The phosphorylation site of Ca2+-dependent protein kinase from alfalfa

A 50 kDa, calcium-dependent protein kinase (CDPK) was purified about 1000-fold from cultured cells of alfalfa (Medicago varia) on the basis of its histone H1 phosphorylation activity. The major polypeptide from bovine histone H1 phosphorylated by either animal protein kinase C (PK-C) or by the alfalfa CDPK gave an identical phosphopeptide pattern. The phosphoamino acid determination showed phosphorylation of serine residues in histone H1 by the plant enzyme. Histone-related oligopeptides known to be substrates for animal histone kinases also served as substrates for the alfalfa kinase. Both of the studied peptides (GKKRKRSRKA; AAASFKAKK) inhibited phosphorylation of H1 histones by bovine and alfalfa kinases. The results of competition studies with the nonapeptide (AAASFKAKK), which is a PK-C specific substrate, suggest common features in target recognition between the plant Ca²⁺-dependent kinase and animal protein kinase C. We also propose that synthetic peptides like AAASFKAKK can be used as a tool to study substrates of plant kinases in crude cell extracts.     

Identification of mitotic (CDC2) and interphase histone H1 kinases by nondenaturing gel electrophoresis and peptide assays

A new method of nondenaturing gel electrophoresis and a specific peptide based assay were used to study the histone kinases in mitotic and interphase mouse fibroblasts. The gels resolved four activities, one of which was shown to be the mitotic (CDC2) H1 kinase by virtue of its antigenicity. A new peptide substrate for the CDC2 kinase was phosphorylated by both S-phase and mitotic cell extracts and reacted with two protein kinases in the gels. Since the interphase enzyme did not react with the antibody, the results suggest that it is either a "masked" form of CDC2 or a second enzyme, functionally related to CDC2, which is responsible for the interphase phosphorylation of H1.     

Comparison of the substrate specificity of adenosine 3':5'-monophosphate- and guanosine 3':5'-monophosphate-dependent protein kinases. Kinetic studies using synthetic peptides corresponding to phosphorylation sites in histone H2B.

The substrate specificities of cyclic GMP-dependent and cyclic AMP-dependent protein kinases have been compared by kinetic analysis using synthetic peptides as substrates. Both enzymes catalyzed the transfer of phosphate from ATP to calf thymus histone H2B, as well as to two synthetic peptides, Arg-Lys-Arg-Ser32-Arg-Lys-Glu and Arg-Lys-Glu-Ser36-Tyr-Ser-Val, corresponding to the amino acid sequences around serine 32 and serine 36 in histone H2B. Serine 38 in the latter peptide was not phosphorylated by either enzyme. Cyclic GMP-dependent kinase and cyclic AMP-dependent kinase catalyzed the incorporation of 1.1 and 2.0 mol of phosphate/mol of histone H2B, respectively. The phosphorylation of histone H2B, respectively. The phosphorylation of histone H2B by cyclic GMP-dependent kinase showed two distinct optima as the magnesium concentration was increased. However, the phosphorylation of either synthetic peptide by this enzyme was depressed at high magnesium concentrations. As the pH of reaction mixtures was elevated from pH 6 to pH 9, the rate of phosphorylation of Arg-Lys-Arg-Ser32-Arg-Lys-Glu by cyclic GMP-dependent kinase continually increased. Acetylation of the NH2 terminus of the peptide did not qualitatively affect this pH profile, but did increase the Vmax value of the enzyme 3-fold. The apparent Km and Vmax values for the phosphorylation of Arg-Lys-Arg-Ser32-Arg-Lys-Glu by cyclic GMP-dependent kinase were 21 microM and 4.4 mumol/min/mg, respectively. The synthetic peptide Arg-Lys-Glu-Ser36-Tyr-Ser-Val was a relatively poor substrate for cyclic GMP-dependent kinase, exhibiting a Km value of 732 microM, although the Vmax was 12 micromol/min/mg. With histone H2B as substrate for the cyclic GMP-dependent kinase, two different Km values were apparent. The Km values for cyclic AMP-dependent kinase for either synthetic peptide were approximately 100 microM, but the Vmax for Arg-Lys-Arg-Ser32-Arg-Lys-Glu was 1.1 mumol/min/mg, while the Vmax for Arg-Lys-Glu-Ser36-Tyr-Ser-Val was 16.5 mumol/min/mg. These data suggest that although the two cyclic nucleotide-dependent protein kinases have similar substrate specificities, the determinants dictated by the primary sequence around the two phosphorylation sites in histone H2B are different for the two enzymes.     

Mammalian growth-associated H1 histone kinase: a homolog of cdc2+/CDC28 protein kinases controlling mitotic entry in yeast and frog cells.

