Jonathan M.G. Higgins

Histone H3 Thr-3 phosphorylation by Haspin positions Aurora B at centromeres in mitosis

Location, Location, Location Cell division is orchestrated by a complex signaling pathway that ensures the correct segregation of newly replicated chromosomes to the two daughter cells. The pathway is controlled in part by restricting the activity of critical regulators to specific subcellular locations. For example, the chromosomal passenger complex (CPC) is recruited to chromosomes during mitosis where it oversees kinetochore activity and cytokinesis (see Perspective by Musacchio). Wang et al. (p. 231, published online 12 August), Kelly et al. (p. 235, published online 12 August), and Yamagishi et al. (p. 239) now show that the phosphorylation of the chromatin protein, histone H3, acts to bring the CPC to chromosomes, thereby activating its aurora B kinase subunit. The Survivin subunit of CPC binds specifically to phosphorylated H3, with the phosphorylation at centromeres being carried out by the mitosis-specific kinase, haspin. Furthermore, Bub1 phosphorylation of histone H2A recruits shugoshin, a centromeric CPC adapter. Thus, these two histone marks in combination define the inner centromere. A critical regulator of cell division is recruited to chromosomes through the specific phosphorylation of a chromatin protein. Aurora B is a component of the chromosomal passenger complex (CPC) required for correct spindle-kinetochore attachments during chromosome segregation and for cytokinesis. The chromatin factors that recruit the CPC to centromeres are unknown, however. Here we show that phosphorylation of histone H3 threonine 3 (H3T3ph) by Haspin is necessary for CPC accumulation at centromeres and that the CPC subunit Survivin binds directly to H3T3ph. A nonbinding Survivin-D70A/D71A mutant does not support centromeric CPC concentration, and both Haspin depletion and Survivin-D70A/D71A mutation diminish centromere localization of the kinesin MCAK and the mitotic checkpoint response to taxol. Survivin-D70A/D71A mutation and microinjection of H3T3ph-specific antibody both compromise centromeric Aurora B functions but do not prevent cytokinesis. Therefore, H3T3ph generated by Haspin positions the CPC at centromeres to regulate selected targets of Aurora B during mitosis.

Phosphorylation of histone H3 at threonine 11 establishes a novel chromatin mark for transcriptional regulation

Posttranslational modifications of histones such as methylation, acetylation and phosphorylation regulate chromatin structure and gene expression. Here we show that protein-kinase-C-related kinase 1 (PRK1) phosphorylates histone H3 at threonine 11 (H3T11) upon ligand-dependent recruitment to androgen receptor target genes. PRK1 is pivotal to androgen receptor function because PRK1 knockdown or inhibition impedes androgen receptor-dependent transcription. Blocking PRK1 function abrogates androgen-induced H3T11 phosphorylation and inhibits androgen-induced demethylation of histone H3. Moreover, serine-5-phosphorylated RNA polymerase II is no longer observed at androgen receptor target promoters. Phosphorylation of H3T11 by PRK1 accelerates demethylation by the Jumonji C (JmjC)-domain-containing protein JMJD2C. Thus, phosphorylation of H3T11 by PRK1 establishes a novel chromatin mark for gene activation, identifying PRK1 as a gatekeeper of androgen receptor-dependent transcription. Importantly, levels of PRK1 and phosphorylated H3T11 correlate with Gleason scores of prostate carcinomas. Finally, inhibition of PRK1 blocks proliferation of androgen receptor-induced tumour cell proliferation, making PRK1 a promising therapeutic target.

Cadherin-11 Provides Specific Cellular Adhesion between Fibroblast-like Synoviocytes

Cadherins are integral membrane proteins expressed in tissue-restricted patterns that mediate homophilic intercellular adhesion. During development, they orchestrate tissue morphogenesis and, in the adult, they determine tissue integrity and architecture. The synovial lining is a condensation of fibroblast-like synoviocytes (FLS) and macrophages one to three cells thick. These cells are embedded within the extracellular matrix, but the structure is neither an epithelium nor an endothelium. Previously, the basis for organization of the synovium into a tissue was unknown. Here, we cloned cadherin-11 from human rheumatoid arthritis (RA)-derived FLS. We developed L cell transfectants expressing cadherin-11, cadherin-11 fusion proteins, and anti–cadherin-11 mAb. Cadherin-11 was found to be expressed mainly in the synovial lining by immunohistologic staining of human synovium. FLS adhered to cadherin-11–Fc, and transfection of cadherin-11 conferred the formation of tissue-like sheets and lining-like structures upon fibroblasts in vitro. These findings support a key role for cadherin-11 in the specific adhesion of FLS and in synovial tissue organization and behavior in health and RA.

T-lymphocyte-epithelial-cell interactions: integrin alpha(E)(CD103)beta(7), LEEP-CAM and chemokines

The epithelia are the avascular layers of cells that cover the environment-exposed surfaces of the body. It appears that T cells localize to selected sites in or adjacent to epithelia via the selective expression of adhesion molecules and chemokine receptors on T cells. These bind to counter-receptors and to chemokines expressed by epithelial cells. Recently, there has been an advance in our understanding of the interaction of the alpha(Ebeta7) integrin with its epithelial cell ligand, E-cadherin. In addition, a new adhesion molecule has been identified on non-intestinal epithelial cells, termed lymphocyte-endothelial-epithelial-cell adhesion molecule (LEEP-CAM). Finally, there have been advances in our understanding of the role of skin- or gut-epithelia-derived chemokines in regulating activated T cell homing to these sites.

