Biochemical characterization of a casein kinase I-like actin kinase responsible for the actin-induced suppression of casein kinase II activity in vitro

By combination of column chromatographies (heparin-agarose, HiTrap heparin and HiTrap SP columns) and gel filtration on a Superdex 200-pg HPLC column, an actin kinase was partially purified from a 1. 5 M NaCl extract of porcine liver. The actin kinase was finally purified, by actin-Sepharose column chromatography (HPLC), as an actin-binding protein kinase. The biochemical properties, such as (1) requirements of divalent cations (10 mM Mg(2+) and 3 mM Mn(2+)) and effective phosphate acceptors (actin and alpha-casein), (2) phosphorylation of both Ser- and Thr-residues on these two phosphate acceptors, (3) autophosphorylation of the catalytic subunit (approximately 37 kDa), and (4) inhibition kinetics by CK-I-7 (a CK-I specific inhibitor), of the purified actin kinase were similar to those reported for CK-I purified from various mammalian cells, but it was distinguishable from three cellular actin kinases (A-kinase, C-kinase and actin-fragmin kinase (approximately 80 kDa)). The 37 kDa actin kinase-mediated phosphorylation of actin did not relate to its polymerizability. Inhibition of CK-II-mediated phosphorylation of functional cellular proteins, including calmodulin (CaM), by actin was significantly stimulated after its full phosphorylation by the purified 37 kDa actin kinase or rCK-I in vitro. These results suggest that: (1) the 37 kDa Ser/Thr actin-binding kinase may be classified as a member of the CK-I family; and (2) specific phosphorylation of actin by the actin kinase may be involved in the suppression mechanism of CK-II-mediated signal transduction at the cellular level.     

Modulation of glycogen synthase kinase activity of skeletal and smooth muscle casein kinase I by spermine

Casein kinase I (CK-I) from skeletal muscle was stimulated 2-3 fold by 0.25-1 mM spermine. The polyamine also stimulated the phosphorylation of glycogen synthase by another casein kinase purified from aortic smooth muscle [DiSalvo et al. (1986) Biochem. Biophys. Res. Commun. 136, 789-796]. Phosphopeptide maps and phosphoamino acid analysis of [32P]glycogen synthase revealed that smooth muscle casein kinase phosphorylated glycogen synthase in the same sites that undergo phosphorylation by CK-I. The stimulatory effect of spermine on glycogen synthase kinase activity of CK-I was accompanied by increased phosphorylation of all peptide sites of glycogen synthase. Increased phosphorylation was observed in both seryl and threonyl residues. Higher concentrations (4 mM) of spermine inhibited CK-I activity by about 50%. These results indicate that aortic smooth muscle casein kinase is a CK-I enzyme and that skeletal and smooth muscle CK-I can be modulated by spermine.     

The modulation of the biological activities of mitochondrial histone Abf2p by yeast PKA and its possible role in the regulation of mitochondrial DNA content during glucose repression.

The mitochondrial histone, Abf2p, of Saccharomyces cerevisiae is essential for the maintenance of mitochondrial DNA (mtDNA) and appears to play an important role in the recombination and copy number determination of mtDNA. Abf2p, encoded by a nuclear gene, is a member of HMG1 DNA-binding protein family and has two HMG1-Box domains, HMG1-Box A and B. To investigate the role of Abf2p in the control of mtDNA copy number, we asked if the in vivo functions of Abf2p are regulated by the possible modification such as phosphorylation. We found that the N-terminal extended segment (KRPT(21)S(22)) of HMG1-Box A is rapidly and specifically phosphorylated by cAMP-dependent protein kinase (PKA) in vitro. The phosphorylation in this region inhibits the binding of Abf2p to all kinds of DNA including four-way junction DNA and the supercoiling activity of Abf2p itself. The abf2 mutant cells with an abf2(T21A/S22A) allele defective in the phosphorylation site have a severe defect in the regulation of mtDNA content during glucose repression in vivo. These observations suggest that the phosphorylation via PKA, that is activated during glucose repression, may regulate the in vivo functions of Abf2p for the control of mtDNA content during shift from gluconeogenic to fermentative growth.     

