Regulation of arrestin-3 phosphorylation by casein kinase II

Arrestins play an important role in regulating the function of G protein-coupled receptors including receptor desensitization, internalization, down-regulation, and signaling via nonreceptor tyrosine kinases and mitogen-activated protein kinases. Previous studies have revealed that arrestins themselves are also subject to regulation. In the present study, we focused on identifying potential mechanisms involved in regulating the function of arrestin-3. Using metabolic labeling, phosphoamino acid analysis, and mutagenesis studies, we found that arrestin-3 is constitutively phosphorylated at Thr-382 and becomes dephosphorylated upon beta(2)-adrenergic receptor activation in COS-1 cells. Casein kinase II (CKII) appears to be the major kinase mediating arrestin-3 phosphorylation, since 1) Thr-382 is contained within a canonical consensus sequence for CKII phosphorylation and 2) wild type arrestin-3 but not a T382A mutant is phosphorylated by CKII in vitro. Functional analysis reveals that mutants mimicking the phosphorylated (T382E) and dephosphorylated (T382A or T382V) states of arrestin-3 promote beta(2)-adrenergic receptor internalization and bind clathrin, beta-adaptin, and Src to comparable levels as wild type arrestin-3. This suggests that the phosphorylation of arrestin-3 does not directly regulate interaction with endocytic (clathrin, beta-adaptin) or signaling (Src) components and is in contrast to arrestin-2, where phosphorylation appears to regulate interaction with clathrin and Src. However, additional analysis reveals that arrestin-3 phosphorylation may regulate formation of a large arrestin-3-containing protein complex. Differences between the regulatory roles of arrestin-2 and -3 phosphorylation may contribute to the different cellular functions of these proteins in G protein-coupled receptor signaling and regulation.     

Regulation of casein kinase 2 by phosphorylation/dephosphorylation.

The effects of various polycation-stimulated (PCS) phosphatases and of the active catalytic subunit of the ATPMg-dependent (AMDc) protein phosphatase on the activity of casein kinase 2 (CK-2) were investigated by using the synthetic peptide substrate Ser-Glu-Glu-Glu-Glu-Glu, whose phosphorylated derivative is entirely insensitive to these protein phosphatases. Previous dephosphorylation of native CK-2 enhances its specific activity 2-3-fold. Such an effect, accounted for by an increase in Vmax, is more readily promoted by the PCS phosphatases than by the AMDc phosphatase. The phosphate incorporated by autophosphorylation could not be removed by the protein phosphatases, suggesting the involvement of phosphorylation site(s) other than the one(s) affected by intramolecular autophosphorylation. The activation of CK-2 by the phosphatase pretreatment is neutralized during the kinase assay; the mechanism of this phenomenon, which is highly dependent on the kinase concentration, is discussed.     

Protein phosphorylation during Rhodnius prolixus embryogenesis: protein kinase casein kinase II activity.

Protein kinase casein kinase II (CK II) activity was assayed during Rhodnius prolixus embryogenesis. Vitellin (VT) is the main endogenous substrate during the whole development. It is maximally phosphorylated at the third day of embryogenesis by CK II and then its phosphorylation decreases to a basal level by the time of first instar eclosion. When dephosphorylated casein was used as an exogenous substrate a different profile of enzyme activity was obtained. CK II activity increases on day 1 after fertilization and reaches a plateau on day 7 and its activity remains elevated until eclosion. Extracts obtained from oocytes or from 3-day old eggs were fractionate through gel filtration chromatography. CK II activity was assayed in each fraction and the enzyme obtained from the 3-day old eggs was shown to be three times more active than that obtained from oocytes, although the amount of enzyme present in the fractions was the same. These enriched CK II fractions were assayed against different effectors, such as: cAMP, H-8, H-89, calphostin C, sphingosine, polylysine and heparin. Heparin was the most effective one. When CK II activity was assayed in non-fertilized eggs, no activation of the enzyme was observed when compared to fertilized eggs. These data indicate that CK II is activated in a fertilization dependent process. The decrease in CK II activity against VT coincides with the beginning of VT proteolysis processing suggesting a possible relationship between protein phosphorylation and yolk degradation.     

Identification of syntaxin-1A sites of phosphorylation by casein kinase I and casein kinase II.

