The lysine-rich H1 histones from the slime moulds, Physarum polycephalum and Dictyostelium discoideum lack phosphorylation sites recognised by cyclic AMP-dependent protein kinase in vitro.

Calcium chloride-extracted histones were prepared from nuclei of the slime moulds, Physarum polycephalum and Dictyostelium discoideum, and phosphorylation by purified preparations of cyclic AMP-dependent protein kinase (cAMP-d PK) and growth-associated H1 histone kinase (HKG) examined and compared. Among the major histone fractions and other proteins in the two preparations, the H1 histones from both organisms were found to be effective and exclusive substrates for HKG. cAMP-d PK, which phosphorylates mammalian H1 histone and certain, in particular H2B, of the mammalian core histones, phosphorylated several of the core histones from both slime moulds but did not phosphorylate H1 histone from either. The slime mould H1s remained ineffective substrates for cAMP-d PK even after extensive alkaline phosphatase treatment of the histone preparations. Additional studies demonstrated that the lack of slime mould H1 phosphorylation by cAMP-d PK was not due to competition of the H1 molecules with the core histones for the kinase. Our studies suggest that H1 histones from these organisms, whilst clearly containing sites for phosphorylation by HKG, apparently lack phosphorylation sites recognised by cAMP-d PK. Thus, the mediation of specific nuclear functions by cAMP-dependent phosphorylation of H1 in higher organisms may not occur or be required in these lower eukaryotes.     

Protein phosphorylation in normal and neoplastic hepatocytes]

Partially purified preparations of protein kinase were isolated from the cytoplasm and nuclei of rat liver and hepatoma 27 and characterized in terms of their substrate specificity. The protein kinases from normal liver and hepatoma revealed some differences in phosphorylating protein substrates. Hepatoma protein kinases were found to phosphorylate arginine-rich histones (H3, H4); differences in phosphorylation of histone H1 were revealed. Hepatoma protein kinases phosphorylated the C-terminal fragment of histone H1, whereas normal liver protein kinases produced no such effect. It was assumed that the phosphorylation of histone H1 in rapidly dividing and resting cells is operated through different channels.     

Phosphorylation of Histone H2A.X by DNA-dependent Protein Kinase Is Not Affected by Core Histone Acetylation,but It Alters Nucleosome Stability and Histone H1 Binding

Phosphorylation of the C-terminal end of histone H2A.X is the most characterized histone post-translational modification in DNA double-stranded breaks (DSB). DNA-dependent protein kinase (DNA-PK) is one of the three phosphatidylinositol 3 kinase-like family of kinase members that is known to phosphorylate histone H2A.X during DNA DSB repair. There is a growing body of evidence supporting a role for histone acetylation in DNA DSB repair, but the mechanism or the causative relation remains largely unknown. Using bacterially expressed recombinant mutants and stably and transiently transfected cell lines, we find that DNA-PK can phosphorylate Thr-136 in addition to Ser-139 both in vitro and in vivo. Furthermore, the phosphorylation reaction is not inhibited by the presence of H1, which in itself is a substrate of the reaction. We also show that, in contrast to previous reports, the ability of the enzyme to phosphorylate these residues is not affected by the extent of acetylation of the core histones. In vitro assembled nucleosomes and HeLa S3 native oligonucleosomes consisting of non-acetylated and acetylated histones are equally phosphorylated by DNA-PK. We demonstrate that the apparent differences in the extent of phosphorylation previously observed can be accounted for by the differential chromatin solubility under the MgCl₂ concentrations required for the phosphorylation reaction in vitro. Finally, we show that although H2A.X does not affect nucleosome conformation, it has a de-stabilizing effect that is enhanced by the DNA-PK-mediated phosphorylation and results in an impaired histone H1 binding.     

VRK1 chromatin kinase phosphorylates H2AX and is required for foci formation induced by DNA damage

All types of DNA damage cause a local alteration and relaxation of chromatin structure. Sensing and reacting to this initial chromatin alteration is a necessary trigger for any type of DNA damage response (DDR). In this context, chromatin kinases are likely candidates to participate in detection and reaction to a locally altered chromatin as a consequence of DNA damage and, thus, initiate the appropriate cellular response. In this work, we demonstrate that VRK1 is a nucleosomal chromatin kinase and that its depletion causes loss of histones H3 and H4 acetylation, which are required for chromatin relaxation, both in basal conditions and after DNA damage, independently of ATM. Moreover, VRK1 directly and stably interacts with histones H2AX and H3 in basal conditions. In response to DNA damage induced by ionizing radiation, histone H2AX is phosphorylated in Ser139 by VRK1. The phosphorylation of H2AX and the formation of γH2AX foci induced by ionizing radiation (IR), are prevented by VRK1 depletion and are rescued by kinase-active, but not kinase-dead, VRK1. In conclusion, we found that VRK1 is a novel chromatin component that reacts to its alterations and participates very early in DDR, functioning by itself or in cooperation with ATM.     