Mammalian growth-associated H1 histone kinase, an enzyme whose activity is sharply elevated at mitosis, is similar to cdc2+ protein kinase from Schizosaccharomyces pombe and CDC28 protein kinase from Saccharomyces cerevisiae with respect to immunoreactivity, molecular size, and specificity for phosphorylation sites in H1 histone. Phosphorylation of specific growth-associated sites in H1 histone is catalyzed by yeast cdc2+/CDC28 kinase, as shown by the in vitro thermal lability of this activity in extracts prepared from temperature-sensitive mutants. In addition, highly purified Xenopus maturation-promoting factor catalyzes phosphorylation of the same sites in H1 as do the mammalian and yeast kinases. The data indicate that growth-associated H1 kinase is encoded by a mammalian homolog of cdc2+/CDC28 protein kinase, which controls entry into mitosis in yeast and frog cells. Since H1 histone is known to be an in vivo substrate of the mammalian kinase, this suggests that phosphorylation of H1 histone or an H1 histone counterpart is an important component of the mechanism for entry of cells into mitosis.     

The assay of the activity of protein kinase C with the synthetic oligopeptide substrate designed for histone kinase II

The activity of histone kinase II was determined on the basis of its ability to phosphorylate the nonapeptide Ala-Ala-Ala-Ser-Phe-Lys-Ala-Lys-Lys-amide designed previously as a specific substrate for this enzyme. Histone kinase II was purified from calf thymus extract by DEAE-cellulose chromatography followed by hydroxylapatite chromatography and high-performance liquid chromatography on a Protein Analysis column (I-125). The Mr value of histone kinase II estimated by the latter method was 50,000-55,000, but several observations indicated that histone kinase II was a product of a proteolytic process. Since the substrate specificity determinants for histone kinase II known from our previous investigations are very similar to those for protein kinase C, it was presumable that histone kinase II was the proteolytic fragment of protein kinase C. Therefore, the nonapeptide was tested as a substrate for protein kinase C prepared from rabbit brain extract by DEAE-cellulose chromatography. The activity of histone kinase II was also detected in brain extract. Histone kinase II was eluted from the DEAE-cellulose in the known position of the proteolytic fragment of protein kinase C. The nonapeptide Ala-Ala-Ala-Ser-Phe-Lys-Ala-Lys-Lys-amide proved to be a better substrate than H1 histone for the detection of the activity of protein kinase C because it was not phosphorylated by the cAMP-dependent protein kinase and the Vmax of protein kinase C was about one order of magnitude higher with the peptide than with H1 histone. The apparent Km of protein kinase C for the peptide was identical with that of histone kinase II (0.2 mM).     

A synthetic peptide substrate for selective assay of protein kinase C.

Among various phosphate acceptor proteins and peptides so far tested, a synthetic peptide having the sequence surrounding Ser(8) of myelin basic protein, Gln-Lys-Arg-Pro-Ser(8)-Gln-Arg-Ser-Lys-Tyr-Leu, (MBP4-14), is the most specific and convenient substrate which can be used for selective assay of protein kinase C. This peptide is not phosphorylated by cyclic AMP-dependent protein kinase, casein kinases I and II, Ca2+/calmodulin-dependent protein kinase II, or phosphorylase kinase, and can be routinely used for the assay of protein kinase C with low background in the crude tissue extracts. The Km value is considerably low (7 microM) with a Vmax value of twice as much as that for H1 histone.     

Purification of a protein histidine kinase from the yeast Saccharomyces cerevisiae. The first member of this class of protein kinases

An enzyme of molecular weight 32,000 comprising a single subunit has been isolated from whole cell extracts of the yeast Saccharomyces cerevisiae. In vitro, the enzyme transfers the gamma phosphate of ATP to a protein substrate, histone H4, to produce an alkali-stable phosphorylation. Modification of the substrate histidine with diethylpyrocarbonate prevented phosphorylation. Phosphoamino acid analysis of the phosphorylated substrate showed the presence of 1-phosphohistidine. Hence, the isolated enzyme is a protein histidine kinase. A novel assay for acid-labile alkali-stable protein phosphorylation was used in the purification of the kinase activity to a final specific activity of 2,700 nmol/15 min/mg. The purified enzyme phosphorylates specifically histidine 75 in histone H4 and does not phosphorylate histidine 18 nor histidine residues in any other core histone. Steady state kinetic data are consistent with an ordered sequential reaction with Km values for Mg-ATP and histone H4 of 60 and 17 microM, respectively. The protein histidine kinase requires a divalent cation such as Mg2+, Co2+, or Mn2+ but will not use Ca2+, Zn2+, Cu2+, Fe2+, spermine, or spermidine. This is the first purification of an enzyme that catalyzes N-linked phosphorylation in proteins.     

