Microsomal cAMP-independent histone H1 kinase activity in plasmacytoma, Morris hepatoma and normal liver

A protein kinase activity with high specificity for histone H1 was isolated from mouse plasmacytoma, Morris hepatoma and normal mouse liver and compared by ion exchange chromatography after DEAE-cellulose, hydroxylapatite and Sephadex G-200 chromatography. This cAMP-independent histone H1 kinase is not affected by the heat-stable cAMP-dependent protein kinase inhibitor. It has the following particular properties: it prefers GTP to ATP as substrate and was found to be present with a great activity only in neoplastic tissues. No phosphatase activity was detected in the partially purified histone H1 kinase fraction from normal and neoplastic cells. These results suggest either an increase amount of histone H1 kinase and/or of its activator in neoplastic cells, or the presence of a strong inhibitor in normal cells. This histone H1 kinase appears to be analogous to the chromatin bound kinase which phosphorylates histone H1 at the NH2 and COOH terminal regions. We might suggest an implication of this kinase in the regulation of cell division.     

Chemical phosphorylation of histidine-containing peptides based on the sequence of histone H4 and their dephosphorylation by protein histidine phosphatase

Using peptides based on the amino acid sequences surrounding the two histidine residues in histone H4, we have investigated the kinetics of the phosphorylation and dephosphorylation reactions of their histidine residues, when reacted with potassium phosphoramidate, by ¹H NMR. We have been able to estimate rate constants for the reactions and have shown that there are differences in the kinetics between the two peptides. The kinetics of hydrolysis of phosphoramidate was measured by ³¹P NMR and protein histidine phosphatase (PHP) was shown to catalyse the reaction. We have shown that the dephosphorylation of the phosphohistidine of the phosphopeptides is catalysed by PHP. In terms of substrate specificity, there is a small preference for 1-phosphohistidine compared to 3-phosphohistidine, although the rate accelerations for hydrolysis induced by the enzyme were 1100- and 33,333-fold, respectively. The kinetics of both the phosphorylation and dephosphorylation reactions depend on the amino acid sequence surrounding the histidine. PHP shows greater substrate specificity for the peptide whose sequence is similar to that around histidine 18 of histone H4. PHP was unable to catalyse the dephosphorylation of histone H4 that had been phosphorylated with a histone H4 histidine kinase.     

Photobleaching of GFP-labeled H2AX in chromatin: H2AX has low diffusional mobility in the nucleus

The Ser-139 phosphorylated form of replacement histone H2AX (gamma-H2AX) is induced within large chromatin domains by double-strand DNA breaks (DSBs) in mammalian chromosomes. This modification is known to be important for the maintenance of chromosome stability. However, the mechanism of gamma-H2AX formation at DSBs and its subsequent elimination during DSB repair remains unknown. gamma-H2AX formation and elimination could occur by direct phosphorylation and dephosphorylation of H2AX in situ in the chromatin. Alternatively, H2AX molecules could be phosphorylated freely in the nucleus, diffuse into chromatin regions containing DSBs and then diffuse out after DNA repair. In this study we show that free histone H2AX can be efficiently phosphorylated in vitro by nuclear extracts and that free gamma-H2AX can be dephosphorylated in vitro by the mammalian protein phosphatase 1-alpha. We made N-terminal fusion constructs of H2AX with green fluorescent protein (GFP) and studied their diffusional mobility in transient and stable cell transfections. In the absence or presence of DSBs, only a small fraction of GFP-H2AX is redistributed after photobleaching, indicating that in vivo this histone is essentially immobile in chromatin. This suggests that gamma-H2AX formation in chromatin is unlikely to occur by diffusion of free histone and gamma-H2AX dephosphorylation may involve the mammalian protein phosphatase 1alpha.     

Ser-10 phosphorylated histone H3 is involved in cytokinesis as a chromosomal passenger

Ser-10 phosphorylation of histone H3 is revealed to be relative to chromosome condensation at prophase during mitosis. In this report, we demonstrate using immunofluorescence microscopy that the subcellular distribution of the Ser-10 phosphorylated histone H3 was similar to that characteristic of chromosomal passenger proteins during the terminal stages of cytokinesis. Co-immunoprecipitation indicates that the Ser-10 phosphorylated histone H3 is associated with the aurora B, and both of the proteins were compacted into a complex with special ternary structure located in the centre of the midbody. When the level of the Ser-10 phosphorylated histone H3 was reduced by RNA interference, the cells formed an aberrant midbody and could not complete cytokinesis successfully. This evidence suggests that Ser-10 phosphorylated histone H3 is a chromosomal passenger protein and plays a crucial role in cytokinesis.     

