Tyrosine-specific dephosphorylation-phosphorylation with alkaline phosphatases and epidermal growth factor receptor kinase as evidenced by 31P NMR spectroscopy

The dephosphorylation of phospho-amino acids with alkaline phosphatase (AlPase) from calf intestine or Escherichia coli and the phosphorylation of bovine serum albumin (BSA) with epidermal growth factor (EGF) receptor kinase from human A431 epidermoid carcinoma cells were investigated by 31P NMR spectroscopy. The initial rates of the dephosphorylation of phospho-tyrosine (P-Tyr) and phosphoserine (P-Ser) with AlPase were essentially the same in the one-substrate system. In the two-substrate system (P-Tyr plus P-Ser), however, the ratio of the initial rate for P-Tyr vs. P-Ser was 2.4 to 4.5 depending on the buffer and pH conditions employed. This substantiates for the first time the specificity of AlPases to P-Tyr over P-Ser at the free amino acid level. In the stationary phase of the overall process, the dephosphorylation of P-Ser became slow compared to that of P-Tyr in the one-substrate system. The decrease in the rate for P-Ser was further pronounced in the two-substrate system. For this remarkable effect, the rephosphorylation of serine was responsible, as demonstrated in the reaction mixture containing serine, Pi, and AlPase. BSA phosphorylated by EGF receptor kinase exhibited sharp 31P resonances around 0 ppm at neutral pH, far distant from the peak positions (4.9 ppm) of histone H1 phosphorylated by cAMP-dependent protein kinase. These NMR data are directed evidence that BSA was phosphorylated exclusively at the tyrosyl residues, whereas the phosphorylation of histone H1 was at the seryl residues.     

Mouse spleen cell nuclear protein kinases and the stimulating effect of dsDNA on NHP phosphorylation by cyclic AMP-independent protein kinase in vitro

cAMP-dependent (designated as enzyme I, about 68,000 daltons) and cAMP-independent protein kinase (designated as enzyme II, about 45,000 daltons) have been partially purified from the nuclei of mouse spleen cells. Both kinases phosphorylated calf thymus histones as well as non-histone proteins (NHP) and required Mg2+ (8 mM) or Mn2+ (2 mM) for maximal activity. NEM (0.5 mM), which is an inhibitor of SH-enzymes, inhibited the histone phosphorylating activity of enzyme II by more than 90%, whereas it inhibited the activity of enzyme I by less than 10%. Moreover, the activity of enzyme II was more sensitive to high temperature than that of enzyme I. Non-histone protein (CM-III protein) served as a more effective substrate for enzyme II than histones; the Km value for CM-III protein was 34.4 micrograms/ml whereas that for histone H2a (14,300 daltons) was 155 micrograms/ml (1.08 x 10(-5) M). CM-III protein phosphorylation by enzyme II in vitro was greatly stimulated by the addition of dsDNA, but not by single-stranded DNA or bacterial ribosomal RNA. However, the phosphorylation of CM-III protein by enzyme I was less than 50% of that of histones, and there was no stimulatory effect. SDS-gel electrophoresis showed that two distinct NHPs (about 13,000 and 19,000 daltons) prepared from calf thymus chromatin were preferentially phosphorylated by enzyme II in vitro in the presence of dsDNA. This finding suggests that these two NHPs may be specific phosphate acceptors of cAMP-independent protein kinase (enzyme II) in the nuclei of mouse spleen cells.     

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.     

Amino acid sequence of histone H1 at the ADP-ribose-accepting site and ADP-ribose X histone-H1 adduct as an inhibitor of cyclic-AMP-dependent phosphorylation

The ADP-ribosylation site of histone H1 from calf thymus by purified hen liver nuclear ADP-ribosyltransferase was determined and effects of the ADP-ribose X histone-H1 adduct on cAMP-dependent phosphorylation of the histone H1 were investigated. ADP-ribosylated histone H1 was prepared by incubation of histone H1, 1 mM [adenylate-32P]NAD and the purified ADP-ribosyltransferase. N-Bromosuccinimide-directed bisection of ADP-ribosylated histone H1 showed that the NH2-terminal fragment (Mr = 6000) was modified and contained serine residue 38, the site of phosphorylation by cAMP-dependent protein kinase. Digestion of the NH2-terminal fragment with cathepsin D and trypsin, and purification of this fragment, using high-performance liquid chromatography, yielded a radiolabelled single peptide corresponding to residues 29-34 of histone H1, containing the arginine residue as the ADP-ribosylation site. These results indicate that ADP-ribosylation of histone H1 occurs at the arginine residue 34, sequenced at the NH2-terminal side of the phosphate-accepting serine residue 38. Phosphorylation of histone H1 from calf thymus by cAMP-dependent protein kinase was markedly reduced when histone H1 was ADP-ribosylated. Kinetic studies of phosphorylation revealed that ADP-ribosylated histone H1 was a linear competitive inhibitor of histone H1 and a linear non-competitive inhibitor of ATP.     