Casein kinases I (CKI) are serine/threonine protein kinases widely expressed in a range of eukaryotes including yeast, mammals and plants. They have been shown to play a role in diverse physiological events including membrane trafficking. CKI alpha is associated with synaptic vesicles and phosphorylates some synaptic vesicle associated proteins including SV2. In this report, we show that syntaxin-1A is phosphorylated in vitro by CKI on Thr21. Casein kinase II (CKII) has been shown previously to phosphorylate syntaxin-1A in vitro and we have identified Ser14 as the CKII phosphorylation site, which is known to be phosphorylated in vivo. As syntaxin-1A plays a key role in the regulation of neurotransmitter release by forming part of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex, we propose that CKI may play a role in synaptic vesicle exocytosis.     

Effect of dexamethasone on nucleolar casein kinase II activity and phosphorylation of nucleolin in lymphosarcoma P1798 cells.

Ribosomal RNA (rRNA) synthesis in murine P1798 lymphosarcoma cells is reversibly inhibited by glucocorticoids. The effects of dexamethasone upon nucleolin phosphorylation and upon the amount and activity of casein kinase II have been examined. P1798 cells were exposed to 0.1 microM dexamethasone for 36 h. Cells were labeled in vivo with [32P]orthophosphate followed by immunoprecipitation with anti-nucleolin antibody. Nucleolin phosphorylation was reduced by 60% in dexamethasone-treated cells. Nucleoli were isolated and labeled with [gamma-32P]ATP in vitro. Nucleolin protein was reduced to 40% of control in nuclei from dexamethasone-treated cells. Nucleolin phosphorylation was reduced to 20% of control. Nucleolar casein kinase II activity and protein were also reduced (30-55% and 35-50% of control, respectively) by treatment with dexamethasone. Cycloheximide (10 micrograms/ml for 3 h) reduced the amount and activity of casein kinase II, but did not cause a decrease in nucleolin protein. These observations are discussed relative to the hypothesis that glucocorticoids regulate the amount or activity of proteins of short biological half-life that are involved in the regulation of rRNA synthesis.     

Mutation of the casein kinase II phosphorylation site abolishes the anti-proliferative activity of p53.

The p53 tumour suppressor protein is phosphorylated by several protein kinases, including casein kinase II. In order to understand the functional significance of phosphorylation by casein kinase II, we have introduced mutations at serine 386 in mouse p53, the residue phosphorylated by this kinase, and investigated their effects on the ability of p53 to arrest cell growth. Replacement of serine 386 by alanine led to loss of growth suppressor activity, while aspartic acid at this position partially retained suppressor function. These data suggest that the anti-proliferative activity of p53 is activated by phosphorylation at serine 386, and establish a direct link between the covalent modification of a growth suppressor protein and regulation of its activity in mammalian cells.     

The development-associated cleavage of lens connexin 45.6 by caspase-3-like protease is regulated by casein kinase II-mediated phosphorylation.

Gap junctions are important in maintaining lens transparency and metabolic homeostasis. In this paper, we report that the gap junction-forming protein, connexin (Cx) 45.6, was specifically truncated during lens development and that the majority of the truncated fragments were located in the differentiated lens fibers. When isolated lens membranes were treated by caspase-3, the truncated fragments of Cx45.6 were reproduced, and this truncation occurred at the COOH terminus of Cx45.6. Moreover, when primary lens cells were treated with apoptosis-inducing reagents, Cx45.6 was cleaved similarly as the in vitro treatment by caspase-3, and this cleavage was blocked by a caspase-3 inhibitor. These results suggest that caspase-3 is responsible for the development-associated cleavage of Cx45.6. The cleavage site of Cx45.6 was identified between amino acid residues Glu(367) and Gly(368). We have shown previously that Ser(363) is an in vivo phosphorylated site by casein kinase II, and this specific phosphorylation leads to a rapid turnover of Cx45.6. Interestingly, we found here that when Ser(363) was phosphorylated by casein kinase II, the cleavage of Cx45.6 catalyzed by caspase-3 was inhibited. This study, for the first time, demonstrates that a connexin can be a direct target of an apoptotic protease and that cleavage by caspase-3-like protease leads to the development-associated truncation of a lens connexin. Finally, caspase-3-mediated cleavage can be regulated by casein kinase II-mediated phosphorylation, suggesting that Cx45.6 turnover and specific cleavage by caspase-3-like protease is alternatively modulated.     

