Mass spectrometric analysis of protein histidine phosphorylation

Summary. Protein histidine phosphorylation is now recognized as an important form of post-translational modification. The acid-lability of phosphohistidine has meant that this phosphorylation has not been as well studied as serine/threonine or tyrosine phosphorylation. We show that phosphohistidine and phosphohistidine-containing phosphopeptides derived from proteolytic digestion of phosphohistone H4 are detectable by ESI-MS. We also demonstrate reverse-phase HPLC separation of these phosphopeptides and their detection by MALDI-TOF-MS.     

Mutational study of human phosphohistidine phosphatase: effect on enzymatic activity

Although protein histidine phosphorylation is estimated to account for about 6% of total protein phosphorylation in eukaryotes, knowledge on histidine phosphorylation and dephosphorylation is still limited. Recently, a few reports have appeared on a mammalian 14-kDa phosphohistidine phosphatase, also named protein histidine phosphatase. Molecular cloning of the protein has opened possibilities for exploring its properties and physiological role. In the present work, we have searched for potential active site residues in the human phosphohistidine phosphatase by point mutations of conserved histidine and arginine residues to alanine. When assayed by the phosphohistidine-containing peptide succinyl-Ala-His(P)-Pro-Phe-p-nitroanilide, mutants H53A and H102A showed no detectable activity. Compared to the wild-type recombinant enzyme, the specific activity of mutant R45A was decreased by one order of magnitude, that of mutant R78A was decreased by about 30%, while that of mutant H81A was essentially unchanged. These results will facilitate future studies of the reaction mechanism, substrate binding, and molecular structure of the phosphohistidine phosphatase.     

Concerted regulation of protein phosphorylation and dephosphorylation by calmodulin

The multiple functions of calmodulin in brain bring to light an apparent paradox in the mechanism of action of this multifunctional regulatory protein: How can the simultaneous calmodulin stimulation of enzymes with opposing functions such as cyclic nucleotide phosphodiesterases and adenylate cyclase, which are responsible for the degradation and synthesis of cAMP, respectively, be physiologically significant? The same question applies to the simultaneous activation of protein kinases (in particular calmodulin kinase II) and a protein phosphatase (calcineurin). One could propose that the protein kinase(s) and the phosphatase may be located in different cells or in different cellular compartments, and are therefore not antagonizing each other. The same result could be achieved if the specific substrates of these enzymes have different cellular localizations. This does not seem to be the case. In many areas of the brain the two enzymes and their substrates coexist in the same cell. For example, the hippocampus is rich in calmodulin kinase II, calcineruin and substrates for the two enzymes. A more general scheme is presented here, based on different mechanisms of the calmodulin regulation of the two classes of enzyme, which helps to solve this apparent inconsistency in the mechanism of action of calmodulin.     

Relevance of glycine and cysteine residues as well as N- and C-terminals for the activity of protein histidine phosphatase

There is increasing evidence that reversible phosphorylation of histidine residues regulates numerous important cellular processes. The first protein histidine phosphatase (PHP) from vertebrates was discovered just recently. Here, we report on amino acids and domains essential for activity of PHP. Point mutations of conserved residues and deletions of the N- and C-termini of PHP were analyzed using [³²P-his]ATP-citrate lyase as a substrate. Individual or joint replacement of all cysteine residues by alanine did not affect PHP activity. Deletion of 9 N-terminal amino acids resulted in inactive PHP. Furthermore, only 4 C-terminal residues could be deleted without losing PHP activity. Single or multiple mutations of the glycine-rich domain (Gly⁷⁵, Gly⁷⁷) of a putative nucleotide binding site of PHP (GxGxxG/S) caused inactivation of PHP. Wildtype PHP could be labeled with [α-³²P]ATP. Such radiolabeling was not detectable for catalytically inactive PHP-G75A and PHP-G77A. These data suggest further studies on the interaction between PHP and ATP.     