CENP-A is phosphorylated by Aurora B kinase and plays an unexpected role in completion of cytokinesis.

Aurora B is a mitotic protein kinase that phosphorylates histone H3, behaves as a chromosomal passenger protein, and functions in cytokinesis. We investigated a role for Aurora B with respect to human centromere protein A (CENP-A), a centromeric histone H3 homologue. Aurora B concentrates at centromeres in early G2, associates with histone H3 and centromeres at the times when histone H3 and CENP-A are phosphorylated, and phosphorylates histone H3 and CENP-A in vitro at a similar target serine residue. Dominant negative phosphorylation site mutants of CENP-A result in a delay at the terminal stage of cytokinesis (cell separation). The only molecular defects detected in analysis of 22 chromosomal, spindle, and regulatory proteins were disruptions in localization of inner centromere protein (INCENP), Aurora B, and a putative partner phosphatase, PP1gamma1. Our data support a model where CENP-A phosphorylation is involved in regulating Aurora B, INCENP, and PP1gamma1 targeting within the cell. These experiments identify an unexpected role for the kinetochore in regulation of cytokinesis.     

Identification of pseudo ‘phosphothreonyl-specific’ protein phosphatase T with a fraction of polycation-stimulated protein phosphatase 2A

Protein phosphatase T from rat liver, so termed due to its activity toward [³²P-Thr]casein and its marked preference for the phosphopeptide Arg-Arg-Ala-Thr(P)-Val-Ala over its phosphoseryl derivative (Donella Deana, A., Marchiori, F., Meggio, F. and Pinna, L.A. (1982) J. Biol. Chem. 257, 8565–8568), is shown here to belong to the family of type 2A protein phosphatase according to Cohen's nomenclature (Ingebritsen, T.S. and Cohen, P. (1983) Eur. J. Biochem. 132, 255–261). In particular, protein phosphatase T is endowed with phosphorylase phosphatase activity that is stimulated by protamine, histone H1 and heparin, it is inhibited by spermine, it does not bind to heparin-Sepharose and it readily dephosphorylates the phosphopeptide Arg-Arg-Leu-Ser(P)-Ile-Ser-Thr-Glu-Ser reproducing the phosphorylation site of the α-subunit of phosphorylase kinase. The M_r of protein phosphatase T determined by gel filtration under non-denaturating conditions is about 150 kDa and its activity ratio toward histone H1 phosphorylated by protein kinase C versus histone H1 phosphorylated by cAMP-dependent protein kinase is unusually high. Some properties of protein phosphatase T, such as its weak binding to DEAE-cellulose and its high stimulation by protamine as compared to a relatively poor stimulation by histone H1, suggest that it may be similar to subtype 2A_o of protein phosphatase 2A.     

Thr 3 phosphorylated histone H3 concentrates at centromeric chromatin at metaphase

Thr 3 was one of the newly characterized phosphorylation sites on histone H3. However, the functional significance of histone H3 Thr 3 phosphorylation during mitosis is unclear. In this study, SDS-PAGE and Western blotting analysis showed that histone H3 Thr 3 was phosphorylated specially during mitosis in MCF-10A and ECV-304 cells. Using indirect immunofluorescence labeling and laser confocal microscopy, we demonstrated that histone H3 Thr 3 phosphorylation occurred from prophase to anaphase and dephosphorylated completely in telophase. Remarkably, Thr 3 phosphorylated histone H3 mostly concentrated at centromeric chromatin at metaphase, which was distinct with Ser 10 phosphorylation aggregated at the telomere, but similar to that characteristic of Thr 11 phosphorylated H3 which is largely restricted to the centromeric chromatin. Using chromatin immunoprecipitation (ChIP) assay, we provided direct evidence that the Thr 3 phosphorylated H3 is associated with centromeric DNA at metaphase. These findings suggested that at metaphase Thr 3 phosphorylated histone H3 may also participate in kinetochore assembly to promote faithful chromosome segregation and serve as another recognition code for kinetochore proteins.     