Synthetic oligopeptide substrates which fail to compete with H1 histone for type II and type III isoenzymes of protein kinase C.

1. The oligopeptide AAASFKAKK which contains recognition motifs similar to that found in the surrounding of the site of H1 histone phosphorylated by protein kinase C is unable to compete with H1 histone for the type II and type III isoenzymes, though it is a good substrate for protein kinase C and it is able to compete with a physiological substrate of the enzyme. 2. Among several oligopeptides tested as an alternative substrate a very basic peptide proved to be the most effective inhibitor of H1 histone phosphorylation. This oligopeptide substrate contains basic recognition motifs at both sides of the phosphorylated residue at variance with the sequence of H1 histone in the surrounding of the phosphorylated site.     

Phosphorylation of calf thymus H1 histone by muscle glycogen phosphorylase kinase

Muscle glycogen phosphorylase kinase [EC 2.7.1.38] has the ability to phosphorylate five fractions of calf thymus histone. H1 histone is the most preferable substrate, and maximally about 1.3 mol of phosphate is incorporated into every mole of this histone. This reaction absolutely depends on CA2+, and the molecular activity is about one third of that of cyclic AMP-dependent protein kinase (protein kinase A). The affinity of phosphorylase kinase for H1 histone is higher than that of protein kinase A. Calmodulin stimulates this histone phosphorylation. Analysis of the N-bromosuccinimide-bisected fragments of fully phosphorylated H1 histone has revealed that the enzyme phosphorylates mostly seryl residues in both amino- and carboxyl-terminal portions, although phosphorylation of the carboxyl-terminal portion is twice as much as that of the amino-terminal portion. Fingerprint analysis indicates that the phosphorylation sites in H1 histone for this enzyme are different from the sites phosphorylated by protein kinase A. This catalytic activity also differs from that of a newly found multifunctional protein kinase which may be activated by the simultaneous presence of Ca2+ and phospholipid.     

Characterization of maturation-activated histone H1 and ribosomal S6 kinases in sea star oocytes

DEAE-Sephacel chromatography of cytosolic extracts from sea star oocytes resolved at least two distinct peaks of maturation-activated protein kinase activity, each of which catalyzed the phosphorylation of histone H1, ribosomal protein S6, and Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala (RRLSSLRA), a synthetic peptide based on the sequence of a phosphorylation site in the latter protein. The first peak (elution conductivity approximately equal to 6 mmho) contained the major activated kinase with respect to the phosphorylation of histone H1, and the second peak (elution conductivity approximately equal to 10.5 mmho) contained the major activated kinase with respect to the phosphorylation of S6 and RRLSSLRA. These kinase activities were barely detectable in extracts from immature oocytes. The major stimulated histone H1 kinase exhibited an apparent Mr of approximately 90 000 on Sephacryl S-300 but eluted from TSK-400 with an apparent Mr of approximately 10 000. After DEAE-Sephacel fractionation, this kinase was shown to utilize both ATP (apparent Km approximately equal to 45 microM) and GTP (apparent Km approximately equal to 10 microM), although the Vmax was 8-fold higher with ATP than with GTP. The enzyme phosphorylated histone H1 with an apparent Km approximately equal to 50 micrograms/mL. Its properties resembled those of the growth-associated histone kinase. The major stimulated RRLSSLRA kinase had an apparent Mr of approximately 84 000 on Sephacryl S-300 and approximately 40 000 on TSK-400. After DEAE-Sephacel chromatography, this kinase selectively utilized ATP (apparent Km approximately equal to 25 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

A phenylalanine in peptide substrates provides for selectivity between cGMP- and cAMP-dependent protein kinases.

