Identification and isolation of casein kinase type II from RNA-binding proteins of amphibian oocytes

Protein kinase previously detected in RNA-binding proteins of amphibian oocytes phosphorylates casein far more efficiently than histones to form phosphoserine and phosphothreonine and utilizes both ATP and GTP. Heparin in concentrations below 1 microgram/ml inhibits protein kinase. This allows to relate the enzyme to casein kinases II. Protein kinase was extensively purified (more than 15000-fold) with respect to proteins of ribosome-free extract. The homogeneous enzyme consists of three polypeptide chains (Mr 43,000, 41,000, and 29,000). The 125I-labelled enzyme possessing casein kinase and RNA-binding activities when injected into amphibian oocytes was detected in the particles identical to free cytoplasmic informosomes in terms of their sedimentation properties.     

Human endothelial cells contain one type of plasminogen activator

RNA-binding protein kinase from amphibian oocytes modifies serine and threonine residues in the molecules of substrates and utilizes both ATP and GTP. Low concentrations of heparin inhibit protein kinase. The foregoing suggests that this enzyme is casein kinase II. It is shown that RNA-binding proteins lack active forms of phosphatases and proteases which may affect the results of phosphorylation of both endogenous and exogenous substrates.     

Inhibition of casein kinase type 2 activity by formation of complexes with mRNA in vivo

It is known that casein kinase 2 possesses, besides the protein kinase, an RNA-binding activity. Using ligand blotting it has been demonstrated that the both activities are localized on the alpha- and alpha'-subunits of the enzyme. Casein kinase 2 is suppressed in vitro by polyuridylic acid. A part of the intracellular pool of casein kinase 2 is found in the informosomes. The informosomes and free proteins were separated by centrifugation in a sucrose density gradient, and each fraction was incubated with casein and [gamma-32P]ATP. The informosome-bound protein kinase is completely inhibited, while the free protein kinases heavily phosphorylate casein. It is concluded that the activity of casein kinase 2 can be regulated by the reversible formation of complexes with RNA.     

Casein kinase II from Rana temporaria oocytes. Intracellular localization and activity during progesterone-induced maturation

A homogeneous preparation of casein kinase II has been isolated from the ribosome-free extracts of Rana temporaria oocytes by means of chromatography on heparin-Sepharose, phosphocellulose and mono Q. The enzyme consists of three subunits with molecular mass of 43 kDa, 41 kDa and 29 kDa. The protein kinase was labelled with radioactive iodine and injected back into oocytes. As shown by histoautoradiography the enzyme forms a diffuse ring around the nucleus in the oocyte cytoplasm. A part of casein kinase II is found in informosomes. During oocytes maturation casein kinase II activity increases 7 h after progesterone administration and at the final stages of maturation (20-23 h). Cycloheximide blocks the second augmentation of kinase activity and does not influence the first one.     

In vivo progesterone regulation of protein phosphatase activity in Xenopus oocytes

Exogenous beta casein, previously phosphorylated in vitro by protein kinase A and casein kinase II, was microinjected into Xenopus oocytes to monitor in vivo protein phosphatase activities. Phosphatase activities were 1.6 and 3.4 fmol/min/oocyte, respectively, for beta casein phosphorylated by casein kinase II and beta casein phosphorylated by protein kinase A. Progesterone induced an early decrease (35% after 10 min) in phosphatase activity restricted to the protein kinase A sites of beta casein.     

pH-dependent changes in structure and RNA-binding activity of casein kinase 2 from Rana temporaria oocytes

It is demonstrated by filter-binding assay that casein kinase 2 from Rana temporaria oocytes binds rRNA in vitro with high affinity. Ligand-blotting shows that rRNA-binding activity is inherent to alpha and alpha' subunits of the enzyme. Increase of pH from 6.5 to 7.5 has little effect on casein kinase but completely suppresses rRNA-binding activity of the enzyme. Sedimentation coefficient of casein kinase 2 also depends on pH: at pH 7.5 it is mainly 10 S, and at pH 6.5-18 S. At pH 6.95 the amounts of both forms are equal. The heavy form of casein kinase 2 practically lacks rRNA-binding activity.     

cAMP-independent protein kinase from amphibian lens: identification, organ distribution and substrates of phosphorylation

