PDlim2 Selectively Interacts with the PDZ Binding Motif of Highly Pathogenic Avian H5N1 Influenza A Virus NS1

The multi-functional NS1 protein of influenza A virus is a viral virulence determining factor. The last four residues at the C-terminus of NS1 constitute a type I PDZ domain binding motif (PBM). Avian influenza viruses currently in circulation carry an NS1 PBM with consensus sequence ESEV, whereas human influenza viruses bear an NS1 PBM with consensus sequence RSKV or RSEV. The PBM sequence of the influenza A virus NS1 is reported to contribute to high viral pathogenicity in animal studies. Here, we report the identification of PDlim2 as a novel binding target of the highly pathogenic avian influenza virus H5N1 strain with an NS1 PBM of ESEV (A/Chicken/Henan/12/2004/H5N1, HN12-NS1) by yeast two-hybrid screening. The interaction was confirmed by in vitro GST pull-down assays, as well as by in vivo mammalian two-hybrid assays and bimolecular fluorescence complementation assays. The binding was also confirmed to be mediated by the interaction of the PDlim2 PDZ domain with the NS1 PBM motif. Interestingly, our assays showed that PDlim2 bound specifically with HN12-NS1, but exhibited no binding to NS1 from a human influenza H1N1 virus bearing an RSEV PBM (A/Puerto Rico/8/34/H1N1, PR8-NS1). A crystal structure of the PDlim2 PDZ domain fused with the C-terminal hexapeptide from HN12-NS1, together with GST pull-down assays on PDlim2 mutants, reveals that residues Arg16 and Lys31 of PDlim2 are critical for the binding between PDlim2 and HN12-NS1. The identification of a selective binding target of HN12-NS1 (ESEV), but not PR8-NS1 (RSEV), enables us to propose a structural mechanism for the interaction between NS1 PBM and PDlim2 or other PDZ-containing proteins

The mechanism and implications of hScrib regulation of ERK

Scribble is a potential tumor suppressor protein, whose loss is a frequent event in late stage cancer development. In both Drosophila and mammalian model systems, Scribble has been shown capable of regulating cell polarity, cell proliferation and apoptosis. Although several interacting partners, including βPiX, have been identified that help to explain how Scribble can regulate cell polarity and migration, little is known about how Scribble can control cell proliferation. Recent work from our laboratory has shown that Scribble can directly regulate the ERK signaling pathway. This is mediated by a direct protein-protein interaction between Scribble and ERK, which has two components. In the first, Scribble appears to anchor ERK at membrane-bound sites, with the loss of Scribble enhancing ERK nuclear translocation. In the second, Scribble can decrease the levels of active phosphorylated ERK, a function that is dependent upon the ability of Scribble to bind ERK directly. One of the consequences of this activity of Scribble is the inhibition of EJ-ras induced cell transformation. These results provide some of the first direct mechanistic information on how Scribble can regulate cell proliferation and, furthermore, they provide indications as to the identity of other signaling intermediates that may be recruited by Scribble to directly regulate mitogenic signaling pathways

E6AP in the Brain: One Protein, Dual Function, Multiple Diseases

E6-Associated Protein (E6AP), the founding member of the HECT (Homologus to E6AP C terminus) family of ubiquitin ligases, has been gaining increased attention from the scientific community. In addition to its ubiquitin ligase function, our laboratory has also identified steroid hormone receptor transcriptional coactivation as yet another essential function of this protein. Furthermore, it has been established that E6AP has a role in numerous diseases including cancers and neurological syndromes. In this review, we delineate genetic and biochemical knowledge of E6AP and we focus on its role in the pathobiology of neuro-developmental and neuro-aging diseases; bringing to light important gaps of knowledge related to the involvement of its well-studied ligase function versus the much less studied nuclear receptor transcriptional coactivation function in the pathogenesis of these diseases. Tackling these gaps of knowledge could reveal novel possible neuro-pathobiological mechanisms and present crucial information for the design of effective treatment modalities for devastating CNS diseases