Nuclear c-Abl-mediated tyrosine phosphorylation induces chromatin structural changes through histone modifications that include H4K16 hypoacetylation

c-Abl tyrosine kinase, which is ubiquitously expressed, has three nuclear localization signals and one nuclear export signal and can shuttle between the nucleus and the cytoplasm. c-Abl plays important roles in cell proliferation, adhesion, migration, and apoptosis. Recently, we developed a pixel imaging method for quantitating the level of chromatin structural changes and showed that nuclear Src-family tyrosine kinases are involved in chromatin structural changes upon growth factor stimulation. Using this method, we show here that nuclear c-Abl induces chromatin structural changes in a manner dependent on the tyrosine kinase activity. Expression of nuclear-targeted c-Abl drastically increases the levels of chromatin structural changes, compared with that of c-Abl. Intriguingly, nuclear-targeted c-Abl induces heterochromatic profiles of histone methylation and acetylation, including hypoacetylation of histone H4 acetylated on lysine 16 (H4K16Ac). The level of heterochromatic histone modifications correlates with that of chromatin structural changes. Adriamycin-induced DNA damage stimulates translocation of c-Abl into the nucleus and induces chromatin structural changes together with H4K16 hypoacetylation. Treatment with trichostatin A, a histone deacetylase inhibitor, blocks chromatin structural changes but not nuclear tyrosine phosphorylation by c-Abl. These results suggest that nuclear c-Abl plays an important role in chromatin dynamics through nuclear tyrosine phosphorylation-induced heterochromatic histone modifications.     

Influence of protein histidine phosphatase overexpression and down-regulation on human umbilical-vein endothelial cell viability

PHP (protein histidine phosphatase) is expressed by mammalian tissues, particularly in blood vessel walls. We investigated whether PHP plays a significant role in endothelial cells. By Western blot and immunofluorescence analysis PHP was found in HUVEC (human umbilical-vein endothelial cells). Overexpression of PHP by the use of a plasmid vector, pIRES2-AcGFP1-PHP, induced apoptosis in HUVEC. To exclude the possibility that increased cellular protein alone unspecifically caused cell damage, the inactive H53A mutant of PHP was also overexpressed as a control; it did not lead to apoptosis. Down-regulation of PHP by the RNAi (RNA interference) technique did not affect cell viability. In conclusion, HUVEC are damaged by overexpression, but not down-regulation, of PHP, suggesting a pronounced impact of the enzyme on the cells when its activity is increased.     

cAMP signaling regulates histone H3 phosphorylation and mitotic entry through a disruption of G2 progression

cAMP signaling is known to have significant effects on cell growth, either inhibitory or stimulatory depending on the cell type. Study of cAMP-induced growth inhibition in mammalian somatic cells has focused mainly on the combined role of protein kinase A (PKA) and mitogen-activated protein (MAP) kinases in regulation of progression through the G1 phase of the cell cycle. Here we show that cAMP signaling regulates histone H3 phosphorylation in a cell cycle-dependent fashion, increasing it in quiescent cells but dramatically reducing it in cycling cells. The latter is due to a rapid and dramatic loss of mitotic histone H3 phosphorylation caused by a disruption in G2 progression, as evidenced by the inhibition of mitotic entry and decreased activity of the CyclinB/Cdk1 kinase. The inhibition of G2 progression induced through cAMP signaling is dependent on expression of the catalytic subunit of PKA and is highly sensitive to intracellular cAMP concentration. The mechanism by which G2 progression is inhibited is independent of both DNA damage and MAP kinase signaling. Our results suggest that cAMP signaling activates a G2 checkpoint by a unique mechanism and provide new insight into normal cellular regulation of G2 progression.     

Variability and inheritance of histone genes H3 and H4 in Vicia faba

Summary We have compared copy numbers and blothybridization patterns of histone genes (H3 plus H4) between and within individuals of broad bean (Vicia faba). Copy number differences among individuals in the population of 200 individuals were as great as 27 fold, and as much as 3.2 fold among separate leaves of the same plant. Among F₂ progeny from genetic crosses, up to a 5.4-fold range was seen (mean=3.5 fold), and among F₁ progeny of self-pollinated plants, up to a 5.9-fold range was observed (mean=2.3 fold). Histone gene blot-hybridization patterns for EcoRI and HindIII were also variable among individuals and indicated that the genes are probably clustered in only a few chromosomal loci. The degree of variation in histone gene copy number per haploid genome (2–55 copies, or 27 fold) was similar to that found previously for ribosomal RNA genes (230–22000, or 95 fold) of V. faba. However, the two gene families change independently, since individuals with a high or low copy number for one gene can have either a high or low copy number for the other. The mechanisms(s) for rapid gene copy number change may be similar for these gene families.     