Eye lens extracts of the frog Rana temporaria contain a cAMP-independent protein kinase which is quantitatively adsorbed on immobilized RNA at physiological salt concentrations. The enzyme activity is maximal in the lenticular cortex, medium in the epithelium and minimal in the lens nuclei. Crude preparations of RNA-binding protein kinase from the epithelium, cortex and nuclei of the eye lens were prepared by affinity chromatography on poly(U)-Sepharose. It was found that these preparations contain no active forms of phosphatases, ATPases or proteases which may interfere with the results of phosphorylation experiments on exogenous and endogenous substrates. The protein kinase under study catalyzes the binding of phosphate groups to threonine and serine residues in casein molecules, does not phosphorylate histones and utilizes GTP alongside with ATP as phosphate donors. Heparin and RNA used at low concentrations inhibit the protein kinase activity. The data obtained allow the identification of lenticular RNA-binding protein kinase(s) as a casein kinase type II. It was shown that incubation of RNA-binding proteins from epithelium and lenticular cortex with [gamma-32P]ATP results in the label incorporation into six to seven polypeptide chains with Mr of 27-130 kDa. Poly(U) and heparin inhibit the self-phosphorylation reaction, cAMP has no stimulating effect on this process, while Ca2+ ions inhibit the self-phosphorylation of RNA-binding proteins.     

Isolation and characterization of casein kinases I and II from rabbit liver. Analysis of their effects on protein biosynthesis in the cell

Two cAMP-independent protein kinases isolated from rabbit liver extracts phosphorylate casein far more effectively than histones. The first protein kinase consists of one polypeptide chain (Mr = 37,000), utilizes exclusively ATP and is not inhibited in the presence of low heparin and RNA concentrations. The second protein kinase consists of three subunits (Mr = 42,000, 40,000 and 25,000 Da), utilizes both ATP and GTP and is inhibited by low heparin and RNA concentrations. The latter enzyme has Mr approximately 140,000 Da and possesses a polyanion-binding activity. These characteristics allow to relate the above enzymes to casein kinases I and II, respectively. Injection of casein kinase I into frog oocytes results in the inhibition of the rate of amino acid incorporations into the soluble and detergent extractable proteins. Casein kinase II has no effect on the amino acid incorporation into the recipient oocytes.     

The adaptor Grb7 links netrin-1 signaling to regulation of mRNA translation

We previously reported a novel biological activity of Netrin-1 in translational stimulation of kappa opioid receptor (KOR). We now identify Grb7 as a new RNA-binding protein that serves as the molecular adaptor for transmitting Netrin-1 signals, through focal adhesion kinase (FAK), to the translation machinery. Grb7 binds specifically to the first stem loop of kor mRNA 5'-UTR through a new RNA-binding domain located in its amino terminus. Upon binding to its capped, target mRNA, Grb7 blocks the recruitment of eIF4E, rendering mRNA untranslatable. The RNA-binding and translation-repressive activity is reduced by FAK-mediated hyperphosphorylation on two tyrosine residues of its carboxyl terminus. This study reports an adaptor protein Grb7 that transmits the stimulating signals of Netrin-1 to the translational machinery to rapidly regulate mRNA translation.     

Purification and characterization of a casein-kinase-II-type enzyme from Xenopus laevis ovary. Biological effects on the meiotic cell division of full-grown oocyte

A casein kinase of type II has been highly purified from Xenopus laevis ovary. A new experimental protocol has been developed for the purification, consisting in four chromatographic steps: hydrophobic on tyrosine-agarose, ion exchange on DEAE-Sepharose, affinity on heparin-Sepharose and fast protein liquid on Mono Q. The purification was greater than 20,000, taking into account an inhibitor present in the starting material which masked the activity in the crude fraction. The overall yield was greater than 20%. Full-grown Xenopus oocytes contain 64 milliunits per oocyte corresponding to an intracellular concentration in the nanomolar range. The enzyme shares the following features with the mammalian casein kinase II: (a) comparable subunit composition (42-kDa doublet, 38 kDa and 26 kDa), (b) autophosphorylation of the 26-kDa subunit, (c) ability to use GTP as well as ATP as phosphate donor, (d) inability to use Mn2+ instead of Mg2+ to support the activity, (e) phosphorylation of both threonine and serine residues of casein, (f) inhibition by low doses of heparin. Biological effects of the highly purified enzyme have been investigated upon microinjection into Xenopus full-grown oocytes. At nanomolar concentrations (approximately 3 nM) the enzyme inhibited progesterone induction of meiotic cell division whereas it facilitates meiotic maturation at the level of maturation-promoting factor. These results suggest a role for the kinase in the phosphorylation cascade involved during the prophase/metaphase transition of meiotic cell division, both in the mechanism of the meiotic prophase arrest and in the activity of the cytoplasmic factor maturation-promoting factor. When microinjected into oocytes above 45 nM, the kinase provoked complex changes in the profile of the in ovo 32P-labelled proteins indicating that its targets could be other kinase/phosphatase regulatory proteins.     