Thyrotropin-stimulated phosphorylation of high mobility group protein 14 in vivo at the site catalyzed by cyclic nucleotide-dependent protein kinases in vitro

Thyrotropin (TSH) treatment of bovine thyroid slices increased 32P-labeling of chromosomal high mobility group 14 (HMG) protein approximately 2-fold. Analogs of cAMP, but not cGMP, also enhanced phosphorylation of HMG 14. The sites of phosphorylation were analyzed by partial acid hydrolysis and by two-dimensional mapping of tryptic digests of 32P-labeled HMG 14 which was purified from control and TSH-treated thyroid tissue. TSH treatment enhanced phosphorylation at serine residues in four prominent tryptic phosphopeptides which were identical with those derived from HMG 14 phosphorylated in vitro with cAMP- and cGMP-dependent protein kinases. The four tryptic phosphopeptides contain serine 6, the major site of in vitro phosphorylation catalyzed by cyclic nucleotide-dependent protein kinases (Walton, G. M., Spiess, J., and Gill, G. N. (1982) J. Biol. Chem. 257, 4661-4668). TSH did not affect phosphorylation of serine 24, a minor site of phosphorylation in vitro. These studies suggest that TSH-stimulated phosphorylation of HMG 14 is catalyzed by cAMP-dependent protein kinase.     

Biochemical characterization of a N-terminal fragment (p5) cleaved from fibroblast growth factor-binding protein (FGF-BP) in bovine milk in vitro

By means of successive gel filtration on a Superdex 30 pg column and Mono S column chromatography, a 5-kDa polypeptide (p5) was highly purified from the low molecular weight (LMW) fraction separated from the partially purified lactoferrin (bLF) fraction of bovine milk, and biochemically characterized as a phosphate acceptor for two protein kinases [cAMP-dependent protein kinase (PKA) and casein kinase 1delta (CK1delta)] in vitro. Purified p5 was identified as a fragment (N-terminal positions 24-51, 28 amino acid residues) cleaved from fibroblast growth factor-binding protein (FGF-BP, p37). Both purified p5 and synthetic p5 (sp5) were effectively phosphorylated by PKA, and also phosphorylated by CK1delta in the presence of two sulfated lipids [sulfatide or cholesterol-3-sulfate (CH-3S), SCS] in vitro. A novel phosphorylation site (RNRRGS) for CK1delta and a potent SCS-binding site (RNRR) on p5 were identified. The PKA-mediated phosphorylation of p5 was highly stimulated when incubated with either acidic FGF (aFGF) or bLF in vitro, but this phosphorylation was more sensitive to SCS than H-89 (a specific PKA inhibitor). Immunoprecipitate experiments revealed p5, but not the phosphorylated p5, to be directly bound to aFGF in vitro. These results show that (i) p5 has a high binding affinity with aFGF as well as bLF; (ii) the binding of SCS to p5 results in the selective inhibition of its phosphorylation by PKA; and (iii) SCS functions as an effective stimulator for the phosphorylation of p5 by CK1delta in vitro. In addition, p5 may play an important physiological role as a trafficking factor for the physiological interaction between aFGF group including endothelial cell growth factors and their binding proteins in vivo.     

High-mobility-group proteins P1, I and Y as substrates of the M-phase-specific p34cdc2/cyclincdc13 kinase

All dividing cells entering the M phase of the cell cycle undergo the transient activation of an M-phase-specific histone H1 kinase which was recently shown to be constituted of at least two subunits, p34cdc2 and cyclincdc13. The DNA-binding high-mobility-group (HMG) proteins 1, 2, 14, 17, I, Y and an HMG-like protein, P1, were investigated as potential substrates of H1 kinase. Among these HMG proteins, P1 and HMG I and Y are excellent substrates of the M-phase-specific kinase obtained from both meiotic starfish oocytes and mitotic sea urchin eggs. Anticyclin immunoprecipitates, extracts purified on specific p34cdc2-binding p13suc1-Sepharose and affinity-purified H1 kinase display strong HMG I, Y and P1 phosphorylating activities, demonstrating that the p34cdc2/cyclincdc13 complex is the active kinase phosphorylating these HMG proteins. HMG I and P1 phosphorylation is competitively inhibited by a peptide mimicking the consensus phosphorylation sequence of H1 kinase. HMG I, Y and P1 all possess the consensus sequence for phosphorylation by the p34cdc2/cyclincdc13 kinase (Ser/Thr-Pro-Xaa-Lys/Arg). HMG I is phosphorylated in vivo at M phase on the same sites phosphorylated in vitro by H1 kinase. P1 is phosphorylated by H1 kinase on sites different from the sites of phosphorylation by casein kinase II. The three thermolytic phosphopeptides of P1 phosphorylated in vitro by purified H1 kinase are all present in thermolytic peptide maps of P1 phosphorylated in vivo in proliferating HeLa cells. These phosphopeptides are absent in nonproliferating cells. These results demonstrate that the DNA-binding proteins HMG I, Y and P1 are natural substrates for the M-phase-specific protein kinase. The phosphorylation of these proteins by p34cdc2/cyclincdc13 may represent a crucial event in the intense chromatin condensation occurring as cells transit from the G2 to the M phase of the cell cycle.     

