Abstract
The steroid receptor coactivator (SRC) proteins comprise a well-characterized family of nuclear receptor (NR) coactivators that increase transcriptional activation by NRs via covalent modification of chromatin proteins and recruitment of other coactivators. We have recently shown that the SRC family member GRIP1 interacts with a class of SUMO-1 (small ubiquitin-like modifier 1) E3 ligases, the PIAS proteins, and that the coactivator is subjected to SUMO-1 modifications (sumoylation). In this work, we demonstrate that lysine residues 239, 731, and 788 of GRIP1 serve as principal attachment sites for SUMO-1. Lys-731 and Lys-788 are located in the NR interaction domain (NID), and their substitution by arginines impairs the ability of GRIP1 to colocalize with androgen receptor (AR) in nuclei. Likewise, Lys-731 and Lys-788 mutants of GRIP1 have attenuated ability to enhance AR-dependent transcription and fail to synergize with PIASx beta-mediated activation of AR function, indicating that sumoylation modifies the ability of GRIP1 to function as a steroid receptor coactivator. The Lys-731 sumoylation site is conserved in SRC-3 and SRC-1, and the NIDs of the latter coactivators harbor one or two additional sites matching with the consensus sites for SUMO-1 attachment, respectively, suggesting a more general role for the modification in the regulation of SRC protein activity.
Highlights
The steroid, retinoic acid, and thyroid hormone receptors belong to a large family of nuclear receptors (NR).1 The NRs are ligand-regulated transcription factors that recognize specific DNA elements and thereby regulate transcription of their target genes [1]
Identification of SUMO-1 Attachment Sites in GRIP1—Four lysine residues (Lys-239, Lys-731, Lys-788, and Lys-1452) in the GRIP1 sequence fulfill the consensus SUMO-1 attachment sequence ⌿KXE found in an overwhelming number of the SUMO-1 substrates [31]. To examine whether these lysine residues serve as SUMO-1 acceptors in mammalian cells, GRIP1 was expressed in COS-1 cells without or with SUMO-1, and the cell lysates were immunoblotted with the anti-GRIP1 antibody
We have characterized SUMO-1 modification of the nuclear receptor coactivator protein GRIP1 and shown that residues Lys-239, Lys-731, and Lys-788 of GRIP1 act as potential SUMO-1 acceptors
Summary
The steroid, retinoic acid, and thyroid hormone receptors belong to a large family of nuclear receptors (NR).1 The NRs are ligand-regulated transcription factors that recognize specific DNA elements and thereby regulate transcription of their target genes [1]. We have recently shown that the SRC family member GRIP1 interacts with a class of SUMO-1 (small ubiquitin-like modifier 1) E3 ligases, the PIAS proteins, and that the coactivator is subjected to SUMO-1 modifications (sumoylation).
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