Casein kinase II is a major protein phosphorylating activity in the nuclei of Xenopus laevis oocytes

The nuclei of Xenopus laevis oocytes contain kinases capable of phosphorylating endogenous and exogenous proteins using either ATP or GTP as phosphoryl donors. These enzymes are much more active with casein and phosvitin as substrates than with histones or protamines. The protein phosphorylating activity of oocyte nuclear extracts is not regulated by cyclic nucleotides, phorbol esters, calmodulin and calcium, or phospholipids. However, the casein phosphorylating activity can be greatly enhanced by the polyamines spermine or spermidine and drastically inhibited by heparin. Fractionation of the nuclear casein kinase activities by DEAE-Sephadex chromatography and glycerol gradient centrifugation indicate that the nuclei contain enzymes with the properties of casein kinases I and II as characterized in other species. Oocyte casein kinase I (Mr 37,000) is specific for ATP as phosphoryl donor, is only slightly inhibited by 10 micrograms/ml heparin, and is not significantly stimulated by polyamines. Casein kinase II (Mr 135,000) can use both ATP and GTP as substrates, and is very sensitive to heparin inhibition and polyamine stimulation. The fact that low concentrations of heparin (10 micrograms/ml) can inhibit a large percentage of the endogenous phosphorylation of nuclear extracts or of whole nuclei indicates that casein kinase II is probably the major protein phosphorylating activity of these oocyte organelles.     

Protein phosphorylation during meiotic maturation of Xenopus oocytes: cdc2 protein kinase targets

M-Phase specific protein kinase or cdc2 protein kinase is a component of MPF (M-Phase promoting factor). During meiotic maturation of Xenopus oocytes, cdc2 protein kinase is activated in correlation with MPF activity. A protein phosphorylation cascade takes place involving several protein kinases, among which casein kinase II, and different changes associated with meiosis occur such as germinal vesicle breakdown, chromosome condensation, cytoskeletal reorganization and increase in protein synthesis. Our results provide a biochemical link between cdc2 protein kinase and protein synthesis since they show that the kinase phosphorylates in vitro a p47 protein identified as elongation factor EF1 (gamma subunit) and that the in vitro site of p47 corresponds to the site phosphorylated in vivo. Immunofluorescence showed that the elongation factor (EF1-beta gamma) is localized in the oocyte cortex. Furthermore, they show that cdc2 kinase phosphorylates and activates casein kinase II in vitro, strongly supporting the view that casein kinase II is involved in the phosphorylation cascade originated by cdc2 kinase.     

Identification of a protein kinase which phosphorylates a subunit of the 26S proteasome and changes in its activity during meiotic cell cycle in goldfish oocytes

The proteasome is involved in the progression of the meiotic cell cycle in fish oocytes. We reported that the alpha4 subunit of the 26S proteasome, which is a component of the outer rings of the 20S proteasome, is phosphorylated in immature oocytes and dephosphorylated in mature oocytes. To investigate the role of the phosphorylation, we purified the protein kinase from immature oocytes using a recombinant alpha4 subunit as substrate. A protein band which well corresponded to the kinase activity was identified as casein kinase Ialpha (CKIalpha). Two-dimensional (2D) PAGE analysis showed that part of the alpha4 subunit was phosphorylated by CKIalpha in vitro. This spot was detected in purified immature 26S proteasome but not in mature 26S proteasome, demonstrate that the alpha4 subunit is phosphorylated by CKIalpha meiotic cell cycle dependently.     

Part of Xenopus translin is localized in the centrosomes during mitosis

During oogenesis, maternal mRNAs are synthesised and stored in a translationally dormant form due to the presence of regulatory elements at the 3' untranslated regions (3'UTR). In Xenopus oocytes, several studies have described the presence of RNA-binding proteins capable to repress maternal-mRNA translation. The testis-brain RNA-binding protein (TB-RBP/Translin) is a single-stranded DNA- and RNA-binding protein which can bind the 3' UTR regions (Y and H elements) of stored mRNAs and can suppress in vitro translation of the mRNAs that contain these sequences. Here we report the cloning of the Xenopus homologue of the TB-RBP/Translin protein (X-translin) as well as its expression, its localisation, and its biochemical association with the protein named Translin associated factor X (Trax) in Xenopus oocytes. The fact that this protein is highly present in the cytoplasm from stage VI oocytes until 48 h embryos and that it has been described as capable to inhibit paternal mRNA translation, indicates that it could play an important role in maternal mRNA translation control during Xenopus oogenesis and embryogenesis. Moreover, we investigated X-translin localisation during cell cycle in XTC cells. In interphase, although a weak and diffuse nuclear staining was observed, X-translin was mostly present in the cytoplasm where it exhibited a prominent granular staining. Interestingly, part of X-translin underwent a remarkable redistribution throughout mitosis and associated with centrosomes, which may suggest a new unknown role for this protein in cell cycle.     