Bovine lung cGMP-binding cGMP-specific phosphodiesterase (cG-BPDE) is a potent and relatively specific substrate for cGMP-dependent protein kinase (cGK) as compared to cAMP-dependent protein kinase (cAK) (Thomas, M. K., Francis, S. H., and Corbin, J. D. (1990) J. Biol. Chem. 265, 14971-14978). A synthetic peptide, RKISASEFDRPLR (BPDEtide), was synthesized corresponding to the sequence surrounding the phosphorylation site in cG-BPDE. BPDEtide retained the cGK/cAK kinase specificity demonstrated by native cG-BPDE: the apparent Km of BPDEtide for cGK was 5-fold lower than that for cAK (Km = 68 and 320 microM, respectively). Vmax values were 11 mumol/min/mg for cGK and 3.2 mumol/min/mg for cAK. The peptide was not phosphorylated to a measurable extent by protein kinase C or by calcium/calmodulin-dependent protein kinase II. Thus, the primary amino acid sequence of the peptide substrate was sufficient to confer kinase specificity. Studies in crude tissue extracts indicated that BPDEtide was the most selective peptide substrate documented for measuring cGK activity. Peptide analogs of BPDEtide were synthesized to determine the contribution of specific residues to cGK or cAK substrate specificity. Substitution of a Lys for the amino-terminal Arg did not reduce cGK/cAK specificity; neither did the exchange of an Ala for the non-phosphorylated Ser nor the removal of the 3 carboxyl-terminal residues. A truncated BPDEtide (RKISASE) served equally well as substrate (Km approximately 90 microM) for both kinases. However, restoration of the Phe, to yield RKISASEF, reproduced the original cGK/cAK specificity for BPDEtide (Km = 120 and 480 microM, respectively), primarily by decreasing the affinity of cAK. Addition of a carboxyl-terminal Phe to the peptide RKRSRAE (derived from the sequence of the cGK phosphorylation site in histone H2B) or to the peptide LRRASLG (derived from the sequence of the cAK phosphorylation site in pyruvate kinase) also improved the cGK/cAK specificity by decreasing the affinity of cAK. These data suggested that the Phe in each substrate tested is a negative determinant for cAK.     

A more sensitive assay for histone deacetylase

The new assay uses as substrate a peptide derived from the amino terminal domain of calf histone H4. The peptide contains all the lysines that are acetylated in H4 in vivo and these lysines are specifically labeled in vitro with acetic anhydride to a high specific activity. This substrate allows histone deacetylase activity to be measured economically and with high sensitivity either with pure enzyme or with crude extracts.     

Studies of histidine phosphorylation by a nuclear protein histidine kinase show that histidine-75 in histone H4 is masked in nucleosome core particles and in chromatin

Histone H4 is a good substrate in vitro for the protein histidine kinase activity found both in Physarum polycephalum nuclear extracts and in Saccharomyces cerevisiae cell extracts. However, histone H4 in nucleosome core particles is not a substrate for these kinases. Isolated chromatin was also not a substrate for the protein histidine kinase. The results significantly limit possible interpretations of histidine phosphorylation on histone H4 in vivo and provide a new, sharper focus for future work. In addition, a polynucleotide kinase activity was identified in the Physarum extracts.     

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.     

Purification and characterization of a novel proline-directed protein kinase from bovine brain.

A novel protein kinase which phosphorylates a synthetic peptide substrate (RRPDAHRTPNRAF) has been purified approximately 200,000-fold from bovine brain. This peptide contains the consensus sequence for phosphorylation by the p34cdc2 kinase. The purification procedure took advantage of the phenomenon that this novel brain kinase, in partially purified extracts, chromatographed on a gel filtration column as a high molecular weight complex which dissociated in buffer containing 1 M NaCl. The purified native enzyme was estimated to be approximately 63,000, and displayed two bands of M(r) = 33,000 and 25,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On Western immunoblot, the M(r) = 33,000 peptide reacted strongly with antibodies specific for a conserved amino-terminal sequence, weakly with antibodies to the conserved PSTAIRE sequence, and not at all with antibodies to the carboxyl terminus, of HeLa cell p34cdc2. The brain kinase and p34cdc2 were similar in displaying good activity toward the parent peptide substrate, but no activity toward peptide analogues in which the -T-P- motif was substituted with either -T-G- or -T-A-. Both kinases showed marked preference in phosphorylating a peptide derived from H1 histone (KTPKKAKKPKTPKKAKKL), and both kinases could be phosphorylated by the src-family tyrosine kinase, p56lyn, purified from bovine spleen. However, the brain kinase did not co-purify with a subunit having a molecular weight corresponding to known cyclins, nor did it undergo specific interaction with p13suc1 beads, suggesting that this enzyme is distinct from p34cdc2.     

pEg2 aurora-A kinase, histone H3 phosphorylation, and chromosome assembly in Xenopus egg extract

In eukaryotes cell division is accompanied by phosphorylation of histone H3 at serine 10. In this work we have studied the kinase activity responsible for this histone H3 modification by using cell-free extracts prepared from Xenopus eggs. We have found that the Xenopus aurora-A kinase pEg2, immunoprecipitated from the extract, is able to phosphorylate specifically histone H3 at serine 10. The enzyme is incorporated into chromatin during in vitro chromosome assembly, and the kinetics of this incorporation parallels that of histone H3 phosphorylation. Recombinant pEg2 phosphorylates efficiently histone H3 at serine 10 in reconstituted nucleosomes and in sperm nuclei decondensed in heated extracts. These data identify pEg2 as a good candidate for mitotic histone H3 kinase. However, immunodepletion of pEg2 does not interfere with the chromosome assembly properties of the extract nor with the pattern of H3 phosphorylation, suggesting the existence of multiple kinases involved in this H3 modification in Xenopus eggs. This hypothesis is supported by in gel activity assay experiments using extracts from Saccharomyces cerevisiae.     

