Identification of a novel LXXLL motif in alpha actinin 4 (ACTN4) spliced isoform that is critical for its interaction with estrogen receptor alpha and co-activators.

August 20, 2012

Alpha actinins (ACTNs) are a family of proteins crosslinking actin filaments to maintain cytoskeletal organization and cell motility. Recently, it has also become clear that ACTN4 can function in the nucleus. In this report, we found that ACTN4 (full-length) and its spliced isoform, ACTN4 (Iso), possess an unusual LXXLL nuclear receptor interacting motif. Both ACTN4 (full-length) and ACTN4 (Iso) potentiate basal transcription activity and directly interact with ER., although ACTN4 (Iso) binds ER. more strongly. We have also found that both ACTN4 (full-length) and ACTN4 (Iso) interact with the ligand-independent and the ligand-dependent activation domains of ER.. While ACTN4 (Iso) interacts efficiently with transcriptional co-activators such as PCAF and SRC-1, the full-length ACTN4 protein either does not or does so weakly. More importantly, the flanking sequences of the LXXLL motif are important not only for interacting with nuclear receptors but also for the association with co-activators. Taken together, we have identified a novel extended LXXLL motif that is critical for interactions with both receptors and co-activators. This motif functions more efficiently in a spliced isoform of ACTN4 than it does in the full-length protein.

Figure 9. Structure models of ACTN LXXLL-containing peptides interacting with synthetic E2- bound ligand-binding domain (LBD) of ERα A-C, Cartoon illustration of the interaction between LBD and co-activating peptides. A co-crystal structure of the LXXLL-containing peptide from GRIP1 NR box II (NH2-KHKILHRLLQD-CO2H) and E2-bound ERα ligand-binding domain (A, 25) was used as a template to model the structure between the LXXLL-containing peptides from ACTN (Iso) (middle, NH2- FRDGLKLMLLLEL-CO2H) (B) or full-length ACTN4 (right, NH2-FRDGLKLMLLLEV-CO2H) (C). D-F, An enlarged view of the interaction between the AF-2 activation pocket and the peptides. H12 is highlighted in red and the AF-2 activation pocket in orange. The LXXLL-containing peptides of GRIP1 (D), ACTN4 (Iso) (E) and ACTN4 (FL) (F) are highlighted in green (left), blue (middle), and cyan (right), respectively."

Results from: Khurana S, Chakraborty S, Zhao X, Liu Y, Guan D, Lam M, Huang W, Yang S, Kao HY.
J Biol Chem. 2012 Aug 20. [Epub ahead of print]