The p53 tumor suppressor stimulates the catalytic activity of human topoisomerase IIalpha by enhancing the rate of ATP hydrolysis

DNA topoisomerase II is an essential nuclear enzyme for proliferation of eukaryotic cells and plays important roles in many aspects of DNA processes. In this report, we have demonstrated that the catalytic activity of topoisomerase IIalpha, as measured by decatenation of kinetoplast DNA and by relaxation of negatively supercoiled DNA, was stimulated approximately 2-3-fold by the tumor suppressor p53 protein. In order to determine the mechanism by which p53 activates the enzyme, the effects of p53 on the topoisomerase IIalpha-mediated DNA cleavage/religation equilibrium were assessed using the prototypical topoisomerase II poison, etoposide. p53 had no effect on the ability of the enzyme to make double-stranded DNA break and religate linear DNA, indicating that the stimulation of the enzyme catalytic activity by p53 was not due to alteration in the formation of covalent cleavable complexes formed between topoisomerase IIalpha and DNA. The effects of p53 on the catalytic inhibition of topoisomerase IIalpha were examined using a specific catalytic inhibitor, ICRF-193, which blocks the ATP hydrolysis step of the enzyme catalytic cycle. Clearly manifested in decatenation and relaxation assays, p53 reduced the catalytic inhibition of topoisomerase IIalpha by ICRF-193. ATP hydrolysis assays revealed that the ATPase activity of topoisomerase IIalpha was specifically enhanced by p53. Immunoprecipitation experiments revealed that p53 physically interacts with topoisomerase IIalpha to form molecular complexes without a double-stranded DNA intermediary in vitro. To investigate whether p53 stimulates the catalytic activity of topoisomerase II in vivo, we expressed wild-type and mutant p53 in Saos-2 osteosarcoma cells lacking functional p53. Wild-type, but not mutant, p53 stimulated topoisomerase II activity in nuclear extract from these transfected cells. Our data propose a new role for p53 to modulate the catalytic activity of topoisomerase IIalpha. Taken together, we suggest that the p53-mediated response of the cell cycle to DNA damage may involve activation of topoisomerase IIalpha.     

Characterization of the catalytic cycle of ATP hydrolysis by human P-glycoprotein. The two ATP hydrolysis events in a single catalytic cycle are kinetically similar but affect different functional outcomes

P-glycoprotein (Pgp) is a plasma membrane protein whose overexpression confers multidrug resistance to tumor cells by extruding amphipathic natural product cytotoxic drugs using the energy of ATP. An elucidation of the catalytic cycle of Pgp would help design rational strategies to combat multidrug resistance and to further our understanding of the mechanism of ATP-binding cassette transporters. We have recently reported (Sauna, Z. E., and Ambudkar, S. V. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 2515-2520) that there are two independent ATP hydrolysis events in a single catalytic cycle of Pgp. In this study we exploit the vanadate (Vi)-induced transition state conformation of Pgp (Pgp.ADP.Vi) to address the question of what are the effects of ATP hydrolysis on the nucleotide-binding site. We find that at the end of the first hydrolysis event there is a drastic decrease in the affinity of nucleotide for Pgp coincident with decreased substrate binding. Release of occluded dinucleotide is adequate for the next hydrolysis event to occur but is not sufficient for the recovery of substrate binding. Whereas the two hydrolysis events have different functional outcomes vis à vis the substrate, they show comparable t(12) for both incorporation and release of nucleotide, and the affinities for [alpha-(32)P]8-azido-ATP during Vi-induced trapping are identical. In addition, the incorporation of [alpha-(32)P]8-azido-ADP in two ATP sites during both hydrolysis events is also similar. These data demonstrate that during individual hydrolysis events, the ATP sites are recruited in a random manner, and only one site is utilized at any given time because of the conformational change in the catalytic site that drastically reduces the affinity of the second ATP site for nucleotide binding. In aggregate, these findings provide an explanation for the alternate catalysis of ATP hydrolysis and offer a mechanistic framework to elucidate events at both the substrate- and nucleotide-binding sites in the catalytic cycle of Pgp.     