Polyamines and heparin do not appreciably influence phosphorylation of chromatin proteins HMG 14 and HMG 17 by nuclear protein kinase II

Phosphorylation of acidic substrates such as casein and phosvitin by nuclear protein kinase II is stimulated by polyamines and inhibited by heparin, which mimics an endogenous proteoglycan inhibitor. The phosphorylation in vitro of the chromatin proteins HMG 14 and HMG 17 by nuclear protein kinase II were examined in this study focusing on the modifying effects of polyamines and heparin. Both HMG proteins were phosphorylated by the enzyme, but polyamines did not appreciably influence the extent of their phosphorylation. In addition, heparin did not inhibit the kinase reaction with the HMG proteins as substrates. These results indicate that the nuclear protein kinase II does actively phosphorylate HMG 14 and HMG 17 in vitro but that in contrast to some model substrates, polyamines and heparin do not appreciably affect their phosphorylation.     

Constitutive phosphorylation of the acidic tails of the high mobility group 1 proteins by casein kinase II alters their conformation, stability, and DNA binding specificity.

The high mobility group (HMG) 1 and 2 proteins are the most abundant non-histone components of chromosomes. Here, we report that essentially the entire pool of HMG1 proteins in Drosophila embryos and Chironomus cultured cells is phosphorylated at multiple serine residues located within acidic tails of these proteins. The phosphorylation sites match the consensus phosphorylation site of casein kinase II. Electrospray ionization mass spectroscopic analyses revealed that Drosophila HMGD and Chironomus HMG1a and HMG1b are double-phosphorylated and that Drosophila HMGZ is triple-phosphorylated. The importance of this post-translational modification was studied by comparing some properties of the native and in vitro dephosphorylated proteins. It was found that dephosphorylation affects the conformation of the proteins and decreases their conformational and metabolic stability. Moreover, it weakens binding of the proteins to four-way junction DNA by 2 orders of magnitude, whereas the strength of binding to linear DNA remains unchanged. Based on these observations, we propose that the detected phosphorylation is important for the proper function and turnover rates of these proteins. As the occurrence of acidic tails containing canonical casein kinase II phosphorylation sites is common to diverse HMG and other chromosomal proteins, our results are probably of general significance.     

A one-step preparative method for separating SER 6-phosphorylated HMG 14 from unphosphorylated HMG 14 and in vitro phosphorylation reaction components

While clear evidence exists for the regulation of the phosphorylation of the very basic high mobility group (HMG) and histone chromatin proteins, the physiological role of their phosphorylation remains poorly understood. Elucidation of these roles has been difficult, in part, because of the inability to obtain sufficient quantities of purified phosphorylated derivatives. We have used Mono S cation-exchange chromatography to prepare milligram quantities of pure Ser 6-phosphorylated HMG 14 (Ser 6-PO4-HMG) from unphosphorylated Mono S-purified calf thymus HMG 14 following in vitro phosphorylation with cAMP-dependent protein kinase (A-kinase). In one step, this technique separates the phosphorylated derivative from A-kinase, ATP, unphosphorylated HMG 14, and a minor phosphorylated by-product which evidence suggests may be the previously reported Ser 6, 24-diphospho-HMG 14. Mono S chromatography also enhances the purity of calf thymus HMG 14 prepared by perchloric acid extraction, acetone and ethanol precipitations, and CM-Sephadex chromatography. In addition, it permits the detection of apparent microheterogenous forms of both unphosphorylated and Ser 6-PO4-HMG 14. The significant reductions in binding affinity resulting from the incorporation of phosphate groups into HMG 14 suggest that Mono S chromatography could have more general application in the isolation of phosphorylated derivatives of other basic proteins, including other chromatin-associated DNA-binding proteins which are known to undergo specific phosphorylation. It would especially be useful when the proteins and their phosphorylated derivatives bind more tightly to Mono S than the kinases used for their phosphorylation.     