Muscle phosphorylase kinase is not a substrate of AMP-activated protein kinase

AMP-activated protein kinase (AMPK) and cAMP-dependent protein kinase (cAMPK) have been reported to phosphorylate sites on phosphorylase kinase (PhK). Their target residues Ser 1018 and Ser 1020, respectively, are located in the so-called multi-phosphorylation domain in the PhK alpha subunit. In PhK preparations, only one of these serines is phosphorylated, but never both of them. The aim of this study was to determine whether phosphorylation by cAMPK or AMPK would influence subsequent phosphorylation by the other kinase. Surprisingly, employing four different PhK substrates, it could be demonstrated that, in contradiction to previous reports, PhK is not phosphorylated by AMPK.     

Mitotic histone H3 phosphorylation by vaccinia-related kinase 1 in mammalian cells

Mitotic chromatin condensation is essential for cell division in eukaryotes. Posttranslational modification of the N-terminal tail of histone proteins, particularly by phosphorylation by mitotic histone kinases, may facilitate this process. In mammals, aurora B is believed to be the mitotic histone H3 Ser10 kinase; however, it is not sufficient to phosphorylate H3 Ser10 with aurora B alone. We show that histone H3 is phosphorylated by vaccinia-related kinase 1 (VRK1). Direct phosphorylation of Thr3 and Ser10 in H3 by VRK1 both in vitro and in vivo was observed. Loss of VRK1 activity was associated with a marked decrease in H3 phosphorylation during mitosis. Phosphorylation of Ser10 by VRK1 is similar to that by aurora B. Moreover, expression and chromatin localization of VRK1 depended on the cell cycle phase. Overexpression of VRK1 resulted in a dramatic condensation of nuclei. Our findings collectively support a role of VRK1 as a novel mitotic histone H3 kinase in mammals.     

Hyperoxia activates the ATR-Chk1 pathway and phosphorylates p53 at multiple sites

Hyperoxia has been shown to cause DNA damage resulting in growth arrest of cells in p53-dependent, as well as p53-independent, pathways. Although H2O2 and other peroxides have been shown to induce ataxia telangiectasia-mutated (ATM)-dependent p53 phosphorylation in response to DNA damage, the signal transduction mechanisms in response to hyperoxia are currently unknown. Here we demonstrate that hyperoxia phosphorylates the Ser15 residue of p53 independently of ATM. Hyperoxia phosphorylated p53 (Ser15) in DNA-dependent protein kinase null (DNA-PK-/-) cells, indicating that it may not depend on DNA-PK for phosphorylation of p53 (Ser15). We show that Ser37 and Ser392 residues of p53 are also phosphorylated in an ATM-independent manner in hyperoxia. In contrast, H2O2 did not phosphorylate Ser37 in either ATM+/+ or ATM-/- cells. Furthermore, H2O2 failed to phosphorylate Ser15 in ATM-/- cells. Additionally, overexpression of kinase-inactive ATM-and-Rad3-related (ATR) in HEK293T cells diminished Ser15, Ser37, and Ser392 phosphorylation compared with vector-only transfected cells. In contrast, wild-type ATR overexpression did not diminish Ser15, Ser37, or Ser392 phosphorylation. We also show that checkpoint kinase 1 (Chk1) is phosphorylated on Ser345 in response to hyperoxia, which could be inhibited by caffeine or wortmannin, potent inhibitors of phosphoinositide 3-kinase-related kinases. Hyperoxia also phosphorylated Chk1 in ATM+/+ as well as in ATM-/- cells, demonstrating an ATM-independent mechanism in Chk1 phosphorylation. Together, our data suggest that hyperoxia activates the ATR-Chk1 pathway and phosphorylates p53 at multiple sites in an ATM-independent manner, which is different from other forms of oxidative stress such as H2O2 or UV light.     