Immunochemical analysis of histone H1 and H5 from pigeon erythroid cells]

An antiserum with the antibody titer of 1 : 4096 was obtained by immunization of rabbits with the tRNA-histone H5 complex from pigeon erythrocytes. The specificity of the antiserum was studied quantitatively from the reaction of the complement binding to a homologous antigen (histone H5) and its modifications (I, II, III), differing in the degree of phosphorylation. It was shown that phosphorylation of histone H5 increases the ability of the antigen to bind to antibodies, which is especially well-pronounced at the antiserum dilutions as high as 20480. The comparison of the antigenic properties of histones H5 from pigeon and chicken erythrocytes revealed beside structural differences of the proteins the presence of common antigenic determinants. A similar observation was made when histones H5 and H1 from pigeon erythrocytes were compared. Histone H1 from chicken erythrocytes and histone H1 from calf thymus did not produce criss-cross reactions with antiserum H5.     

ADP-ribosylation regulates the phosphorylation of histones by the catalytic subunit of cyclic AMP-dependent protein kinase

Phosphorylation of whole histones from calf thymus by the catalytic subunit of cyclic AMP-dependent protein kinase was markedly reduced when the histones were ADP-ribosylated. NAD, nicotinamide or free ADP-ribose molecule did not suppress the phosphorylation. Urea gel electrophoretic analyses of the phosphorylated histones which had already been ADP-ribosylated revealed that the suppression of phosphorylation occurred in both H1 and core histones. Therefore, the possibility that ADP-ribosylation may regulate the phosphorylation of histones phosphorylation in nuclei warrants further investigation.     

Modification of the lysine residues of histones H1 and H5: Effects on structure and on the binding to chromatin

The extensive modification of histone H1 from calf thymus with the amino-group reagent dimethylmaleic anhydride (over 35 lysine residues modified per molecule) produces no effect on its secondary structure detectable by circular dichroism (far UV). Fluorescence and circular dichroism (near-UV) of the modified histone show variations in the local environment of its sole tyrosine residue. These changes are reversed on regeneration of the modified amino groups. While histone H1 is easily dissociated with this reagent from calf thymus or chicken erythrocyte chromatin, a much stronger treatment is needed to liberate histone H5 from erythrocyte chromatin. This difference appears to be related to the higher arginine content of histone H5.     

Manifold effects of sodium butyrate on nuclear function. Selective and reversible inhibition of phosphorylation of histones H1 and H2A and impaired methylation of lysine and arginine residues in nuclear protein fractions

In addition to its known effect in suppressing the deacetylation of the nucleosomal core histones, sodium butyrate in the concentration range 0.5 to 15 mM causes a selective inhibition of [32P]phosphate incorporation into histones H1 and H2A of cultured HeLa S3 cells. No commensurate general inhibition of phosphorylation is seen in the non-histone nuclear proteins of butyrate-treated cells, but phosphorylation patterns are altered and 32P-uptake may be stimulated, as well as inhibited, depending upon the protein fraction analyzed. The degree of inhibition of histone phosphorylation in intact cells increases progressively as the butyrate concentration is raised from 0.5 to 15 mM. The effect is time-dependent and fully reversible. Butyrate has no effect on the kinetics of phosphate release from previously phosphorylated histones of cultured cells, nor does it significantly alter the rate of dephosphorylation of 32P-labeled histone H1 by endogenous phosphatases in vitro. Despite the suppression of [32P]phosphate incorporation into histones H1 and H2A of butyrate-treated cells, Na-butyrate does not inhibit the in vitro activities of either type I or type II cyclic AMP-dependent protein kinases, or the cAMP-independent H1 kinase associated with cell cycle progression. This suggests that the butyrate effect on histone phosphorylation in vivo is indirect and may involve an alteration in substrate accessibility or a modulation of systems affecting kinase activities. The poly(ADP)-ribosylation of HeLa histones is not inhibited by 5 mM Na-butyrate. Cells exposed to butyrate show an impaired methylation of lysine and arginine residues in their histones and nuclear hnRNP particles, respectively.     

Phosphorylation of keratin intermediate filaments by protein kinase C, by calmodulin-dependent protein kinase and by cAMP-dependent protein kinase.