Phosphorylation of protein phosphatase type-1 inhibitory proteins by integrin-linked kinase and cyclic nucleotide-dependent protein kinases

Protein phosphatases play key roles in cellular regulation and are subjected to control by protein inhibitors whose activity is in turn regulated by phosphorylation. Here we investigated the possible regulation of phosphorylation-dependent type-1 protein phosphatase (PP1) inhibitors, CPI-17, PHI-1, and KEPI, by various kinases. Protein kinases A (PKA) and G (PKG) phosphorylated CPI-17 at the inhibitory site (T38), but not PHI-1 (T57). Phosphorylated CPI-17 inhibited the activity of both the PP1 catalytic subunit (PP1c) and the myosin phosphatase holoenzyme (MPH) with IC(50) values of 1-8 nM. PKA predominantly phosphorylated a site distinct from the inhibitory T73 in KEPI, whereas PKG was ineffective. Integrin-linked kinase phosphorylated KEPI (T73) and this dramatically increased inhibition of PP1c (IC(50)=0.1 nM) and MPH (IC(50)=8 nM). These results suggest that the regulatory phosphorylation of CPI-17 and KEPI may involve distinct kinases and signaling pathways.     

On the interactions of histone H4 and H4 peptides with DNA. Electrooptical, hydrodynamic and electron microscopy studies

The interactions of DNA with histone H4 and with its fragments N-H4 (1-84) and C-H4 (85-102) have been studied by using electrooptical techniques, viscosity and electron microscopy. Electron microscopy reveals that histone H4 induces a large folding of DNA molecules : this is in agreement with electrooptical measurements which indicate that, with the increase of their ratio, H4/DNA complexes undergo a gradual process of condensation. Viscosity measurements show that complexes at ratios up to 0.20-0.25 become more rigid as compared to DNA. It appears that C-H4, and not the N-H4 fragment, causes a great distorsion to the structure of DNA, accompanied by an increase of rigidity at ratios up to 0.20-0.25, as occurs for H4/DNA complexes. Electrooptical studies of C-H4/DNA complexes show, along a range of histone/DNA ratios, an important permanent dipole component. These effects reveal a particular mode of interaction of C-H4 with DNA, indicating that some charged residues of the peptide are kept distant enough from the DNA backbone. As no dipole character, in addition to that shown for DNA, has been detected for H4/DNA complexes, it is concluded that the conformation of the H4 molecule modifies to some extent the interaction of the C-terminal region. Our results show that this histone, and particularly its C-terminal region, is important as a determinant factor in the folding of DNA within artificial complexes.     

Src kinase regulation by phosphorylation and dephosphorylation

Src and Src-family protein-tyrosine kinases are regulatory proteins that play key roles in cell differentiation, motility, proliferation, and survival. The initially described phosphorylation sites of Src include an activating phosphotyrosine 416 that results from autophosphorylation, and an inhibiting phosphotyrosine 527 that results from phosphorylation by C-terminal Src kinase (Csk) and Csk homologous kinase. Dephosphorylation of phosphotyrosine 527 increases Src kinase activity. Candidate phosphotyrosine 527 phosphatases include cytoplasmic PTP1B, Shp1 and Shp2, and transmembrane enzymes include CD45, PTPalpha, PTPepsilon, and PTPlambda. Dephosphorylation of phosphotyrosine 416 decreases Src kinase activity. Thus far PTP-BL, the mouse homologue of human PTP-BAS, has been shown to dephosphorylate phosphotyrosine 416 in a regulatory fashion. The platelet-derived growth factor receptor protein-tyrosine kinase mediates the phosphorylation of Src Tyr138; this phosphorylation has no direct effect on Src kinase activity. The platelet-derived growth factor receptor and the ErbB2/HER2 growth factor receptor protein-tyrosine kinases mediate the phosphorylation of Src Tyr213 and activation of Src kinase activity. Src kinase is also a substrate for protein-serine/threonine kinases including protein kinase C (Ser12), protein kinase A (Ser17), and CDK1/cdc2 (Thr34, Thr46, and Ser72). Of the three protein-serine/threonine kinases, only phosphorylation by CDK1/cdc2 has been demonstrated to increase Src kinase activity. Although considerable information on the phosphoprotein phosphatases that catalyze the hydrolysis of Src phosphotyrosine 527 is at hand, the nature of the phosphatases that mediate the hydrolysis of phosphotyrosine 138 and 213, and phosphoserine and phosphothreonine residues has not been determined.     