Characterization of dual specificity protein kinase from maize seedlings

A protein kinase of 57 kDa, able to phosphorylate tyrosine in synthetic substrates pol(Glu4,Tyr1) and a fragment of Src tyrosine kinase, was isolated and partly purified from maize seedlings (Zea mays). The protein kinase was able to phosphorylate exogenous proteins: enolase, caseins, histones and myelin basic protein. Amino acid analysis of phosphorylated casein and enolase, as well as of phosphorylated endogenous proteins, showed that both Tyr and Ser residues were phosphorylated. Phosphotyrosine was also immunodetected in the 57 kDa protein fraction. In the protein fraction there are present 57 kDa protein kinase and enolase. This co-purification suggests that enolase can be an endogenous substrate of the kinase. The two proteins could be resolved by two-dimensional electrophoresis. Specific inhibitors of typical protein-tyrosine kinases had essentially no effect on the activity of the maize enzyme. Staurosporine, a nonspecific inhibitor of protein kinases, effectively inhibited the 57 kDa protein kinase. Also, poly L-lysine and heparin inhibited tyrosine phosphorylation by 57 kDa maize protein kinase. The substrate and inhibitor specificities of the 57 kDa maize protein kinase phosphorylating tyrosine indicate that it is a novel plant dual-specificity protein kinase.     

Substitution of the autophosphorylation site Thr516 with a negatively charged residue confers constitutive activity to mouse 3-phosphoinositide-dependent protein kinase-1 in cells

3-Phosphoinositide-dependent protein kinase-1 (PDK-1)is a serine/threonine kinase that has been found to phosphorylate and activate several members of the AGC protein kinase family including protein kinase B (Akt), p70 S6 kinase, and protein kinase Czeta. However, the mechanism(s) by which PDK-1 is regulated remains unclear. Here we show that mouse PDK-1 (mPDK-1) undergoes autophosphorylation in vitro on both serine and threonine residues. In addition, we have identified Ser(399) and Thr(516) as the major mPDK-1 autophosphorylation sites in vitro. Furthermore, we have found that these two residues, as well as Ser(244) in the activation loop, are phosphorylated in cells and demonstrated that Ser(244) is a major in vivo phosphorylation site. Abolishment of phosphorylation at Ser(244), but not at Ser(399) or Thr(516), led to a significant decrease of mPDK-1 autophosphorylation and kinase activity in vitro, indicating that autophosphorylation at Ser(399) or Thr(516) is not essential for mPDK-1 autokinase activity. However, overexpression of mPDK-1(T516E), but not of mPDK-1(S244E) or mPDK-1(S399D), in Chinese hamster ovary and HEK293 cells was sufficient to induce Akt phosphorylation at Thr(308) to a level similar to that of insulin stimulation. Furthermore, this increase in phosphorylation was independent of the Pleckstrin homology domain of Akt. Taken together, our results suggest that mPDK-1 undergoes autophosphorylation at multiple sites and that this phosphorylation may be essential for PDK-1 to interact with and phosphorylate its downstream substrates in vivo.     

Partial displacement of histone H1 from chromatin is required before it can be phosphorylated by mitotic H1 kinase in vitro.

The massive nonselective and reversible phosphorylation of histone H1 during mitosis is a universal phenomenon among eukaryotes. The growth-associated kinase responsible for this phosphorylation is identical to the maturation promoting factor, a key regulator of the cell cycle. Here we showed that growth-associated kinase, isolated from mitotic HeLa cells which were capable of phosphorylating HeLa H1 in vitro with high activity and mostly at the same sites phosphorylated during mitosis in vivo (assayed by two-dimensional analysis of tryptic phosphopeptides), did not significantly phosphorylate chromatin-bound or nuclear H1 in vitro. Its inability to phosphorylate chromatin-bound H1 did not change when the amount of kinase was increased or the incubation was prolonged. The resistance of chromatin-bound H1 to phosphorylation did not result from chromatin aggregation. Rapid phosphorylation of H1 in vitro, as well as in a nuclear system, was restored when NaCl concentrations were raised above 200 mM where H1:DNA interactions are weakened. At 300 mM NaCl, chromatin-bound H1 was phosphorylated in a subset of the sites observed for free H1 phosphorylated in vitro. These results suggest that active displacement of H1 from chromatin DNA may take place before H1 can be fully phosphorylated during mitosis.     