Keratins, constituent proteins of intermediate filaments of epithelial cells, are phosphoproteins containing phosphoserine and phosphothreonine. We examined the in vitro phosphorylation of keratin filaments by cAMP-dependent protein kinase, protein kinase C and Ca2+/calmodulin-dependent protein kinase II. When rat liver keratin filaments reconstituted by type I keratin 18 (molecular mass 47 kDa; acidic type) and type II keratin 8 (molecular mass 55 kDa; basic type) in a 1:1 ratio were used as substrates, all the protein kinases phosphorylated both of the constituent proteins to a significant rate and extent, and disassembly of the keratin filament structure occurred. Kinetic analysis suggested that all these protein kinases preferentially phosphorylate keratin 8, compared to keratin 18. The amino acid residues of keratins 8 and 18 phosphorylated by cAMP-dependent protein kinase or protein kinase C were almost exclusively serine, while those phosphorylated by Ca2+/calmodulin-dependent protein kinase II were serine and threonine. Peptide mapping analysis indicated that these protein kinases phosphorylate keratins 8 and 18 in a different manner. These observations gave the way for in vivo studies of the role of phosphorylation in the reorganization of keratin filaments.     

Histone H5 in nucleated erythrocytes of fish as determined by radioimmunoassay

Tissue-specific histone H5 in the nucleated erythrocytes of dogfish, scup, skate, tautog, sea robin and toad fish was studied. The presence of this histone was inferred by its electrophoretic mocility on polyacrylamide gels containing either acid-urea or sodium dodecyl sulfate. By radioimmunoprecipitation assays, cross reaction was observed between fish histones and an anti-H5chicken antibody. The antibody was specific to chicken histone H5; purified chicken histone H1 and calf thymus total histones did not cross react. It is concluded that fish histone H5 shares common antigenic determinants with the chicken H5 histone.     

Phenylalanine metabolism in isolated rat liver cells. Effects of glucagon and diabetes.

Tryptic digestion of histone H1 from the sperm of the sea urchin Sphaerechinus granularis leaves a limiting peptide of approx. 80 residues that is of similar size to the limit peptide from calf thymus H1 or chicken erythrocyte H5. The S. granularis limit peptide folds to form tertiary structure similar to that of the intact parent histone H1 (shown by n.m.r. spectra), but the helical content is decreased by the digestion from 64 residues to 28. In contrast, intact calf thymus H1 and chicken erythrocyte H5 histones have only about 28 helical residues, which are preserved in their limit peptides. The extra helix in S. granularis is shown to be rapidly digested away by trypsin, and its location in histone H1 is discussed. A possible relationship of this structural feature to the length of linker DNA is proposed.     

Histone H1 kinase in exponential and synchronous populations of Chinese hamster fibroblasts

Nuclear histone kinase activity, specifically histone H1 phosphotransferase activity, was shown to increase in synchronous Chinese hamster cells from the G1/S boundary to late G2/early M phase. Chromatin extracts purified by DEAE-Sephacel chromatography showed a cAMP-independent kinase activity that demonstrated cell cycle dependence and high specificity for histone H1 as the phosphate acceptor in the presence of [gamma-32P] ATP. This activity was purified approximately 40-fold. Using as substrates calf thymus histone H1 subfractions resolved by Bio-Rex 70 ion exchange chromatography, phosphorylation by the nuclear histone H1 kinase indicated that 32P incorporation into H1-2 was at least twice that for H1-1 and H1-3 subfractions. Both amino- and carboxy-terminal fragments generated by N-bromosuccinimide cleavage were phosphorylated. Phosphoamino acid analysis showed phosphothreonine to be approximately twice as abundant as phosphoserine. Histone H1 kinase activity was not activated by cyclic nucleotides, nor inhibited by cAMP-dependent protein kinase inhibitors or regulatory subunits. There was no effect on activity by Ca2+ alone or in the presence of calmodulin or diacylglycerol. Kinase activity was inhibited by nonhydrolyzable analogs of ATP such as adenyl-5'-yl imidodiphosphate, by 5'-p-fluorosulfonylbenzoyladenosine which binds to the ATP binding site of the enzyme, and by quercetin. Column fractions enriched in histone H1 kinase were labeled with 5'-p-fluorosulfonylbenzoyl[8-14C]adenosine, and peptides were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One band, Mr 67,000, was specifically labeled and may represent the H1 kinase catalytic subunit.     

Identification of the phosphorylation sites of H2B histone by a catalytic fragment of p72syk from porcine spleen.

Phosphorylated sites of calf thymus H2B histone were investigated with a catalytic fragment of 72 kDa protein-tyrosine kinase (p72syk). Three of five tyrosine residues in H2B histone can be phosphorylated by this kinase. In this analysis, H2B histone was thoroughly phosphorylated in vitro with [gamma-32P]ATP and the kinase, and then digested with a lysylendopeptidase. The resulting radioactive phosphopeptides were separated by a reverse-phase column on high performance liquid chromatography. Subsequent sequential Edman degradation of the purified phosphopeptides revealed that 40Y, 83Y and 121Y were phosphorylated. 121Y is the major phosphorylated residue in H2B histone. No phosphorylation was detected in 37Y and 42Y. Although the consensus sequence was not defined from these analyses, our data suggest that higher-order structure(s) in addition to primary one may participate in recognition of H2B histone by this protein kinase.     