Role of Caenorhabditis elegans protein phosphatase type 1, CeGLC-7 beta,in metaphase to anaphase transition during embryonic development

In Caenorhabditis elegans embryogenesis, phosphorylation events are critical to chromosomal changes. To investigate the dephosphorylation of chromosome behavior, we cloned and characterized the cDNA that encodes C. elegans protein phosphatase type 1 (CeGLC-7 beta), which is composed of 333 amino acids. CeGLC-7 beta possesses a highly conserved amino acid sequence with mammalian and Drosophila protein phosphatase 1. Here, we report on the contribution of CeGLC-7 beta to the dephosphorylation of histone H3 at anaphase. At the embryonic stage, CeGLC-7 beta is associated with the nuclear membrane and chromosomes. The deletion of the Ceglc-7 beta gene and a microinjection of double-stranded RNA produce a disorganized embryogenesis. The Ceglc-7 beta gene mutation causes an abnormal accumulation of phosphorylated histone H3 and delays the mitotic process after anaphase. We propose that CeGLC-7 beta is involved in chromosome dynamics including histone H3 dephosphorylation.     

Structural basis for the specificity of PPM1H phosphatase for Rab GTPases

LRRK2 serine/threonine kinase is associated with inherited Parkinson’s disease. LRRK2 phosphorylates a subset of Rab GTPases within their switch 2 motif to control their interactions with effectors. Recent work has shown that the metal‐dependent protein phosphatase PPM1H counteracts LRRK2 by dephosphorylating Rabs. PPM1H is highly selective for LRRK2 phosphorylated Rabs, and closely related PPM1J exhibits no activity towards substrates such as Rab8a phosphorylated at Thr72 (pThr72). Here, we have identified the molecular determinant of PPM1H specificity for Rabs. The crystal structure of PPM1H reveals a structurally conserved phosphatase fold that strikingly has evolved a 110‐residue flap domain adjacent to the active site. The flap domain distantly resembles tudor domains that interact with histones in the context of epigenetics. Cellular assays, crosslinking and 3‐D modelling suggest that the flap domain encodes the docking motif for phosphorylated Rabs. Consistent with this hypothesis, a PPM1J chimaera with the PPM1H flap domain dephosphorylates pThr72 of Rab8a both in vitro and in cellular assays. Therefore, PPM1H has acquired a Rab‐specific interaction domain within a conserved phosphatase fold.     

Identification of pseudo 'phosphothreonyl-specific' protein phosphatase T with a fraction of polycation-stimulated protein phosphatase 2A

Protein phosphatase T from rat liver, so termed due to its activity toward [32P-Thr]casein and its marked preference for the phosphopeptide Arg-Arg-Ala-Thr(P)-Val-Ala over its phosphoseryl derivative (Donella Deana, A., Marchiori, F., Meggio, F. and Pinna, L.A. (1982) J. Biol. Chem. 257, 8565-8568), is shown here to belong to the family of type 2A protein phosphatase according to Cohen's nomenclature (Ingebritsen, T.S. and Cohen, P. (1983) Eur. J. Biochem. 132, 255-261). In particular, protein phosphatase T is endowed with phosphorylase phosphatase activity that is stimulated by protamine, histone H1 and heparin, it is inhibited by spermine, it does not bind to heparin-Sepharose and it readily dephosphorylates the phosphopeptide Arg-Arg-Leu-Ser(P)-Ile-Ser-Thr-Glu-Ser reproducing the phosphorylation site of the alpha-subunit of phosphorylase kinase. The Mr of protein phosphatase T determined by gel filtration under non-denaturating conditions is about 150 kDa and its activity ratio toward histone H1 phosphorylated by protein kinase C versus histone H1 phosphorylated by cAMP-dependent protein kinase is unusually high. Some properties of protein phosphatase T, such as its weak binding to DEAE-cellulose and its high stimulation by protamine as compared to a relatively poor stimulation by histone H1, suggest that it may be similar to subtype 2Ao of protein phosphatase 2A.     