IQGAP1 and Calmodulin Modulate E-cadherin Function

Ca2+-dependent cell-cell adhesion is mediated by the cadherin family of transmembrane proteins. Adhesion is achieved by homophilic interaction of the extracellular domains of cadherins on adjacent cells, with the cytoplasmic regions serving to couple the complex to the cytoskeleton. IQGAP1, a novel RasGAP-related protein that interacts with the cytoskeleton, binds to actin, members of the Rho family, and E-cadherin. Calmodulin binds to IQGAP1 and regulates its association with Cdc42 and actin. Here we demonstrate competition between calmodulin and E-cadherin for binding to IQGAP1 both in vitro and in a normal cellular milieu. Immunocytochemical analysis in MCF-7 (E-cadherin positive) and MDA-MB-231 (E-cadherin negative) epithelial cells revealed that E-cadherin is required for accumulation of IQGAP1 at cell-cell junctions. The cell-permeable calmodulin antagonist CGS9343B significantly increased IQGAP1 at areas of MCF-7 cell-cell contact, with a concomitant decrease in the amount of E-cadherin at cell-cell junctions. Analysis of E-cadherin function revealed that CGS9343B significantly decreased homophilic E-cadherin adhesion. On the basis of these data, we propose that disruption of the binding of calmodulin to IQGAP1 enhances the association of IQGAP1 with components of the cadherin-catenin complex at cell-cell junctions, resulting in impaired E-cadherin function.

A Positive Feedback Loop Involving Haspin and Aurora B Promotes CPC Accumulation at Centromeres in Mitosis

Haspin phosphorylates histone H3 at Thr3 (H3T3ph) during mitosis [1, 2], providing a chromatin binding site for the chromosomal passenger complex (CPC) at centromeres to regulate chromosome segregation [3-5]. H3T3ph becomes increasingly focused at inner centromeres during prometaphase [1, 2], but little is known about how its level or location and the consequent chromosomal localization of the CPC are regulated. In addition, CPC binding to shugoshin proteins contributes to centromeric Aurora B localization [5, 6]. Recruitment of the shugoshins to centromeres requires the phosphorylation of histone H2A at Thr120 (H2AT120ph) by the kinetochore kinase Bub1 [7], but the molecular basis for the collaboration of this pathway with H3T3ph has been unclear. Here, we show that Aurora B phosphorylates Haspin to promote generation of H3T3ph and that Aurora B kinase activity is required for normal chromosomal localization of the CPC, indicating an intimate linkage between Aurora B and Haspin functions in mitosis. We propose that Aurora B activity triggers a CPC-Haspin-H3T3ph feedback loop that promotes generation of H3T3ph on chromatin. We also provide evidence that the Bub1-shugoshin-CPC pathway supplies a signal that boosts the CPC-Haspin-H3T3ph feedback loop specifically at centromeres to produce the well-known accumulation of the CPC in these regions.

Structure and Functional Characterization of the Atypical Human Kinase Haspin.

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 γ-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.

A phospho/methyl switch at histone H3 regulates TFIID association with mitotic chromosomes

Histone methylation patterns are correlated with eukaryotic gene transcription. High‐affinity binding of the plant homeodomain (PHD) of TFIID subunit TAF3 to trimethylated lysine‐4 of histone H3 (H3K4me3) is involved in promoter recruitment of this basal transcription factor. Here, we show that for transcription activation the PHD of TAF3 can be replaced by PHDs of other high‐affinity H3K4me3 binders. Interestingly, H3K4me3 binding of TFIID and the TAF3‐PHD is decreased by phosphorylation of the adjacent threonine residue (H3T3), which coincides with mitotic inhibition of transcription. Ectopic expression of the H3T3 kinase haspin repressed TAF3‐mediated transcription of endogenous and of reporter genes and decreased TFIID association with chromatin. Conversely, immunofluorescence and live‐cell microscopy studies showed an increased association of TFIID with mitotic chromosomes upon haspin knockdown. Based on our observations, we propose that a histone H3 phospho–methyl switch regulates TFIID‐mediated transcription during mitotic progression of the cell cycle.

Integrin αE(CD103)β7 Mediates Adhesion to Intestinal Microvascular Endothelial Cell Lines Via an E-Cadherin-Independent Interaction

Integrins are important for T cell interactions with endothelial cells. Because the integrin αEβ7 is expressed on some circulating gut-homing T cells and as T cell numbers are reduced in the intestinal lamina propria of αE-deficient mice, we evaluated whether αEβ7 mediates binding to intestinal endothelial cells. We found that anti-αEβ7 mAbs partially blocked the binding of cultured intraepithelial T cells to human intestinal microvascular endothelial cells (HIMEC). Furthermore, αEβ7-transfected K562 cells bound more efficiently than vector-transfected K562 cells to HIMEC. Finally, HIMEC bound directly to an αEβ7-Fc fusion protein. These interactions were partially blocked by anti-αEβ7 mAbs, and endothelial cell binding to the αEβ7-Fc was dependent upon the metal ion-dependent adhesion site within the αE A domain. Of note, the HIMEC lacked expression of E-cadherin, the only known αEβ7 counterreceptor as assessed by functional studies, flow cytometry, and RT-PCR. Thus, HIMEC/αEβ7 binding was independent of E-cadherin. In addition, this interaction appeared to be tissue selective, as HIMEC bound to the αEβ7-Fc, whereas microvascular endothelial cells from the skin did not. Finally, there was evidence for an αEβ7 ligand on intestinal endothelial cells in vivo, as αEβ7 expression enhanced lymphocyte binding around vessels in the lamina propria in tissue sections. Thus, we have defined a novel interaction for αEβ7 at a nonepithelial location. These studies suggest a role for αEβ7 in interactions with the intestinal endothelium that may have implications for intestinal T cell homing or functional responses.

Haspin inhibitors reveal centromeric functions of Aurora B in chromosome segregation

Haspin inhibitors reveal that Aurora B at centromeres is required for metaphase chromosome alignment and spindle checkpoint signaling.