CK2 Phosphorylates SSRP1 and Inhibits Its DNA-binding Activity

Abstract

We have previously shown that CK2 associates with the human high-mobility group protein SSRP1 and that this association increases in response to UV irradiation. CK2 also phosphorylates SSRP1 in vitro. Here we extend this work by investigating CK2 regulation of SSRP1 function through phosphorylation. Phosphorylation of SSRP1 by CK2 inhibited the nonspecific DNAbinding activity of SSRP1 and FACT (facilitating chromatin-mediated transcription) complex in vitro. Using a serine/threonine-scanning Auto-spot peptide array coupled with a filter-based kinase assay with synthetic peptides as substrates, we identified serines 510, 657, and 688 as phosphorylation targets of CK2 in vitro. Mutagenesis of the three serines revealed that serine 510 was more important for the regulation of SSRP1 DNA-binding activity. Furthermore, we found that SSRP1 was phosphorylated in cells in response to UV (but not γ) irradiation. These results suggest that CK2 regulates the DNA-binding ability of SSRP1 and that this regulation may be responsive to specific cell stresses.CK2 is a ubiquitous and evolutionarily conserved kinase. This enzyme is a heterotetrameric protein complex consisting of two regulatory β subunits (28 kDa) and two catalytic α (42 kDa) or α′ (38 kDa) subunits with the stoichiometry of either α2β2, α′2β2, or αα′β2 as the holoenzyme (1). Genetic studies in yeast (2) and in mice (3) demonstrate that this enzyme is essential for viability and animal embryogenesis. Biochemical and functional analyses of this enzyme reveal that it can phosphorylate a broad spectrum of protein substrates in vitro and regulate a variety of cellular functions including transcription in the nucleus. Consistent with its regulatory role in the nucleus, CK2 associates with a number of chromatin and nuclear matrix proteins in the cell (4-6).One of the mammalian CK2-interacting nuclear proteins is the previously identified DNAbinding protein called SSRP1 (structure-specific recognition protein 1) (7). SSRP1 (named T160 in mice) (8) is a member of the high-mobility group (HMG) 1 family of proteins (9), * This work was supported in part by NCI, National Institutes of Health Grants CA93614, CA095441, and CA079721 (to H. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‖ Supported by National Institutes of Health Grant GM48231. [623][624][625][626][627][628][629][630][631][632][633][634][635][636][637][638][639][640], and a mixed charge domain at the extreme C-terminal region (aa 661-709). In yeast, Pob3 and Nhp6 form a bipartite SSRP1 analog (12). Pob3 is highly homologous with the N terminus of SSRP1, whereas Nhp6 is an HMG protein resembling the C terminus of SSRP1 (12). These domains are crucial for the functions of SSRP1. For example, the highly conserved N-terminal region of SSRP1 has been shown to directly interact with its partner, Spt16 (7), forming a tight het...

Drosophila MCRS2 Associates with RNA Polymerase II Complexes To Regulate Transcription

“…Notably, many of these proteins have previously been reported to copurify with each other and with RNAP II in humans. Thus, Cdk11 has been found to associate with both subunits of TFIIF, CK2, the FACT complex, and hypo-and hyperphosphorylated RNAP II (47), and CK2 has been isolated in complex with TFIIF, Cdk11 p110 , RNAP II, and SSRP1, all of which it is capable of phosphorylating (14,26,33,48).…”

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“…SSRP1 may have SPT16-independent functions as well (24,53). In mitosis, we found that SPT16 did not colocalized with SSRP1 to the spindle and midbody (see Fig.…”

Coupling caspase cleavage and ubiquitin–proteasome-dependent degradation of SSRP1 during apoptosis

“…Recent works suggest that SSRP1 phosphorylation by casein kinase 2 (CK2) alters its DNA-binding properties. 22,23 During our studies attempting to understand the significance of this post-translational modification, we observed a reproducible decrease of SSRP1 levels after UV treatment. This prompted us to study SSRP1 turnover and elucidate the mechanism of SSRP1 degradation.…”

“…17,22 However, human SSRP1 is unable to bind mononucleosomes by itself and requires interaction with Spt16, 6 which occurs via the SSRP1 N-terminal. 13 These in vitro data suggest that the N-terminal part of SSRP1 is critical for its interaction with nucleosomes.…”