Oligomerization activity of a double-stranded RNA-binding domain

Xenopus laevis RNA-binding protein A (Xlrbpa) is a highly conserved, ubiquitously expressed hnRNP- and ribosome-associated RNA-binding protein that contains three double stranded RNA-binding domains (dsRBDs) in tandem arrangement. A two-hybrid screen with Xlrbpa as a bait recovered Xlrbpa itself as the strongest interaction partner, indicating multimerization of this protein. To search for regions responsible for the observed interaction, we conducted two-hybrid assays with Xlrbpa deletion constructs and identified the third dsRBD of Xlrbpa as the exclusive interacting domain. Additionally, these results were confirmed by coimmunoprecipitation experiments with truncated proteins expressed both in yeast and Xenopus oocytes. In PACT, the human homologue of Xlrbpa, we could demonstrate that the third dsRBD displays the same multimerization activity. Interestingly, this domain is essential for the activation of the dsRNA-activated protein kinase PKR. Addition of RNAses to coimmunoprecipitation experiments did not affect the dimerization, suggesting that the interaction is independent of RNA-binding. We report here a homomultimerization activity of a type B dsRBD and suggest possible implications that include a model for PKR activation by PACT.     

Maturation hormone induced an increase in the translational activity of starfish oocytes coincident with the phosphorylation of the mRNA cap binding protein,eIF-4E,and the activation of several kinases

The stimulation of translation in starfish oocytes by the maturation hormone, 1-methyladenine (1-MA), requires the activation or mobilization of both initiation factors and mRNAs [Xu and Hille, Cell Regul. 1:1057, 1990]. We identify here the translational initiation complex, eIF-4F, and the guanine nucleotide exchange factor for eIF-2, eIF-2B, as the rate controlling components of protein synthesis in immature oocytes of the starfish, Pisaster orchraceus. Increased phosphorylation of eIF-4E, the cap binding subunit of the eIF-4F complex, is coincident with the initial increase in translational activity during maturation of these oocytes. Significantly, protein kinase C activity increased during oocyte maturation in parallel with the increase in eIF-4E phosphorylation and protein synthesis. An increase in the activities of cdc2 kinase and mitogen-activated myelin basic protein kinase (MBP kinase) similarly coincide with the increase in eIF-4E phosphorylation. However, neither cdc2 kinase nor MBP kinase phosphorylates eIF-4E in vitro. Casein kinase II activity does not change during oocyte maturation, and therefore, cannot be responsible for the activation of translation. Treatment of oocytes with phorbol 12-myristate 13-acetate, an activator of protein kinase C, for 30 min prior to the addition of 1-MA resulted in the inhibition of 1-MA-induced phosphorylation of eIF-4E, translational activation, and germinal vesicle breakdown. Therefore, protein kinase C may phosphorylate eIF-4E, after very early events of maturation. Another possibility is that eIF-4E is phosphorylated by an unknown kinase that is activated by the cascade of reactions stimulated by 1-MA. In conclusion, our results suggest a role for the phosphorylation of eIF-4E in the activation of translation during maturation, similar to translational regulation during the stimulation of growth in mammalian cells. © 1993 Wiley-Liss, Inc.     

Protein phosphorylation in Rhodnius prolixus oocytes: identification of a type II casein kinase

A protein kinase activity in chorionated oocytes of Rhodnius prolixus phosphorylates in vitro vitellin (VT), the major yolk protein. Phosphatase inhibitors including NaF, sodium vanadate, beta-glycerophosphate and okadaic acid did not alter the protein phosphorylation profile to a visible extent. Among the exogenous protein substrates tested, casein was readily phosphorylated, but histones were not. Several different protein kinase activators, including cAMP, Ca2+ plus calmodulin, Ca2+ plus diolein and phosphatidylserine, were added to the reaction media but spermidine was the only effective one, inducing a 2-fold increase in the phosphorylation of VT. A strong inhibition was obtained with nanomolar levels of heparin. The enzyme could also accept GTP as the phosphate donor instead of ATP. These properties identify the major protein kinase activity as a type II casein kinase (CK II). The pH dependence and the effects of mono- and divalent cations on VT phosphorylation were also studied. Gel filtration revealed only one peak of protein kinase activity, with a molecular mass of 170 K, similar to values previously reported in the literature for CK IIs from other organisms.     

The effects of exogenous actin-binding casein kinase injected into the oocytes and ova of the clawed toad

The effects of microinjections of exogenous casein kinase 2 on the structural organization of the maturing oocytes and eggs were studied in Xenopus laevis. Kinase inhibited the progesterone-stimulated oocyte maturation and induced dislocation of pigment granules. The morphological effect was shown to be dose-dependent. The results obtained are discussed in the light of the possible influence of casein kinase 2 on the organization of the cortical actin cytoskeleton through phosphorylation of actin-binding proteins.