Phosphorylation Of Phosvitin Research Articles

The Golgi 'casein kinase' Fam20C is a genuine 'phosvitin kinase' and phosphorylates polyserine stretches devoid of the canonical consensus.

Egg yolk phosvitins, generated through the fragmentation of vitellogenins (VTGs), are among the most heavily phosphorylated proteins ever described. Despite the early discovery in 1900 that chicken phosvitin is a phosphoprotein and its subsequent employment as an artificial substrate for a number of protein kinases, the identity of the enzyme(s) responsible for its phosphorylation remained a matter of conjecture until present. Here, we provide evidence that phosvitin phosphorylation is catalyzed by a family with sequence similarity 20, member C (Fam20C), an atypical protein kinase recently identified as the genuine casein kinase and responsible for the phosphorylation of many other secreted proteins at residues specified by the S-x-E/pS consensus. Such a conclusion is grounded on the following observations: (a) the levels of Fam20C and phosphorylated VTG rise in parallel upon treatment of zebrafish with oestrogens; (b) zebrafish phosvitin is readily phosphorylated upon coexpression in U2OS cells with Fam20C, but not with its catalytically inactive mutant; (c) a peptide reproducing a stretch of 12 serines, which are phosphorylated in chicken phosvitin despite lacking the C-terminal priming motif S-x-E, is efficiently phosphorylated by both recombinant and native Fam20C. The last finding expands the repertoire of potential targets of Fam20C to include several proteins known to harbor (p-Ser)n clusters not specified by any known kinase consensus.

Phosphorylation of phosvitin plays a crucial effects on the protein-induced differentiation and mineralization of osteoblastic MC3T3-E1 cells

This study investigated the effects of phosvitin (PV), one of the major proteins from egg yolk, with different degree of phosphorylation on the physiology of an osteoblast (MC3T3-E1) cell line. The proliferation and differentiation of MC3T3-E1 were analyzed using the CCK-8 and the alkaline phosphatase (ALP) assay, respectively. The effect of PV on the mineralization of MC3T3-E1 was monitored using the Alizarin-red staining. PV at 100 μg/mL increased the ALP activity by 145% of the control after 7 days of incubation. PV also stimulated the proliferation and differentiation of MC3T3-E1 in a phosphorylation level-dependent manner. The RT-PCR reactions indicated that PV stimulated the expression of BMP-2 and OPG mRNA in a phosphorylation-dependent manner, but inhibited RANKL mRNA expression in MC3T3-E1. This result suggested that the phosphate groups in PV not only stimulated the proliferation and differentiation of MC3T3-E1, but also controlled the mineralization by regulating the expression of BMP-2, RANKL and OPG mRNA in the osteoblast cell.

Phosphorylated serine clusters of phosvitin plays a crucial role in the regulatory mineralization

Phosphorylation of phosvitin has been proved to play an important role in the mineralization, but the active region of phosvitin is still not known yet. Four phosvitin phosphopeptides (PPPs) were obtained after separating from ion exchange column and desalting from gel filtration, and named as PPP0, PPP1, PPP3 and PPP4, respectively. The effect of PPP on the mineralization was investigated by pH-stat system, Fourier transform infrared spectroscopy, X-ray diffraction and scanning electron microscope. SDS-PAGE and circular dichroism were applied to study the composition and the structure of PPP. Results showed that the order of promoting mineralization was as follows: PPP3 > phosvitin > PPP4 > PPP1 > control > PPP0. PPP0 and PPP1 was not involved in the mineralization, while the structure of PPP4 was too compact to promote mineralization because of its high β-sheet conformation. PPP3, which contained a 10 kDa peptide, is the most effective promoter for mineralization. LTQ-MS/MS results indicated that the phosphorylated serine clusters of phosvitin was the active region for promoting mineralization.

