Modifications of Human Histone H3 Variants during Mitosis

Garcia, Benjamin A.; Barber, Cynthia M.; Ptak, Celeste; Turner, Fiona B.; Busby, Scott A.; Shabanowitz, Jeffrey; Moran, Richard G.; Allis, C. David; Hunt, Donald F.

doi:10.1021/bi050906n

Cited by 84 publications

(64 citation statements)

References 55 publications

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“…The bulkiness of K4 methylation likely compromises H3 tail interactions with the narrow substrate-binding groove of haspin. This finding is consistent with the observation that H3T3ph was detected adjacent to H3K4me, but not to H3K4me2 or H3K4me3, among histone peptides from mitotic cells (26). In mitosis, H3K4me2, H3K4me3, and H3T3ph have distinct localizations at centromeres (7,27).…”

Section: Discussionsupporting

confidence: 91%

Structure and functional characterization of the atypical human kinase haspin

Eswaran

Patnaik

Filippakopoulos

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

154

167

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The protein kinase haspin/Gsg2 plays an important role in mitosis, where it specifically phosphorylates Thr-3 in histone H3 (H3T3). Its protein sequence is only weakly homologous to other protein kinases and lacks the highly conserved motifs normally required for kinase activity. Here we report structures of human haspin in complex with ATP and the inhibitor iodotubercidin. These structures reveal a constitutively active kinase conformation, stabilized by haspin-specific inserts. Haspin also has a highly atypical activation segment well adapted for specific recognition of the basic histone tail. Despite the lack of a DFG motif, ATP binding to haspin is similar to that in classical kinases; however, the ATP ␥-phosphate forms hydrogen bonds with the conserved catalytic loop residues Asp-649 and His-651, and a His651Ala haspin mutant is inactive, suggesting a direct role for the catalytic loop in ATP recognition. Enzyme kinetic data show that haspin phosphorylates substrate peptides through a rapid equilibrium random mechanism. A detailed analysis of histone modifications in the neighborhood of H3T3 reveals that increasing methylation at Lys-4 (H3K4) strongly decreases substrate recognition, suggesting a key role of H3K4 methylation in the regulation of haspin activity.ATP binding ͉ histone modification ͉ phosphorylation mechanism ͉ germ cell-specific gene 2 (Gsg2) ͉ mitosis E ukaryotic protein kinases (ePKs) constitute a large group of enzymes that regulate a vast diversity of cellular processes. Kinase activity depends on a number of highly conserved sequence motifs required for ATP/Mg 2ϩ binding and catalysis. About 10% of human ePKs lack one or more essential catalytic motifs and have been initially classified as inactive pseudokinases (1). However, a number of such proteins are active kinases, including haspin [haploid germ cell-specific nuclear protein kinase, encoded by Germ cell-specific gene 2 (Gsg2)]. Haspin lacks both the conserved ATP/Mg 2ϩ binding motif Asp-Phe-Gly (DFG), which is replaced by Asp-Tyr-Thr (DYT), and the Ala-Pro-Glu (APE) motif usually found at the C terminus of the activation segment (2). In addition, haspin shares only weak sequence homology with ePKs and contains a highly divergent kinase domain with several unique inserts (2, 3). Haspin mRNA is abundant in testis and also present in somatic cells (4, 5), and orthologues are found throughout eukaryotic phyla (2). Ectopically expressed haspin localizes to the nucleus in interphase and to the chromosomes in mitosis (4, 6), while depletion of haspin leads to premature chromatid separation, failure of chromosome alignment, and a block in mitosis in a prometaphase-like state (6, 7).Haspin autophosphorylates in vitro (4, 6, 8) and phosphorylates its only currently known substrate, histone H3, at threonine-3 (H3T3ph) both in vitro and in cells (6). H3T3 phosphorylation occurs specifically during mitosis and is particularly prominent at inner centromere regions (6, 7, 9). Recently, H3T3ph has been suggested to relieve an inhibitory effect of ...

