphorylation and regulation of beta-catenin by casein kinase I epsilon

beta-Catenin transduces cytosolic signals to the nucleus in the Wnt pathway. The Wnt ligand stabilizes cytosolic beta-catenin protein, preventing its phosphorylation by inhibiting glycogen synthase kinase 3 (GSK3). Serine-33 and -37 of beta-catenin are GSK3 phosphorylation sites that serve as recognition sites for the beta-TRCP-ubiquitin ligase complex, which ultimately triggers beta-catenin degradation. Mutations at those two sites, as well as in Ser-45, stabilize beta-catenin. Recently, casein kinase I epsilon (CKI epsilon) has been shown to be a positive regulator of the Wnt pathway. Its action mechanism, however, remains unknown. Here I show that Ser-45 is phosphorylated not by GSK3 but by CKI epsilon. Axin, a scaffold protein that binds CKI epsilon and beta-catenin, enhances this CKI epsilon-mediated phosphorylation. Overexpression of CKI epsilon in cells increases the amount of beta-catenin phosphorylated at Ser-45. Ser-45 phosphorylated beta-catenin is a better substrate for GSK3, which suggests that CKI epsilon and GSK3 may co-operate in destabilizing beta-catenin. In spite of the fact that CKI epsilon was found as a positive regulator of the Wnt pathway, mutational analysis suggests that mutation of Ser-45 regulates beta-catenin stability by inhibiting the ability of GSK3 to phosphorylate Ser-33 and -37, thereby disrupting the interaction between beta-catenin, beta-TRCP and Axin. I propose that phosphorylation of Ser-45 by CKI epsilon plays an important role in regulating beta-catenin stability.     

Phosphorylation of murine caspase-9 by the protein kinase casein kinase 2 regulates its cleavage by caspase-8

Previous studies from our laboratory had indicated that cytochrome c-independent processing and activation of caspase-9 by caspase-8 contributed to early amplification of the caspase cascade in tumor necrosis factor (TNF)-alpha-treated murine cells. Here we show that murine caspase-9 is phosphorylated by casein kinase 2 (CK2) on a serine near the site of caspase-8 cleavage. CK2 has been shown to regulate cleavage of the pro-apoptotic Bid protein by phosphorylating serine residues near its caspase-8 cleavage site. Similarly, CK2 modification of Ser(348) on caspase-9 appears to render the protease refractory to cleavage by active caspase-8. This phosphorylation did not affect the ability of caspase-9 to autoprocess. Substitution of Ser(348) abolished phosphorylation but not cleavage, and a phospho-site mutant promoted apoptosis in TNF-alpha-treated caspase-9 knock-out mouse embryo fibroblasts. Furthermore, inhibition of CK2 activity and RNA interference-mediated knockdown of the kinase accelerated caspase-9 activation, whereas phosphatase inhibition delayed both caspase-9 activation and death in response to TNF receptor occupation. Taken together, these studies show that TNF receptor cross-linking promotes dephosphorylation of caspase-9, rendering it susceptible to processing by activated caspase-8 protein. Thus, our data suggest that modification of procaspase-9 to protect it from inappropriate cleavage and activation is yet another mechanism by which the oncogenic kinase CK2 promotes survival.     

Phosphorylation of the human ubiquitin-conjugating enzyme, CDC34, by casein kinase 2

The ubiquitin-conjugating enzyme, CDC34, has been implicated in the ubiquitination of a number of vertebrate substrates, including p27(Kip1), IkappaBalpha, Wee1, and MyoD. We show that mammalian CDC34 is a phosphoprotein that is phosphorylated in proliferating cells. By yeast two-hybrid screening, we identified the regulatory (beta) subunit of human casein kinase 2 (CK2) as a CDC34-interacting protein and show that human CDC34 interacts in vivo with CK2beta in transfected cells. CDC34 is specifically phosphorylated in vitro by recombinant CK2 and HeLa nuclear extract at five sites within the carboxyl-terminal 36 amino acids of CDC34. Importantly, this phosphorylation is inhibited by heparin, a substrate-specific inhibitor of CK2. We have also identified a kinase activity associated with CDC34 in proliferating cells, and we show that this kinase is sensitive to heparin and can utilize GTP, strongly suggesting it is CK2. Phosphorylation of CDC34 by the associated kinase maps predominantly to residues 203 and 222. Mutation of CDC34 at CK2-targeted residues, Ser-203, Ser-222, Ser-231, Thr-233, and Ser-236, abolishes the phosphorylation of CDC34 observed in vivo and markedly shifts nuclearly localized CDC34 to the cytoplasm. These results suggest a potential role for CK2-mediated phosphorylation in the regulation of CDC34 cell localization and function.     

Phosphorylation of RIG-I by casein kinase II inhibits its antiviral response

RIG-I is an intracellular RNA virus sensor that mediates a signaling pathway that triggers the alpha/beta interferon (IFN-α/β) immune defenses. However, the mechanism for regulation of RIG-I activity remains largely unknown. Here we show that RIG-I activity is regulated by phosphorylation and dephosphorylation in its repressor domain (RD). Threonine at amino acid (aa) 770 and serine at aa 854 to 855 of RIG-I are phosphorylated by casein kinase II (CK2) in the resting state of the cell and dephosphorylated when cells are infected by RNA virus. Mutation at aa position 770 or 854 to 855 of RIG-I renders it constitutively active. Pharmacological inhibition of CK2 enhances virus-induced expression of IFN-β and suppresses virus proliferation, while inhibition of phosphatase reduces virus-induced expression of IFN-β. Overexpression of CK2 suppresses RIG-I-mediated signaling, while silencing of CK2 results in the increased suppression of virus proliferation. Our results reveal a novel mechanism of the regulation of RIG-I activity during RNA virus infection.