Role of phosphorylation of phosvitin in the phase transformation of mineralization

Phosvitin is a unique highly phosphorylated protein that plays a role in the regulation of mineralization. This study investigated the role of phosphorylation of phosvitin in the phase transformation of calcium phosphate in the mineralization solution. Partially dephosphorylated phosvitins (T1, T2, T3 and T4) were prepared with 2.98, 19.46, 43.39 and 71.07% of phosphate released from native phosvitin, respectively. And their effect on regulating the phase transformation was investigated, the characterization and composition analysis was performed by circular dichroism, Fourier transform infrared spectroscopy, X-ray diffraction and scanning electron microscopy. Results showed that phosvitin in the acceleration effect of phase transformation was dose-concentration effect by pH-stat titration. With lower dephosphorylation degree of phosvitin (<20%), the acceleration effect was weaker than native phosvitin, since phosphorylation and random structure of phosvitin were reduced. However, with higher dephosphorylation degree of phosvitin (>40%), the acceleration effect was enhanced compared to native phosvitin, in which the β-sheet structure was increased and phosvitin was partially hydrolyzed to phosphopeptides. The acceleration effect of phase transformation was as follows: T4>T3>phosvitin>T1>T2> Control. This study clearly demonstrated that phosphorylation of phosvitin played an important role in the regulation of mineralization.

The effect of heparin on the activity of Trichosporon cutaneum casein kinase I.

Casein kinase I from Trichosporon cutaneum ribosome-free extracts was purified. Its molecular mass was calculated for 33 kDa. It was shown that casein, phosvitin and Trichosporon cutaneum ribosomal protein of 15 kDa were preferable substrates for the enzyme. It was found that heparin can stimulate or inhibit CKI activity depending on the substrate used. Stimulation of casein and inhibition of phosvitin phosphorylation was observed. In addition it was shown that ribosomal proteins of 19 kDa and 38 kDa were phosphorylated by CKI only in the presence of heparin.

Ectoprotein kinase activities on non-differentiated and differentiated U-937 cells

Incubation of intact U-937 cells with 1 μm [γ- 32P] ATP resulted in rapid (10 min) incorporation of radioactivity into phosvitin, kemptide and protein kinase C (PKC)-peptide. The amount of incorporation was dependent on substrate type and concentration, and on incubation time. Staurosporine, H-7 and Mg 2+-exclusion abolished phosphorylation of kemptide and PKC-peptide but not phosvitin. Cyclic AMP and phorbol ester enhanced kemptide and PKC-peptide phosphorylation. Protein kinase inhibitor (PKI) inhibits only kemptide phosphorylation. Cell differentiation enhanced 2-fold the phosphorylation of phosvitin and PKC-peptide without significant effect on kemptide phosphorylation. ATP concentrations sufficient to trigger changes in intracellular Ca 2+ were sufficient to support extracellular phosphorylation reactions. The results suggest the presence of at least three ectokinase activities on U-937 cells that may play important roles in regulating membrane associated specific functions of developing and mature monocytes.

Recombinant rabbit muscle casein kinase I α is inhibited by heparin and activated by polylysine

The casein kinase I (CKI) family consists of widely distributed monomeric Ser/Thr protein kinases that have a preference for acidic substrates. Four mammalian isoforms are known. A full length cDNA encoding the CKIα isoform was cloned from a rabbit skeletal muscle cDNA library and was utilized to construct a bacterial expression vector. Active CKIα was expressed in Escherichia coli as a polypeptide of Mr 36,000. The protein kinase phosphorylated casein, phosvitin and a specific peptide substrate (D4). The enzyme was inhibited by the isoquinolinesulfonamide CKI-7, half-maximally at 70 μM. Heparin inhibited phosphorylation of the D4 peptide or phosvitin by CKIα. Polylysine activated when the D4 peptide was the substrate but had no effect on phosvitin phosphorylation. It is becoming clear that the individual CKI isoforms have different kinetic properties and hence could have quite distinct cellular functions.