The phosphorylation of high mobility group proteins 14 and 17 and their distribution in chromatin

The phosphorylation of the high mobility group (HMG) proteins has been investigated in mouse Ehrlich ascites, L1210 and P388 leukemia cells, human colon carcinoma cells (HT-29), and Chinese hamster ovary cells. HMG 14 and 17, but not HMB 1 and 2, were phosphorylated in the nuclei of all cell lines with a serine being the site of modification for both proteins in Ehrlich ascites cells. Phosphorylation of HMG 14 and 17 was greatly reduced in cultured cells at plateau phase in comparison to log phase cells, suggesting that modification of HMG 14 and 17 is growth-associated. However, phosphorylation was not linked to DNA synthesis, since incorporation of 32P did not vary through G1 and S phase in synchronized Chinese hamster ovary cells. Treatment of HT-29 or Ehrlich ascites cells with sodium butyrate reduced HMG phosphorylation by 30 and 70%, respectively. The distribution of the phosphorylated HMG proteins in chromatin was examined using micrococcal nuclease and DNase I. 32P-HMG 14 and 17 were preferentially associated with micrococcal nuclease-sensitive regions as demonstrated by the release of a substantial fraction of the phosphorylated forms of these proteins under conditions which solubilized less than 3% of the DNA. Short digestions with DNase I did not show a marked release of 32P-HMG 14 or 17.     

Is high mobility group protein 17 phosphorylated in vivo? re-examination of the HeLa cell cycle data

When in vivo [32P] phosphate labeled HMG proteins from unsynchronized HeLa cells are separated by electrophoresis in acid-urea polyacrylamide gels, as opposed to separation in SDS-polyacrylamide, HMG 17 does not show any 32P incorporation. Likewise, no 32P radioactivity was found in HMG 17 protein isolated at different stages of the cell cycle from synchronized cells. By contrast, HMG 14 reveals a previously reported (Bhorjee, J.S. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 6944-6948) cell cycle stage-specific dependent phosphorylation with maximum 32P radioactivity in the G2 phase relative to G1. Furthermore, HMG 14 is resolved into multiple electrophoretic forms as phosphoprotein in the acid-urea system. The results presented seriously question the data on the in vivo phosphorylation of HMG 17, and suggest that these be reevaluated.     

Binding of high-mobility-group proteins HMG 14 and HMG 17 to DNA and histone H1 as influenced by phosphorylation

We have used affinity chromatography to study the effects of phosphorylation of calf thymus high-mobility-group proteins HMG 14 and HMG 17 on their binding properties towards calf thymus single- and double-stranded DNA and histone H1. Without in vitro phosphorylation, HMG 14 and HMG17 eluted from doble-stranded DNA-columns at 200 mM NaCl. HMG 14 was released from single-stranded DNA-column at 300 mM NaCl and from H1-column at 130 mM NaCl, whereas the corresponding values for HMG 17 were 230 mM and 20 mM, respectively. Phosphorylation of HMG 14 and HMG 17 by cAMP-dependent protein kinase (A-kinase) decreased markedly their affinity (270 mM and 200 mM NaCl, respectively) for single-stranded DNA, whereas HMG 14 phosphorylated by nuclear protein kinase II (NII-kinase) eluted only slightly (290 mM NaCl) ahead of the unphosphorylated protein. HMG 14 phosphorylated by both A-kinase and NII-kinase eluted from double-stranded DNA-columns almost identically (190 mM NaCl) with the unphosphorylated protein. Interestingly, phosphorylation of HMG 14 by NII-kinase increased considerably its affinity for histone H1 and the phosphorylated protein eluted at 200 mM NaCl. Phosphorylation of HMG 14 by A-kinase did not alter its interaction towards histone H1. These results indicate that modification of HMG 14 by phosphorylation at specific sites may have profound effects on its binding properties towards DNA and histone H1, and that HMG 17 has much weaker affinity for single-stranded DNA and histone H1 than HMG 14.     

Phosphorylation of human high mobility group N1 protein by protein kinase CK2

High mobility group (HMG) N1 protein, formerly known as HMG 14, is a member of the chromosomal HMG protein family. Protein kinase CK2 was previously reported to be able to phosphorylate bovine HMGN1 in vitro; Ser89 and Ser99, corresponding to Ser88 and Ser98 in human HMGN1, were shown to be major and minor recognition sites, respectively. In this report, we employed mass spectrometry and examined both the extent and the sites of phosphorylation in HMGN1 protein catalyzed by recombinant human protein kinase CK2. We found that five serine residues, i.e., Ser6, Ser7, Ser85, Ser88, and Ser98, in HMGN1 can be phosphorylated by the kinase in vitro. All five sites were previously shown to be phosphorylated in MCF-7 human breast cancer cells in vivo. Among these five sites, Ser6, Ser7, and Ser85 were new sites of phosphorylation induced by protein kinase CK2 in vitro.     