Phosphorylation of histone H1 during the cell cycle of artichoke

Abstract. Histones have been extracted from tuber and cultured tuber explant material and separated by gel electrophoresis. Histone H1 is heterogenous with 3–4 components in addition to the widely recognized histone H1a and H1b. Using labelling procedures and alkaline phosphatase treatment, histone H1 has been shown to be phosphorylated on both serine and threonine residues and possibly other acid-labile linkages. Variations in histone H1 phosphorylation have been measured through the cell cycle and the evidence indicates enhanced phosphorylation occurring during the G2/M phase as in animal systems.     

Protein kinase A-mediated serine 35 phosphorylation dissociates histone H1.4 from mitotic chromosome

Global histone H1 phosphorylation correlates with cell cycle progression. However, the function of site-specific H1 variant phosphorylation remains unclear. Our mass spectrometry analysis revealed a novel N-terminal phosphorylation of the major H1 variant H1.4 at serine 35 (H1.4S35ph), which accumulates at mitosis immediately after H3 phosphorylation at serine 10. Protein kinase A (PKA) was found to be a kinase for H1.4S35. Importantly, Ser-35-phosphorylated H1.4 dissociates from mitotic chromatin. Moreover, H1.4S35A substitution mutant cannot efficiently rescue the mitotic defect following H1.4 depletion, and inhibition of PKA activity increases the mitotic chromatin compaction depending on H1.4. Our results not only indicate that PKA-mediated H1.4S35 phosphorylation dissociates H1.4 from mitotic chromatin but also suggest that this phosphorylation is necessary for specific mitotic functions.     

Comparison of two forms of pig heart phosphoprotein phosphatase.

A phosphoprotein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) was partially purified from pig heart using as substrate H2B histone which had been phosphorylated at Ser-32 and Ser-36 by adenosine 3',5'-monophosphate-dependent protein kinase (EC 2.7.1.37). The enzyme had a molecular weight of approx. 250 000 and was converted to a smaller form with a molecular weight of approx. 30 000 upon treatment with ethanol. Phosphorylase alpha (EC 2.4.1.1) and phosphorylated H1 histone also served as substrates for both forms of the enzyme. The conversion of the large form of the enzyme to the small form decreased the phosphohistone phosphatase activity to 25-50% with a concomitant 7-fold increase in the phosphorylase alpha phosphatase activity. Ser-36 phosphate was removed 6- and 15-fold more rapidly than was Ser-32 phosphate by the large and small forms of the enzyme, respectively. Among Ser-36-containing tryptic phosphopeptides derived from phosphorylated H2B histone, Lys-Glu-Ser(P)-Tyr-Ser-Val-Tyr was the shortest phosphopeptide which was dephosphorylated at a significant reaction rate with the phosphoprotein phosphatase. The Km values for phosphorylated H2B histone and the tryptic phosphopeptide were 23.7 micron and 187.1 micron, respectively, with the large form, and 81.4 micron and 90.0 micron, respectively, with the small form of the enzyme.     

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.     

The Equilibrative Nucleoside Transporter (ENT1) can be phosphorylated at multiple sites by PKC and PKA

Abstract

Equilibrative Nucleoside Transporters (SLC29) are a family of proteins that transport nucleosides, nucleobases and nucleoside analogue drugs across cellular membranes. ENT1 is expressed ubiquitously in mammalian tissues and responsible for a significant portion of nucleoside analog drug uptake in humans. Despite the important clinical role of ENT1, many aspects of the regulation of this protein remain unknown. A major outstanding question in this field is the whether ENT1 is phosphorylated directly. To answer this question, we overexpressed tagged human (h) and mouse (m) ENT1, affinity purified protein using the tag, conducted phosphoamino acid analysis and found that m/hENT1 is predominantly phosphorylated at serine residues. The large intracellular loop of ENT1, between transmembrane domains 6 and 7, has been suggested to be a site of regulation by phosphorylation, therefore we generated His/Ubiquitin tagged peptides of this region and used them for in vitro kinase assays to identify target serines. Our data support a role for PKA and PKC in the phosphorylation of ENT1 within the intracellular loop and show that PKA can phosphorylate multiple sites within this loop while PKC specifically targets serines 279 and 286 and threonine 274. These data demonstrate, for the first time, that ENT1 is a phosphoprotein that can be directly phosphorylated at several sites by more than one kinase. The presence of multiple kinase targets within the loop suggests that ENT1 phosphorylation is considerably more complex than previously thought and thus ENT1 may be subject to phosphorylation by multiple pathways.     