On the interaction of phosvitins with ferric ion: Solubility of the Fe(III)-phosphoprotein complex under acidic conditions is a function of the iron/phosphate ratio and the degree of phosvitin phosphorylation

The interaction of phosvitins, the polyphosphoproteins of the eggs of egg-laying vertebrates, with ferric chloride was investigated under acidic conditions at iron-to-protein phosphorus ratios ranging up to 10. Phosvitins of which all or nearly all serine residues are phosphorylated (P/ser > 0.8) precipitate when titrated with the iron salt. As the total Fe/P ratio reaches the value of about 0.5, precipitation becomes maximal. At Fe/P ratios above 0.5, the Fe(III)-phosvitin complex becomes increasingly soluble. At ratios above 2, solubility is essentially fully restored, Phosvitins with an appreciable portion of their serine residues non-phosphorylated (P/ser < 0.7) show a different dependence of solubility on the Fe/P ratio. The Fe/P ratios of all precipitated complexes themselves vary within a narrow range between about 0.4 and about 1.0; the total Fe/P ratio is varied between 0 and 10. The results imply that phosvitin iron binding sites are non-uniform and that, overall, phosvitin is capable of accommodating iron in different ways, depending on the relative magnitude of the iron load and the availability of phosphoserine clusters in the phosphoprotein.

Phosvitin kinase activity in Acholeplasma axanthum

Incubating the soluble fraction derived from A. axanthum with phosvitin and [γ- 32P]ATP results in the phosphorylation of phosvitin. Casein, histone and kemptide were practically ineffective substrates, whereas a 55 kDa protein of M. gallisepticum was efficiently phosphorylated. The enzymatic activity has an optimal pH in the pH range 6.0–6.2 and requires divalent cations. The activity was inhibited by ammonium sulfate, heparin and sulphydryl blocking reagents, but was not affected by calmodulin with or without Ca 2+ or by cyclic AMP.

Phosphorylation of phosvitin by casein kinase-2 provides the evidence that phosphoserines can replace carboxylic amino acids as specificity determinants

The consensus sequence of casein kinase-2 consists of a serine (threonine) followed by a cluster of glutamic and/or aspartic acids, the one at position +3 playing an especially crucial role (Marin et al., (1986) Eur. J. Biochem. 160, 239–244 and Kuenzel et al. (1987) J. Biol. Chem. 262, 9136–9140). None of the 123 serines of the main phosvitin component (34 kDa) fulfils such a requirement (Byrne et al. (1984) Biochemistry 23, 4275–4279), rather, most of them are clustered into stretches of up to 14 entirely phosphorylated residues. Three out of the four threonines lie close to the N-terminal side of such phosphoseryl blocks. Here we show that native 34 kDa phosvitin is a poor substrate of casein kinase-2, its radiolabeling occurring mostly at threonine residue(s); a very slight (1%) previous dephosphorylation with acid phosphatase converts phosvitin into an excellent substrate for casein kinase-2, its phosphorylation occurring almost exclusively at serine residues. Extensive dephosphorylation however (greater than 40%) reduces the phosphorylation efficiency of casein kinase-2. These results show that phosphoserine residues can replace carboxylic residues as specificity determinants for casein kinase-2.

Phosphorylation of phosvitin by casein kinase-2 provides the evidence that phosphoserines can replace carboxylic amino acids as specificity determinants

The consensus sequence of casein kinase-2 consists of a serine (threonine) followed by a cluster of glutamic and/or aspartic acids, the one at position +3 playing an especially crucial role (Marin et al., (1986) Eur. J. Biochem. 160, 239-244 and Kuenzel et al. (1987) J. Biol. Chem. 262, 9136-9140). None of the 123 serines of the main phosvitin component (34 kDa) fulfils such a requirement (Byrne et al. (1984) Biochemistry 23, 4275-4279), rather, most of them are clustered into stretches of up to 14 entirely phosphorylated residues. Three out of the four threonines lie close to the N-terminal side of such phosphoseryl blocks. Here we show that native 34 kDa phosvitin is a poor substrate of casein kinase-2, its radiolabeling occurring mostly at threonine residue(s); a very slight (1%) previous dephosphorylation with acid phosphatase converts phosvitin into an excellent substrate for casein kinase-2, its phosphorylation occurring almost exclusively at serine residues. Extensive dephosphorylation however (greater than 40%) reduces the phosphorylation efficiency of casein kinase-2. These results show that phosphoserine residues can replace carboxylic residues as specificity determinants for casein kinase-2.