Activation of mammalian DNA ligase I through phosphorylation by casein kinase II.

Mammalian DNA ligase I has been shown to be a phosphoprotein. Dephosphorylation of purified DNA ligase I causes inactivation, an effect dependent on the presence of the N-terminal region of the protein. Expression of full-length human DNA ligase I in Escherichia coli yielded soluble but catalytically inactive enzyme whereas an N-terminally truncated form expressed activity. Incubation of the full-length preparation from E. coli with purified casein kinase II (CKII) resulted in phosphorylation of the N-terminal region and was accompanied by activation of the DNA ligase. Of a variety of purified protein kinases tested, only CKII stimulated the activity of calf thymus DNA ligase I. Tryptic phosphopeptide analysis of DNA ligase I revealed that CKII specifically phosphorylated a major peptide also apparently phosphorylated in cells, implying that CKII is a protein kinase acting on DNA ligase I in the cell nucleus. These data suggest that DNA ligase I is negatively regulated by its N-terminal region and that this inhibition can be relieved by post-translational modification.     

Specific phosphorylation of the acidic central region of the N-myc protein by casein kinase II.

The central region of the N-myc protein has a characteristic amino acid sequence EDTLSDSDDEDD, which is very similar to those of particular domains of adenovirus E1A, human papilloma virus E7, Simian virus 40 large T, c-myc and L-myc proteins. Domains of these three viral oncoproteins have recently been shown to be specific binding sites for the tumor-suppressor gene retinoblastoma protein. We have noted that the sequence of serine followed by a cluster of acidic amino acids is exactly the same as that of a typical substrate of casein kinase II (CKII). Therefore, we investigated whether these nuclear oncoproteins are phosphorylated by CKII. For this purpose, we fused the beta-galactosidase and N-myc genes including this domain and expressed it in Escherichia coli cells. Several mutant N-myc genes, containing single amino acid substitutions in this domain, were also used to produce fused proteins. Strong phosphorylation by CKII was detected with the fused protein of wild-type N-myc. However, no phosphorylation of beta-galactosidase itself was observed and the phosphorylations of fused mutant proteins were low. Another fused N-myc protein containing most of the C-terminal region downstream of this acidic region was not phosphorylated by CKII. Analysis of phosphorylation sites in synthetic peptides of this acidic region identified the major sites phosphorylated by CKII as Ser261 and Ser263. On two-dimensional tryptic mapping of phosphorylated N-myc proteins, major spots of in vitro-labeled and in-vivo-labeled N-myc proteins were detected in the same positions. These results suggest that two serine residues of the acidic central region of the N-myc protein are phosphorylated by CKII in vivo as well as in vitro. The functional significance of this acidic domain is discussed.     

Regulation of the deubiquitinating enzyme CYLD by IkappaB kinase gamma-dependent phosphorylation

Tumor suppressor CYLD is a deubiquitinating enzyme (DUB) that inhibits the ubiquitination of key signaling molecules, including tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2). However, how the function of CYLD is regulated remains unknown. Here we provide evidence that inducible phosphorylation of CYLD is an important mechanism of its regulation. Under normal conditions, CYLD dominantly suppresses the ubiquitination of TRAF2. In response to cellular stimuli, CYLD undergoes rapid and transient phosphorylation, which is required for signal-induced TRAF2 ubiquitination and activation of downstream signaling events. Interestingly, the CYLD phosphorylation requires IκB kinase gamma (IKKγ) and can be induced by IKK catalytic subunits. These findings suggest that CYLD serves as a novel target of IKK and that the site-specific phosphorylation of CYLD regulates its signaling function.     