An H3 histone-specific kinase isolated from bovine thymus chromatin.

A substantial portion of the histone phosphorylating activity of bovine thymus chromatin can be isolated by extraction in 0.2 M NaCl. The specificity of this extract for either free histones or washed chromatin substrates was compared. The salt-extracted kinase enzymes favor H2b as the major acceptor when whole free histone is the substrate and H3 when the substrate is intact chromatin. The H3 kinase activity of bovine thymus tissue has been purified free from other detectable histone kinase activities by ammonium sulfate fractionation and is highly specific for H3 histone when assayed either with chromatin or isolated whole histone. The activity is cAMP-independent. Tryptic peptide mapping of the labeled H3 histone reveals a single site of phosphorylation. This site appears to be identical with the major site of metaphase-associated H3 phosphorylation in hepatoma tissue culture cells. No corresponding H3 phosphorylation has been detected in thymus tissue in vivo.     

Protein kinase C phosphorylates the synthetic peptide Arg-Arg-Lys-Ala-Ser-Gly-Pro-Pro-Val in the presence of phospholipid plus either Ca2+ or a phorbol ester tumor promoter

The synthetic nonapeptide Arg-Arg-Lys-Ala-Ser-Gly-Pro-Pro-Val is a substrate for in vitro phosphorylation by a partially purified preparation of rat brain protein kinase C, with Kmapp of about 130 microM. The closely related peptide kemptide was a much weaker substrate, bovine serum albumin was not a substrate and the peptide Arg-Arg-Lys-Ala-Ala-Gly-Pro-Pro-Val was a weak inhibitor of the enzyme. Protein kinase C-catalyzed phosphorylation of histone III-S and the nonapeptide are regulated by identical mechanisms since with both substrates the reaction required added phospholipid and either Ca2+ (1mM) or TPA (200 nM TPA). Our findings show that polypeptides containing multiple basic residues followed by the sequence Ala-Ser can be substrates for TPA-stimulated phosphorylation by protein kinase C.     

Two methods to avoid the effect of endogenous protein inhibitors during the assay of protein kinase C activity in tissue extracts.

Using H1 as substrate the protein kinase C activity of rat liver cell sap was increased about fourfold by treatment with DEAE-cellulose at pH 7.5 at an intermediate ionic strength due to removal of protein inhibitors. The activity of cell sap from rat spleen, brain or muscle was about doubled by the same treatment. In contrast, when a specific synthetic peptide substrate was used the corresponding increase of enzyme activity was not obtained when the inhibitors were removed. This shows that this type of substrates should be preferred for reliable assays of protein kinase C in crude extracts. The possible role of the protein inhibitors for the substrate specificity of protein kinase C is briefly discussed.     

Activation of multiple protein kinases during the burst in protein phosphorylation that precedes the first meiotic cell division in Xenopus oocytes

A number of different protein and peptide substrates were used to identify and characterize stimulated kinase activities in Xenopus oocyte extracts prepared during the major burst in protein phosphorylation that precedes meiotic cell division. While total cAMP-dependent protein kinase activity in the cytosol was not stimulated, this kinase was the major kinase phosphorylating a number of the substrates and consequently had to be inhibited to prevent its masking cAMP-independent protein kinase activities. Sizable stimulations of kinase activities were then observed in extracts from progesterone-treated oocytes as compared to controls when the following substrates were utilized: Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) (8-fold); the synthetic peptide, Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala, the sequence of which is based on that of a phosphorylation site in ribosomal protein S6 (8-fold); ribosomal protein S6 (8-fold); histone H1 (5-fold); skeletal muscle glycogen synthase (3-fold); and myelin basic protein (30-fold). When these substrates were used to assay extracts fractionated on DEAE-Sephacel, at least three distinct peaks of stimulated kinase activity were detected, eluting at 0.12, 0.17, and 0.21 M NaCl. These peaks were tentatively designated M-phase Activated Kinases(s), MAK-H, MAK-S, and MAK-M, respectively. Using histone H1 as a selective probe for MAK-H and S6 peptide or Kemptide as probes for MAK-S, the kinase activities comprising these peaks were found to cycle with the meiotic cell cycle.