A multisubstrate Ca 2+ and cyclic nucleotide independent kinase from vascular smooth muscle: Modulation of activity by polycations

A multisubstrate Ca 2+ and cyclic nucleotide independent kinase (Mr = 47,000) was purified from bovine aortic smooth muscle. Phosphorylation of glycogen synthase by this enzyme was polycation modulable. Low concentrations of polylysine (0.04–0.16 μM) stimulated phosphorylation 2–7 fold, whereas higher concentrations suppressed phosphorylation. Glycogen synthase converted to its glucose 6-PO 4 dependent form following phosphorylation in either the presence (7 mol 32P/mol synthase) or absence (4 mol 32P/mol synthase) of polylysine: extent of conversion correlated to extent of phosphorylation. Seven of 14 potential substrates tested were phosphorylated: kinase activity was greatest for phosvitin followed by casein, the receptor protein from type 2 cAMP-kinase, histone H 2b, phosphorylase kinase, glycogen synthase, and myocardial myosin light chains. Phosphorylation of phosvitin or synthase was inhibited by heparin ( 1 2 maximally by 0.5 μg/ml without salt and 37 μg/ml with 150 mM NaCl). The results suggest that the enzyme may participate in regulating arterial glycogen metabolism and that such regulation may be modulated by polycationic and polyanionic effectors.

Properties of protein kinases in brain coated vesicles.

Coated vesicles prepared from bovine brain contained cyclic nucleotides- and Ca2+-calmodulin-independent protein kinases which in the presence of Mg2+ catalyzed the phosphorylation of an endogenous 48,000 Mr protein of coated vesicles (C-48), phosvitin and troponin T. Phosvitin was phosphorylated either in the presence of ATP or GTP. The phosphorylation of C-48, on the other hand, was specific for ATP. Heparin inhibited the phosphorylation of phosvitin but not that of C-48. Mn2+ inhibited the phosphorylation of phosvitin, while Mn2+ substituted for Mg2+ in the phosphorylation of C-48. When the coated vesicles were prepared in the presence of NaF, C-48 contained 2.5-2.8 mol of phosphate/mol. On incubation with Mg2+ and ATP, C-48 incorporated 1.2-1.6 mol of phosphate/mol. With C-48 as a substrate, the value of its apparent Km for ATP was 6 microM. With phosvitin as a substrate, the value of its apparent Km was 20 microM. The phosphorylated amino acid residues in the phosvitin were identified as serine and threonine. Phosphothreonine was detected in C-48. These results suggest that brain coated vesicles possess two different classes of protein kinase, a casein kinase II and C-48 kinase.

Reversible phosphorylation of T-substrate by wheat germ, human erythrocyte, and rabbit skeletal muscle protein kinases.

The reversibility of the reactions catalyzed by the wheat germ kinase and the cyclic AMP independent protein kinases isolated from human erythrocytes (casein kinases A and G) and rabbit skeletal muscle (casein kinases I and II) has been investigated. The reverse reaction requires ADP, Mg2+, phosphoprotein, and kinase and results in the formation of ATP from the phosphoprotein and ADP. The requirement for ADP in the wheat germ kinase and casein kinases II and G catalyzed reactions appears to be nonspecific. These kinases can also utilize GDP, IDP, and UDP as phosphoryl acceptors. Studies with the wheat germ protein T-substrate indicate that the phosphorylation of this protein substrate by the kinases is fully reversible. By contrast, the phosphorylation of phosvitin and casein is only partially reversible. Since the T-substrate is found to contain multiple phosphorylation sites and can serve as phosphoryl acceptor for the various kinases, the specificity of the phosphorylation of the substrate by the kinases is examined by way of the reverse reaction. The wheat germ kinase, casein kinase G, and casein kinase II appear to phosphorylate the same sites on the T-substrate as they are capable of completely dephosphorylating each other's 32P-T-substrate. Each of these kinases can catalyze the incorporation of 12 mol of 32P/48 000 g of T-substrate. In contrast, casein kinases A and I can incorporate only 6 mol of 32P/48 000 g of T-substrate. Studies on the reverse reactions suggest that these phosphorylation sites may be the same for both enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

An endogenous, Mg2+-dependent, protein kinase in coated vesicles from synaptosomes of bovine brain