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Section: Discussionsupporting

confidence: 91%

Structure and functional characterization of the atypical human kinase haspin

Eswaran

Patnaik

Filippakopoulos

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

154

167

View full text Add to dashboard Cite

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“…Mass spectrometry analyses of human histone H3 during mitosis revealed that phosphorylation and methylation can be found on adjacent residues of the same H3 molecule such as Lys9/Ser10 and Lys27/Ser28, 11,12 providing support for the "methylation/phosphorylation" binary switch hypothesis. 26 This hypothesis proposed that post-translational modifications on adjacent amino acids, such as methylation of Lys9 and phosphorylation of Ser10 on histone H3, could modulate the binding of effector molecules to histones.…”

Section: Histone H3 Phosphorylation During Mitosismentioning

confidence: 90%

“…4,8,10 In mammalian cells, histone H3 is phosphorylated at several sites during mitosis, including serines 10 and 28 (H3S10ph and H3S28ph) and threonines 3 and 11 (H3T3ph and H3T11ph). 8,[11][12][13] The mitotic phosphorylation of at least some of these residues is conserved in a variety of metazoa, fungi, plants and protozoa. [8][9][10][11] Moreover, several kinases implicated in phosphorylating histone H3 during mitosis in mammalian cells, Aurora B for H3S10 and H3S28 and Haspin for H3T3, 2,8,14 appear to have orthologs in a wide spectrum of eukaryotes (Fig.…”

Section: Histone H3 Phosphorylation During Mitosismentioning

confidence: 99%

“…13,15 The histone variant H3.3 also undergoes phosphorylation at serine 31 in mitotic mammalian cells, predominantly in regions bordering centromeres during metaphase. 11,21 In higher plants with monocentric chromosomes, like Hordeum vulgare and Secale cereale, phosphorylation of H3S10 and H3S28 during mitosis remains limited to pericentromeric regions. 8,9,22,23 In contrast, H3T3ph and H3T11ph start near centromeres but then disperse along entire chromosome arms.…”

Section: Histone H3 Phosphorylation During Mitosismentioning

confidence: 99%

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Histone H3 phosphorylation: Universal code or lineage specific dialects?

Cerutti¹,

Casas-Mollano²

2009

Epigenetics

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Post-translational modifications of histones modulate the functional landscape of chromatin and impinge on many DNA-mediated processes. Phosphorylation of histone H3 plays a role in the regulation of gene expression and in chromosome condensation/ segregation. Certain evolutionarily conserved residues on histone H3, namely Thr3, Ser10, Thr11 and Ser28, are phosphorylated during interphase or mitosis in both metazoa and plants. However, many of the kinases involved in these events appear to have evolved independently in different lineages. Likewise, the mechanistic function of specific phosphorylated amino acids, although poorly understood, also seems to differ among eukaryotes. Moreover, some modifications, such as phosphorylation of histone H3 Ser10, appear to have both a positive and a negative connotation and only become meaningful in combination with other histone marks within a particular chromatin context. Thus, a detailed understanding of the influence of histone H3 phosphorylation on biological processes may require learning organismal dialects of the histone code.

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“…Despite the availability of an array of techniques for the elucidation of post-translational modifications [4][5][6], their study continues to present a formidable analytical challenge. Mass spectrometry (MS) is playing an increasingly important role in such analyses because it provides a fast, sensitive means to specifically localize and characterize covalent modifications [7][8][9].…”

Section: Introductionmentioning

confidence: 99%

A novel high-capacity ion trap-quadrupole tandem mass spectrometer

Krutchinsky¹,

Cohen

Chait

2007

International Journal of Mass Spectrometry

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We describe a prototype tandem mass spectrometer that is designed to increase the efficiency of linked-scan analyses by >100-fold over conventional linked-scan instruments. The key element of the mass spectrometer is a novel high ion capacity ion trap, combined in tandem configuration with a quadrupole collision cell and a quadrupole mass analyzer (i.e. a TrapqQ configuration). This ion trap can store >10 6 ions without significant degradation of its performance. The current mass resolution of the trap is 100-450 full width at half maximum for ions in the range 800-4000 m/z, yielding a 10-20 m/z selection window for ions ejected at any given time into the collision cell. The sensitivity of the mass spectrometer for detecting peptides is in the low femtomole range. We can envisage relatively straightforward modifications to the instrument that should improve both its resolution and sensitivity. We tested the tandem mass spectrometer for collecting precursor ion spectra of all the ions stored in the trap and demonstrated that we can selectively detect a phosphopeptide in a mixture of non-phosphorylated peptides. Based on this prototype instrument, we plan to construct a fully functional model of the mass spectrometer for detecting modification sites on proteins and profiling their abundances with high speed and sensitivity.

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Modifications of Human Histone H3 Variants during Mitosis

Cited by 84 publications

References 55 publications

Structure and functional characterization of the atypical human kinase haspin

Structure and functional characterization of the atypical human kinase haspin

Histone H3 phosphorylation: Universal code or lineage specific dialects?

A novel high-capacity ion trap-quadrupole tandem mass spectrometer

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