Activation of protein phosphatase 2A by cAMP-dependent protein kinase-catalyzed phosphorylation of the 74-kDa B″ (δ) regulatory subunit in vitro and identification of the phosphorylation sites

Human erythrocyte protein phosphatase 2A, which comprises a 34-kDa catalytic C subunit, a 63-kDa regulatory A subunit and a 74-kDa regulatory B″ (δ) subunit, was phosphorylated at serine residues of B″ in vitro by cAMP-dependent protein kinase (A-kinase). In the presence and absence of 0.5 μM okadaic acid (OA), A-kinase gave maximal incorporation of 1.7 and 1.0 mol of phosphate per mol of B″, respectively. The K_m value of A-kinase for CAB″ was 0.17±0.01 μM in the presence of OA. The major in vitro phosphorylation sites of B″ were identified as Ser-60, -75 and -573 in the presence of OA, and Ser-75 and -573 in the absence of OA. Phosphorylation of B″ did not dissociate B″ from CA, and stimulated the molecular activity of CAB″ toward phosphorylated H1 and H2B histones, 3.8- and 1.4-fold, respectively, but not toward phosphorylase a.     

Regulation of aflatoxin production by Ca(2+)/calmodulin-dependent protein phosphorylation and dephosphorylation

To elucidate Ca(2+)-mediated regulation of aflatoxin production, the status of Ca(2+)/calmodulin-dependent protein phosphorylation and dephosphorylation was investigated employing toxigenic and non-toxigenic strains of Aspergillus parasiticus. Incubation of cytoplasmic extracts with [gamma-(32)P]ATP followed by SDS-PAGE and autoradiography revealed total absence of protein phosphorylation during periods corresponding to aflatoxin production in the toxigenic strain (NRRL 2999). In contrast, protein phosphorylation was unaffected in the non-toxigenic strain (SRRC 255). Aflatoxin production in the toxigenic strain was also accompanied by enhanced (26-fold) activity of calcineurin (calmodulin-dependent protein phosphatase 2B) concomitant with a lowered (6-fold) activity of calmodulin-dependent protein kinase. In addition, the in vitro activity of Ca(2+)/calmodulin-dependent protein kinase was susceptible to dose-dependent inhibition by aflatoxin. Since calcineurin remains active in the absence of phosphorylation by calmodulin-dependent protein kinase, it is suggested that calcineurin-mediated dephosphorylation of regulatory enzymes ensures continued production of aflatoxins.     

Isolation and characterization of histone H3 lysine 4 demethylase-containing complexes

Histone methylation is involved in the regulation of many cellular processes. In the past 2 years, several histone demethylases including BHC110/LSD1 have been characterized. BHC110, the first known histone lysine demethylase, removes methyl groups from methylated histone H3 lysine 4 and has been found in many multi-protein complexes. Using one-step affinity purification, we have isolated enzymatically active BHC110-containing complexes. Here, we detail the methods used for the isolation and characterization of these histone demethylase complexes from a human stable cell line.     

Regulation of Chk2 phosphorylation by interaction with protein phosphatase 2A via its B' regulatory subunit

Chk2 is a key player of the DNA damage signalling pathway. To identify new regulators of this kinase, we performed a yeast two-hybrid screen and found that Chk2 associated with the B' regulatory subunit of protein phosphatase PP2A. In vitro GST-Chk2 pulldowns demonstrated that B'gamma isoforms bound to Chk2 with the strongest apparent affinity. This was confirmed in cellulo by co-immunoprecipitation after overexpression of the respective partners in HEK293 cells. The A and C subunits of PP2A were present in the complexes, suggesting that Chk2 was associated with a functionnal PP2A. In vitro kinase assays showed that B'gamma3 was a potent Chk2 substrate. This phosphorylation increased the catalytic phosphatase activity of PP2A measured on MAP kinase-phosphorylated myelin basic protein as well as on autophosphorylated Chk2. Finally, we demonstrated that overexpressing B'gamma3 in HEK293 suppressed the phosphorylation of Chk2 induced by a genotoxic treatment, suggesting that PP2A may counteract the action of the checkpoint kinase in living cells.