MAP kinases mediate UVB-induced phosphorylation of histone H3 at serine 28

Histone H3 phosphorylation is related closely to chromatin remodeling and chromosome condensation. H3 phosphorylation at serine 28 is coupled with mitotic chromosome condensation in diverse mammalian cell lines. However, the pathway that mediates phosphorylation of H3 at serine 28 is unknown. In the present study, ERK1, ERK2, or p38 kinase strongly phosphorylated H3 at serine 28 in vitro. JNK1 or JNK2 was able also to phosphorylate H3 at serine 28 in vitro but to a lesser degree. UVB irradiation markedly induced phosphorylation of H3 at serine 28 in JB6 Cl 41 cells. PD 98059, a MEK1 inhibitor, and SB 202190, a p38 kinase inhibitor, efficiently repressed UVB-induced H3 phosphorylation at serine 28. Expression of dominant negative mutant (DNM) ERK2 in JB6 Cl 41 cells totally blocked UVB-induced phosphorylation of H3 at serine 28. Additionally, DNM p38 kinase or DNM JNK1 partially blocked UVB-induced H3 phosphorylation at serine 28. Furthermore, UVB-induced H3 phosphorylation at serine 28 was inhibited in Jnk1(-/-) cells but not in Jnk2(-/-) cells. These results suggest that UVB-induced H3 phosphorylation at serine 28 may be mediated by mitogen-activated protein kinases.     

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.     

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.     

Cell cycle-dependent chromatin shuttling of HBO1–JADE1 histone acetyl transferase (HAT) complex

HAT HBO1 interacts with 2 isoforms of JADE1: JADE1S and JADE1L. JADE1 promotes acetylation of nucleosomal histones by HBO1. HBO1–JADE1 complex facilitates cell proliferation by unclear mechanisms. Here we report intracellular chromatin shuttling of HBO1–JADE1 complex during mitosis coupled to phosphorylation of JADE1. In interphase of dividing cells JADE1S was localized to the nucleus and associated with chromatin. As cells approached mitosis, specifically prophase, JADE1S dissociated from chromatin and associated with cytoplasm. JADE1S chromatin re-association began in telophase and paralleled nuclear envelope membrane reassembly. By early G₁, JADE1S was re-associated with chromatin and localized to the nucleus. Importantly, cytoplasmic but not chromatin-associated JADE1 protein was phosphorylated. Mass-Spectrometric analysis of JADE1S protein isolated from G₂/M-arrested cells identified 6 phosphorylated amino acid residues: S89, T92, S102, S121, S392, and T468, including 3 novel sites. Temporally, JADE1S phosphorylation and dephosphorylation during mitosis correlated with JADE1S chromatin dissociation and recruitment. JADE1S chromatin recruitment was accompanied by the global histone H4 acetylation. Pharmacological inhibitor of Aurora A kinase prevented JADE1S protein band shift and chromatin dissociation, suggesting regulatory function for phosphorylation. In vivo experiments supported our in vitro results. In mouse kidneys, JADE1S transiently accumulated in the cytoplasm of tubular epithelial cells during kidney regeneration. The transient increase in the number of cells with cytoplasmic JADE1S directly correlated with activation of tubular cell proliferation and inversely correlated with the number of cells with nuclear JADE1S staining, supporting biological role of HBO1–JADE1 shuttling during organ regeneration.     

Studies on the sites in histones phosphorylated by adenosine 3':5'-monophosphate-dependent and guanosine 3':5'-monophosphate-dependent protein kinases.

Adenosine 3':5'-monophosphate-dependent protein kinase (protein kinase A) purified from silkworm pupae phosphorylated five major fractions of calf thymus histone, whereas guanosine 3':5'-monophosphate-dependent protein kinase (protein kinase G) purified from the same organism reacted preferentially with H1, H2A, and H2B histones. Amino acid analysis of the phosphopeptides which were obtained by proteolytic digestion revealed that both protein kinases A and G showed the abilities to phosphorylate the same serine hydroxyl groups in H1 and H2B histones. Both protein kinases reacted with Ser-38 in H1 histone. With H2B histone as substrate protein kinase A phosphorylated Ser-32 as well as Ser-36, whereas protein kinase G reacted preferentially with Ser-32 and the reaction with Ser-36 was very slow. H3 and H4 histones were practically inactive substrates for protein kinase G. Although H2A histone has not been analyzed, the evidence has raised a possibility that protein kinase G utilizes a portion of the substrate proteins for protein kinase A.