Coated vesicles (CV) extracted from bovine brain synaptosomes were purified by differential and continuous sucrose density gradient centrifugation. An endogenous protein kinase, dependent upon Mg2+, was found in the vesicles. The kinase catalysed, independently of cyclic nucleotides and Ca2+ plus calmodulin, the incorpotation of the radioactive phosphate of [γ-32p]-ATP and -GTP into a protein, 48,000 in molecular weight, termed C-48. The CV-protein kinase mediated the phosphorylation of Phosvitin, but poorly that of casein, histone, or protamine. The enzyme activity was inhibited by heparin sulfate, an inhibitor in the receptor-mediated endocytosis in fibroblasts in culture. Spermidine slightly suppressed the activity of the kinase. The results showed that the endogenous protein kinase in CV from brain synaptosomes, inhibited by heparin sulfate, agreed in many respects with the Phosvitin kinase reported to be distributed in a variety of tisssues.

Change in phosvitin kinase activity during early development of the sea urchin

AbstractProtein kinase, which phosphorylated phosvitin at the expense of ATP but did not phosphorylate casein, protamine, and histone mixture, was obtained by DEAE‐cellulose column chromatography of the extract from the embryos of the sea urchin, Strongylocentrotus intermedius. This enzyme, partially purified by DEAE‐cellulose column, reversibly catalyzed the reaction of phosvitin phosphorylation. This indicates that the sea urchin embryos contain phosvitin kinase. Phosvitin kinase in sea urchin embryos is somewhat different from that found in the other types of cells, which are able to phosphorylate casein as well as phosvitin. In unfertilized eggs, the activity of this enzyme was found only in the supernatant fraction obtained by centrifuging the homogenate at 10,000g for 20 min. The activity in the embryos at the swimming and the mesenchyme blastula stage was higher than in unfertilized eggs, and was localized in the sedimentable fraction obtained by centrifuging the homogenate of the embryos at 10,000g for 20 min. The highest activity of phosvitin kinase was observed in the embryos at the mesenchyme blastula stage, and the enzyme activity became quite low at the late gastrula stage. The activity and the intracellular distribution of phosvitin kinase changed during the development. The enzyme in this sedimentable fraction was not solubilized with 1% Triton X‐100 but was extracted by 1 M NaCl.

Thermolabile protein kinase molecules in a temperature-sensitive murine sarcoma virus pseudotype.

Murine sarcoma virus-associated protein kinases that bind to actin have been purified by affinity chromatography on actin coupled to Sepharose. Heat inactivation studies showed the presence of thermolabile enzyme activity in pseudotypes containing a temperature-sensitivity mutant of murine sarcoma virus (MSV) but not in two independent wild-type MSV pseudotypes. Studies with Sephadex G-75 column fractions showed that a low molecular weight form, approximately 15,000, is the major thermolabile kinase in the temperature-sensitive MSV virions. Antibodies raised against the MSV-coded p60 protein, when added to the in vitro reaction mixtures, showed specific phosphorylation of the IgG heavy chain and a simultaneous reduction in the extent of phosvitin phosphorylation catalyzed by the various MSV pseudotype kinases. Thus a transforming retrovirus-coded enzyme activity that interacts directly with a major cytoskeletal protein and whose activity parallels the transforming ability of a conditional MSV mutant has now been identified.

Enzymatic activities associated with a purified simian virus 40 T antigen-related protein

