SUMMARY FACT, a heterodimer of Spt16 and Pob3, is an essential histone chaperone. We show that the H2A-H2B binding activity that is central to FACT function resides in short acidic regions near the C-termini of each subunit. Mutations throughout these regions impact binding and cause correlated phenotypes that range from mild to lethal, with the largest individual contributions unexpectedly coming from an aromatic residue and a nearby carboxylate residue within each domain. Spt16 and Pob3 bind overlapping sites on H2A-H2B, and Spt16-Pob3 heterodimers simultaneously bind two H2A-H2B dimers, the same stoichiometry as the components of a nucleosome. An Spt16:H2A-H2B crystal structure explains the biochemical and genetic data, provides a model for Pob3 binding, and implies a mechanism for FACT reorganization that we confirm biochemically. Moreover, unexpected similarity to binding of ANP32E and Swr1 with H2A.Z-H2B reveals that diverse H2A-H2B chaperones use common mechanisms of histone binding and regulating nucleosome functions.

Accumulating evidence suggests that protein tyrosine phosphorylation-based signaling pathways are under the regulation of reactive oxygen species. Although protein tyrosine phosphatases are directly regulated by reversible oxidation, it is not clear whether protein tyrosine kinases (PTKs) are also directly regulated by reduction/oxidation (redox). In this study we report a mechanism of direct oxidative inactivation specific for the PTKs in the Src and fibroblast growth factor receptor (FGFR) families, key enzymes in mammalian signal transduction. Src is fully active when reduced and retains 8 -25% of the full activity toward various substrates when oxidized. This inactivation is caused by oxidation of a specific cysteine residue (Cys-277), which results in homodimerization of Src linked by a disulfide bridge. Cys-277 is located in the Gly loop in the catalytic domain. This cysteine residue is conserved only in 8 of the >90 PTKs in the human kinome, including 3 of the 10 Src family kinases and all 4 kinases of the FGFR family. FGFR1 is also reversibly regulated by redox because of this cysteine residue, whereas Csk, a PTK that lacks a cysteine residue at the corresponding position, is not similarly regulated. These results demonstrate a mechanism of direct redox regulation conserved in certain specific PTKs. R eactive oxygen species (ROS), such as hydrogen peroxide and superoxide, can alter the function of proteins by oxidizing free sulfhydryl groups to sulfenic, sulfinic, or sulfonic acids (1, 2). Cellular responses to ROS are historically considered a damage-control mechanism to certain pathological situations that lead to oxidative stress. However, recent studies indicate that certain growth factors and cell adhesion also stimulate the production of ROS, which serve as secondary messengers to regulate downstream signaling pathways (3, 4). Numerous protein phosphorylation pathways respond to ROS (5-9), and identifying the proteins and the residues sensitive to oxidation will help elucidate the mechanism of cross-talk between redox and protein tyrosine phosphorylation.The level of protein tyrosine phosphorylation is the function of opposing actions of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). All PTPs contain a catalytic Cys residue in the active site, and oxidation of this residue leads to the inactivation of the PTP activity (10). This response is recognized as a major mechanism by which ROS regulate the level of protein tyrosine phosphorylation. However, whether PTKs also directly respond to ROS is not established. PTK Src is a key regulator of cell survival, cytoskeleton reorganization, DNA synthesis, and cell division (11,12). A number of studies suggest that Src also plays an important role in cellular response to ROS, because Src specific inhibitors and dominantnegative Src mutants strongly attenuate cellular response to ROS (13-16). However, how ROS regulate Src activity has been controversial, likely reflecting the complexity of Src regulation. Src contains regulatory str...

Background: Spt16-M is a functionally important region of the essential histone chaperone FACT. Results: The Spt16-M crystal structure was determined, and mutations that affect FACT function were mapped to this double PH domain. Conclusion: Spt16-M resembles the Pob3 and Rtt106 histone chaperones and binds histones H3-H4. Significance: Mechanistic models of FACT are advanced, published genetic data are explained, and functionally important surfaces are identified.

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Protein tyrosine kinases are key enzymes of mammalian signal transduction. Substrate specificity is a fundamental property that determines the specificity and fidelity of signaling by protein tyrosine kinases. However, how protein tyrosine kinases recognize the protein substrates is not well understood. C-terminal Src kinase (Csk) specifically phosphorylates Src family kinases on a C-terminal Tyr residue, which down-regulates their activities. We have previously determined that Csk recognizes Src using a substrate-docking site away from the active site. In the current study, we identified the docking determinants in Src recognized by the Csk substrate-docking site and demonstrated an interaction between the docking determinants of Src and the Csk substrate-docking site for this recognition. A similar mechanism was confirmed for Csk recognition of another Src family kinase, Yes. Although both Csk and MAP kinases used docking sites for substrate recognition, their docking sites consisted of different substructures in the catalytic domain. These results helped establish a docking-based substrate recognition mechanism for Csk. This model may provide a framework for understanding substrate recognition and specificity of other protein tyrosine kinases.The human genome contains ϳ500 genes for protein kinases, including ϳ100 protein tyrosine kinases (PTKs) 3 (1). PTKs are important mediators of signal transduction and key targets of anticancer drug discovery (2). They mediate cellular signaling by responding to upstream signals and phosphorylating protein substrates on Tyr residues. To send a regulatory signal to specific protein targets, each PTK phosphorylates only one or a few protein substrates on specific tyrosine residues. Thus, substrate specificity determines signaling specificity and fidelity and distinguishes one PTK from another (3).One of the best understood PTK regulatory systems is the regulation of Src family protein tyrosine kinases (SFKs) by phosphorylation of a Tyr on its C-terminal tail (4, 5). There are nine kinases in the Src family, and each one contains, from the N to the C terminus, a myristoylation motif, a unique region, an SH3 domain, an SH2 domain, a catalytic domain, and a regulatory C-terminal tail. The C-terminal tail contains a Tyr residue (Tyr-527 in avian Src) for phosphorylation by the C-terminal Src kinase (Csk) (6) and the Csk-homologous kinase (7). The phosphorylated tail Tyr binds to the SH2 domain intramolecularly (8, 9), which leads to inactivation of SFKs (10). Ever since the PTK-substrate relationship between Csk and Src families was established about 15 years ago, how Csk specifically recognizes Src family kinases and phosphorylates them on the C-terminal tail Tyr has been an intriguing question (11,12). This exemplifies the largely unanswered question of how PTKs recognize protein substrates in general.Although the Tyr residue for phosphorylation is located on the C-terminal tail of SFKs, SFKs are ϳ50,000-fold better substrates than peptides, mimicking the C-terminal tails (k...