Primary organization of nucleosomes. Interaction of non-histone high mobility group proteins 14 and 17 with nucleosomes, as revealed by DNA-protein crosslinking and immunoaffinity isolation

The binding sites for histones and high mobility group proteins (HMG) 14 and 17 have been located on DNA in the nucleosomal cores and H1/H5-containing nucleosomes. The nucleosomes were specifically associated with two molecules of the non-histone proteins HMG 14 and/or HMG 17 when followed by DNA-protein crosslinking and immunoaffinity isolation of the crosslinked HMG-DNA complexes. HMGs 14 and 17 were shown to be crosslinked in a similar manner to each core DNA strand at four sites: to both 3' and 5' DNA ends and also at distances of about 25 and 125 nucleotides from the 5' termini of the DNA. These sites are designated as HMG(143), (0), (25) and (125). The site HMG(125) is located at the place where no significant histone-DNA crosslinking was observed. The HMG(125) and HMG(25) sites lie opposite one another on the complementary DNA strands across the minor DNA groove and are placed, similarly to histones, on the inner side of the DNA superhelix in the nucleosome. The crosslinking of HMG 17 to the 3' ends of the DNA is much weaker than that of HMG 14. These data indicate that each of two molecules of HMG 14 and/or HMG 17 is bound to the double-stranded core DNA at two discrete sites: to the 3' and 5' ends of the DNA and at a distance of 20 to 25 base-pairs from each DNA terminus inside the nucleosome on a histone-free DNA region. Binding of HMG 14 or 17 does not induce any detectable rearrangement of histones on DNA and both HMGs seem to choose the same sites for attachment in nucleosomal cores and H1/H5-containing nucleosomes.     

Phosphorylation of Mitogen-Activated Protein Kinase in Cultured Rat Cortical Glia by Stimulation of Metabotropic Glutamate Receptors

Abstract: Activation of metabotropic glutamate receptors (mGluRs) in glia results in significant physiological effects for both the glia and the neighboring neurons; but in many cases, the mGluR subtypes and signal transduction mechanisms mediating these effects have not been determined. In this study, we report that mGluR activation in primary cultures of rat cortical glia results in tyrosine phosphorylation of several proteins, including p44/p42 mitogen-activated protein kinases, also referred to as extracellular signal-regulated kinases (ERK1/2). Incubation of glial cultures with the general mGluR agonist 1-aminocyclopentane-1 S ,3 R -dicarboxylate and the mGluR group I-selective agonists ( RS )-3,5-dihydroxyphenylglycine (DHPG) and l -quisqualate resulted in increased tyrosine phosphorylation of ERK1/2. The group II-selective agonist (2 S ,2' R ,3' R )-2-(2',3'-dicarboxycyclopropyl)glycine and group III-selective agonist l (+)-2-amino-4-phosphonobutyric acid had no effect on tyrosine phosphorylation. DHPG-induced ERK1/2 phosphorylation could be inhibited by an antagonist that acts at group I or group II mGluRs but not by antagonists for group II and group III mGluRs. Protein kinase C (PKC) activators also induced ERK1/2 phosphorylation, but the PKC inhibitor bisindolylmaleimide I did not inhibit DHPG-induced ERK1/2 phosphorylation at a concentration that inhibited the response to phorbol 12,13-dibutyrate. These data suggest that mGluR activation of ERK1/2 in cultured glia is mediated by group I mGluRs and that this effect is independent of PKC activation. Furthermore, immunoblots with antibodies against various mGluR subtypes show expression of mGluR5, but no other mGluRs in our cultures. Taken together, these results suggest that mGluR5 stimulation results in tyrosine phosphorylation of ERK1/2 and other glial proteins.     

The phosphorylation of nuclear proteins in normal and transformed cells.

Normal (Rat 1) and transformed cells (middle T antigen-transformed derivative 3C3) were grown in the presence of 32P-orthophosphate. 32P-labelled nuclear proteins were fractionated by means of two-dimensional gel electrophoresis and detected by autoradiography. The comparative analysis of the autoradiographs of the normal and transformed cells revealed differences in the phosphorylation patterns of histone and low-molecular-mass high mobility group proteins (HMG). Three of the HMG proteins were highly phosphorylated in the transformed cells, and the analysis of their phosphorylation sites showed that these HMG proteins were phosphorylated on serine and threonine but not on tyrosine residues.