A protein antigenically related to the simian virus (SV 40) A gene product has been purified to near homogeneity from cells infected with the adenovirus-SV 40 hybrid virus Ad2(+)D2 and shown to contain ATPase (ATP phosphohydrolase, EC 3.6.1.3) and protein kinase (ATP:phosphotransferase, EC 2.7.1.37) activity. Both enzymatic activities copurify with the protein through six stages including one gel filtration column, two ion exchange columns, and a heparin affinity column. Analogous fractions from extracts of cells uninfected or infected with adenovirus 2 alone do not contain these enzymatic activities. The D2 hybrid protein resolves into two forms (I and II) during ion exchange chromatography. Form I, the major species (85%) of the D2 hybrid protein, elutes from DEAE-Sephadex in 0.37 M NaCl and is able to catalyze the hydrolysis of ATP to ADP + P(i) at a rate of 3 mumol/hr per mg. The remaining 10-15% of the D2 hybrid protein consists of form II which elutes from DEAE-Sephadex in 0.29 M NaCl and is able to hydrolyze ATP as well as to incorporate phosphorus from ATP into either the D2 hybrid protein itself or other protein acceptors such as phosvitin. Although both forms are able to bind DNA, the ATPase activity of form I cosediments with SV 40 DNA more efficiently than does the protein kinase activity of form II during glycerol gradient centrifugation. The ATPase activity of form I is efficiently inhibited by addition of anti-T gamma globulin to the reaction mixture whereas control gamma globulin has no effect. Similarly, the phosphorylation of the D2 hybrid protein by form II is inhibited by anti-T gamma globulin. By contrast, phosphorylation of phosvitin is specifically inhibited by antibody only when the immune complex is removed from the reaction mixture. Thus, it appears likely that one and possibly two enzymatic activities are carried out by the D2 hybrid protein. These findings are discussed in terms of mechanisms of SV 40 DNA replication and virally induced transformation.

Effects of polyamines and polyanions on a cyclic nucleotide-independent and a cyclic AMP-dependent

The phosphorylation of phosvitin in vitro by a cyclic nucleotide-independent protein kinase (phosvitin kinase) derived from rooster liver is markedly stimulated by the divalent cation, Mg 2+. In addition, the activity is further stimulated by low concentrations of the polyamines putrescine, spermidine and spermine leading to higher rates of phosphate incorporation than could be obtained at any concentration of Mg 2+. Spermine is inhibitory at higher concentrations. The polyamines shift the Mg 2+ requirement for maximal activity to lower concentrations. The activity of a cyclic AMP-dependent histone kinase from beef heart is not altered by the presence of polyamines. Heparin is a potent inhibitor of phosvitin kinase but has no effect on histone kinase. Polyribonucleotides (polyadenylic acid and transfer RNA) inhibit both types of kinases, but the degree of inhibition of phosvitin kinase is variable and depends upon the type of the polyanion present. Spermidine and spermine, but not Mg 2+, efficiently counteract the inhibitory action of heparin and tRNA. The results suggest that, also in vivo, naturally occurring polyamines and polyanions such ass tRNA may have a regulatory function on protein kinases.

Protein kinase associated with peripheral nerve myelin. 1. Phosphorylation of endogenous myelin proteins and exogenous substrates

When highly purified myelin from rat sciatic nerve was incubated with [γ- 32P]ATP, protein components of the membrane were phosphorylated indicating the presence of both the substrate (receptor protein) and an endogenous kinase in the membrane. Polyacrylamide gel electrophoresis of the phosphorylated membrane proteins followed by scintillation counting of gel slices and autoradiography showed that the polypeptides of molecular weights 28000, 23000 and 19000 were phosphorylated, and 32P from [γ- 32P]ATP having been incorporated into serine residues of the substrate proteins. Phosphorylation of purified myelin was Mg 2+-dependent, was optimal at pH 6.5 and was not stimulated by adenosine 3′,5′-monophosphate. We found that proteins other than those in myelin, such as phosvitin, casein, protamine and histones, can also act as a substrate for the membrane associated kinase. Muscle protein kinase inhibitor had no effect on the endogenous phosphorylation of myelin proteins or on the phosphorylation of phosvitin by peripheral nerve myelin protein kinase. However, the phosphorylation of histone by peripheral nerve myelin protein kinase was inhibited by the protein kinase inhibitor. After washing the membrane with 150 mM KCl the protein kinase that utilizes histone as substrate was found in the supernatant. In contrast, the endogenous phosphorylation of membrane proteins or the phosphorylation of phosvitin by the membrane associated kinase was not affected by washing. From these findings we conclude that at least two protein kinase systems exist in purified peripheral nerve myelin. One system is not inhibited by muscle kinase inhibitor, is tightly bound to the membrane and utilizes as its receptor proteins either exogenous phosvitin or endogenous membrane proteins. The second system is inhibited by muscle kinase inhibitor, is removable from the membrane and utilizes histones as its receptor proteins.