Np95 Is a Histone-Binding Protein Endowed with Ubiquitin Ligase Activity

Abstract

Np95 is an important determinant in cell cycle progression. Its expression is tightly regulated and becomes detectable shortly before the entry of cells into S phase. Accordingly, Np95 is absolutely required for the G 1 /S transition. Its continued expression throughout the S/G 2 /M phases further suggests additional roles. Indeed, Np95 has been implicated in DNA damage response. Here, we show that Np95 is tightly bound to chromatin in vivo and that it binds to histones in vivo and in vitro. The binding to histones is direct and shows a remarkable preference for histone H3 and its N-terminal tail. A novel protein domain, the SRA-YDG domain, contained in Np95 is indispensable both for the interaction with histones and for chromatin binding in vivo. Np95 contains a RING finger. We show that this domain confers E3 ubiquitin ligase activity on Np95, which is specific for core histones, in vitro. Finally, Np95 shows specific E3 activity for histone H3 when the endogenous core octamer, coimmunoprecipitating with Np95, is used as a substrate. Histone ubiquitination is an important determinant in the regulation of chromatin structure and gene transcription. Thus, the demonstration that Np95 is a chromatin-associated ubiquitin ligase suggests possible molecular mechanisms for its action as a cell cycle regulator.Ubiquitination is a frequent posttranslational modification with a vast impact on cell physiology. Ubiquitin (Ub) is a conserved 76-amino-acid polypeptide that is covalently attached to target proteins via an isopeptide bond between its carboxyl-terminal glycine and the ε-amino group of a lysine in substrate proteins (23). A complex enzymatic cascade leads to ubiquitination. Ub is first activated through the formation of a thiol ester bond with the Ub-activating enzyme (E1) and then transferred to a Ub-conjugating enzyme (E2). Finally, Ub is transferred to the substrate through the action of a Ub ligase (E3). Two major families of E3s exist, the HECT type and the RING type. In HECT-E3-mediated catalysis, Ub is transferred from E2 to HECT-E3 and then by E3 to the substrate. In RING-E3-mediated catalysis, E3 mediates the direct transfer of Ub from E2 to the substrate. The E3 ligases, therefore, are the substrate recognition components of the system and confer specificity on the process (23, 47).The best-characterized type of ubiquitination is polyubiquitination, in which the substrate-bound Ub serves as an acceptor for further cycles of ubiquitination (23). By and large, polyubiquitin functions as a general device for targeting of the polyubiquitinated substrate to the proteasome, with ensuing proteolytic degradation (25,48). An emerging body of evidence indicates, however, that when Ub is appended as a single moiety to a target protein (monoubiquitination), the posttranslational modification has a completely different biological impact and serves primarily to modulate protein function and/or interaction(s) (10,24,57).Histones are among the major monoubiquitinated proteins in the cell, and histones H2A, H2B, and H3...

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