Histone H1 phosphorylated by protein kinase C is a selective substrate for the assay of protein phosphatase 2A in the presence of phosphatase 1

A protein phosphatase assay, selective for protein phosphatase 2A, has been developed. Bovine histone H1 phosphorylated by protein kinase C and [gamma-32P]ATP, designated H1(C), was tested as the substrate for various preparations of protein phosphatases 1 and 2A. The phosphatase 2A preparations were 10-60-times more active with H1(C) as the substrate when compared to phosphorylase a. The phosphatase 1 enzymes showed very little dephosphorylation of the H1(C) substrate, the activity being less than 5% of the phosphorylase phosphatase activity. This preference and selectivity was demonstrated for purified phosphatase preparations in addition to fresh tissue extracts. The assay provides a rapid, simple assay for the routine analysis of phosphatase 2A in the presence of phosphatase 1, without the use of heat-stable inhibitor proteins.     

Novel in vitro and in vivo phosphorylation sites on protein phosphatase 1 inhibitor CPI-17

CPI-17 is a protein phosphatase 1 (PP1) inhibitor that has been shown to act on the myosin light chain phosphatase. CPI-17 is phosphorylated on Thr-38 in vivo, thus enhancing its ability to inhibit PP1. Thr-38 has been shown to be the target of several protein kinases in vitro. Originally, the expression of CPI-17 was proposed to be smooth muscle specific. However, it has recently been found in platelets and we show in this report that it is endogenously phosphorylated in brain on Ser-128 in a domain unique to CPI-17. Ser-128 is within a consensus phosphorylation site for protein kinase A (PKA) and calcium calmodulin kinase II. However, these two kinases do not phosphorylate Ser-128 in vitro but phosphorylate Ser-130 and Thr-38, respectively. The kinase responsible for Ser-128 phosphorylation remains to be identified. CPI-17 has strong sequence similarity with PHI-1 (which is also a phosphatase inhibitor) and LimK-2 kinase. The novel in vivo and in vitro phosphorylation sites (serines 128 and 130) are in a region/domain unique to CPI-17, suggesting a specific interaction domain that is regulated by phosphorylation.     

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.     

Stimulation of a histone H4 protein kinase in Triton X-100 lysates of rabbit peritoneal neutrophils pretreated with chemotactic factors. Effect of fMet-Leu-Phe and partial characterization of the protein kinase

Rabbit peritoneal neutrophils were stimulated with either the chemotactic factor, fMet-Leu-Phe (10(-8) M, 10 s) or the protein kinase C activator, phorbol-12-myristate-13-acetate (PMA), (0.1 microgram/ml, 3 min) at 37 degrees C, lysed with Triton X-100 at the indicated times and the histone H4 kinase activity of the lysate measured. The histone H4 protein kinase activity was increased severalfold by fMet-Leu-Phe but not PMA. The inclusion of the potent protein kinase C inhibitor, 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine (50 microM) inhibited little if any of the histone H4 protein kinase activity. The effect of fMet-Leu-Phe was transient, maximum stimulation occurring within 10 s and decaying thereafter. The soluble fraction (extract) of the Triton X-100 lysates from control and fMet-Leu-Phe-treated cells was found to contain both histone H4 protein kinase and calcium-phospholipid-activated protein kinase (protein kinase C) activities. The histone H4 protein kinase activity obtained after fMet-Leu-Phe treatment was very little affected by calcium, phospholipid, and PMA and preferred histone H4 but not H1 or H2A as its substrate. In contrast, the calcium-phospholipid-activated protein kinase activity of the extract preferred histones H1 or H2A as substrates and was strongly inhibited by 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine. The histone H4 protein kinase was partially separated from kinase C by DEAE-cellulose and phenyl-Sepharose 4B chromatography. It phosphorylated mostly serine in histone H4. The results indicate that the chemotactic factor, fMet-Leu-Phe, stimulates a protein kinase with substrate specificity and biochemical properties distinct from calcium-phospholipid-activated protein kinase C.