Amino acid sequence of in vivo phosphorylation sites in the main intrinsic protein (MIP) of lens membranes

The main intrinsic membrane protein of the lens fiber cell, MIP, has been previously shown to be phosphorylated in preparations of lens fragments. Phosphorylation occurred on serine residues near the cytoplasmic C-terminus of the molecule. Since MIP is thought to function as a channel protein in lens plasma membranes, possibly as a cell-to-cell channel protein, phosphorylation could regulate the assembly or gating of these channels. We sought to identify the specific serines which are phosphorylated in order to help identify the kinases involved in regulating MIP function. To this end we purified a peptide fragment from native membranes that had not been subjected to any exogenous kinases or kinase activators. Any phosphorylation detected in these fragments must be due to cellular phosphorylation and thus is termed in vivo phosphorylation. Purified membranes were also phosphorylated with cAMP-dependent protein kinase to determine the mobility of phosphorylated and unphosphorylated MIP-derived peptides on different HPLC columns and to determine possible cAMP-dependent protein kinase phosphorylation sites. Lens membranes, which contain 50-60% of the protein as MIP, were digested with lysylendopeptidase C. Peptides were released from the C-terminal region of MIP and a major product of 21-22 kDa remained membrane-associated. Separation of the lysylendopeptidase-C-released peptides on C8 reversed-phase HPLC demonstrated that one of these fragments, corresponding to residues 239-259 in MIP, was partially phosphorylated. The phosphorylated and nonphosphorylated forms of this peptide were separated on QAE HPLC. In vivo phosphorylation sites were found at residues 243 and 245 through phosphoserine modification via ethanethiol and sequence analysis. Phosphorylation was never detected on serine 240. The phosphorylation level of serine 243 could be increased by incubation of membranes with cAMP-dependent protein kinase under standard assay conditions. Other kinases that phosphorylate serines found near acidic amino acids must be responsible for the in vivo phosphorylation demonstrated at serine 245.     

Amino acid sequence of rat thymus histone H2B and identification of the in vitro phosphorylation sites.

The amino acid sequence of rat thymus histone obtained in highly purified form by preparative electrophoresis, was determined. This sequence is identical to the sequence of calf thymus histone H2B. The in vitro phosphorylation of the rat histone with a cyclic AMP-dependent protein kinase isolated from rat pancreas led to the identification of four sites of phosphorylation: two major ones, at serine residues 32 and 36, and two minor ones, specific of the rat protein kinase, at serine residues 87 and 91.     

Synergistic phosphorylation of rabbit muscle glycogen synthase by cyclic AMP-dependent protein kinase and casein kinase I. Implications for hormonal regulation of glycogen synthase

The phosphorylation of rabbit skeletal muscle glycogen synthase by casein kinase I is markedly enhanced if the enzyme has previously been phosphorylated by cAMP-dependent protein kinase. The presence of phosphate in the primary cAMP-dependent protein kinase sites, sites 1a, 1b, and 2 (serine 7), increases the activity of casein kinase I toward residues in the vicinity of these sites. This synergistic phosphorylation correlates with potent inactivation of the glycogen synthase. Analysis of the NH2 terminus of the enzyme subunit indicated that phosphorylation at serine 7 caused serine 10 to become a preferred casein kinase I site and that phosphoserine can be an important recognition determinant for casein kinase I. This finding can also explain how epinephrine stimulation of skeletal muscle provokes significant increases in the phosphorylation state of serine residues, in particular serine 10, not recognized by cAMP-dependent protein kinase.     

Identification of the phosphoserine residue in histone H1 phosphorylated by protein kinase C

The site-specific phosphorylation of bovine histone H1 by protein kinase C was investigated in order to further elucidate the substrate specificity of protein kinase C. Protein kinase C was found to phosphorylate histone H1 to 1 mol per mol. Using N-bromosuccinimide and thrombin digestions, the phosphorylation site was localized to the globular region of the protein, containing residues 71-122. A tryptic peptide containing the phosphorylation site was purified. Modification of the phosphoserine followed by amino acid sequence analysis demonstrated that protein kinase C phosphorylated histone H1 on serine 103. This sequence, Gly97-Thr-Gly-Ala-Ser-Gly-Ser(PO4)-Phe-Lys105, supports the contention that basic amino acid residues C-terminal to the phosphorylation site are sufficient determinants for phosphorylation by protein kinase C.