Modulation of radiation response by inhibiting topoisomerase II catalytic activity

Due to the essential role played by DNA topoisomerases (topos) in cell survival, the use of topoisomerase inhibitors as chemotherapeutic drugs in combination with radiation has become a common strategy for the treatment of cancer. Catalytic inhibitors of these enzymes would be promising to improve the effectiveness of radiation and therefore, it appears reasonable to incorporate them in combined modality trials. In this work, we have investigated the capacity of both ICRF-193 and Aclarubicin (ACLA), two catalytic inhibitors of topoisomerase II (Topo II), to modulate radiation response in Chinese hamster V79 cell line and its radiosensitive mutant irs2. We also have explored potential mechanisms underlying these interactions. Experiments were performed in the presence and absence of either ICRF-193 or ACLA, and topo II activity was measured using an assay based upon decatenation of kinetoplast DNA (kDNA). For the combined experiments cells were incubated for 3 h in the presence of various inhibitor concentrations and irradiated 30 min prior to the end of treatments and cell survival was determined by clonogenic assay. DNA-damaging activity was measured by single-cell gel electrophoresis. Our results demonstrate that combinations of catalytic inhibitors of topo II and radiation produce an increase in cell killing induced by ionising radiation. The mechanism of radiation enhancement may involve a direct or indirect participation of topo II in the repair of radiation-induced DNA damage.     

Regulation of catalytic activity of S6 kinase 2 during cell cycle

Ribosomal S6 kinase 2 (S6K2) is one of the kinases regulated by the mammalian target of rapamycin (mTOR) signaling pathway. Although it has been identified as a kinase homologous to S6K1, evidence suggests that the two kinases have non-overlapping functions, and the biological function of S6K2 still remains unknown. In order to identify the cell cycle stage(s) during which S6K2 plays a role, we assessed changes in the catalytic activity of S6K2 throughout the cell cycle. Our data show that S6K2 is active throughout the cell cycle with higher activity in G2 and M phases. We also show that S6K1 activity peaks sharply during M phase. Our data suggest that S6K1 and S6K2 likely play yet-unknown roles in G2 and M phases.     

Regulation of type II PIP kinase by PKD phosphorylation

The type II PIP kinases phosphorylate the poorly understood inositol lipid PtdIns5P, producing the multi-functional lipid product PtdIns(4,5)P(2). To investigate the regulation of these enzymes by phosphorylation, we partially purified a protein kinase from pig platelets that phosphorylated type IIalpha PIP kinase on an activation loop threonine residue, T376. Pharmacological studies suggested this protein kinase was protein kinase D (PKD), and in vitro experiments confirmed this identification. A phospho-specific antibody was developed and used to demonstrate phosphorylation of T376 in living cells, and its enhancement under conditions in which PKD was activated. Although we were unable to determine the effects of phosphorylation on PIP kinase activity directly, mutation of T376 to aspartate significantly inhibited enzyme activity. We conclude that the type II PIP kinases are physiological targets for PKD phosphorylation, and that this modification is likely to regulate inositol lipid turnover by inhibition of these lipid kinases.     

Regulation of enzyme activity in plants by reversible phosphorylation

This paper reviews the seven specific plant enzymes which have been shown or suggested, to date, to undergo reversible covalent modification by regulatory phosphorylation, including mitochondrial pyruvate dehydrogenase (EC 1.2.4.1), chloroplastic pyruvate, orthophosphate dikinase (EC 2.7.9.1) and ribulose bisphosphate carboxylase/oxygenase (EC 4.1.1.39), cytoplasmic phosphoenolpyruvate carboxylase (EC 4.1.1.31) and 6-phosphofructo-2-kinase (EC 2.7.1.105), microsomal hydroxymethylglutaryl - CoA reductase (EC 1.1.1.34), and quinate: NAD+ oxidoreductase (EC 1.1.1.24).     

Regulation of CD163 associated casein kinase II activity is haptoglobin genotype dependent

Free hemoglobin is now recognized as a major mediator of a variety of vascular diseases. The abundant serum protein haptoglobin irreversibly binds to hemoglobin and promotes the uptake of hemoglobin via the macrophage CD163 receptor. The haptoglobin gene is polymorphic in man with two common alleles denoted 1 and 2. The haptoglobin genotype specifies the nature of the response of the macrophage to free hemoglobin. Hp 1-Hb complexes stimulate an anti-inflammatory macrophage phenotype while Hp 2-Hb complexes do not. We have previously demonstrated that Hp 1-Hb induced anti-inflammatory cytokine production is critically dependent on casein kinase II. In this study we set out to determine whether the amount or the activity of casein kinase II associated with CD163 was altered by the binding of Hp 1-1-Hb to CD163. Our results indicate that casein kinase II activity is increased by the binding of Hp 1-1-Hb to CD163.