Abstract
Hub proteins participate in cellular regulation by dynamic binding of multiple proteins within interaction networks. The hub protein LC8 reversibly interacts with more than 100 partners through a flexible pocket at its dimer interface. To explore the diversity of the LC8 partner pool, we screened for LC8 binding partners using a proteomic phage display library composed of peptides from the human proteome, which had no bias toward a known LC8 motif. Of the identified hits, we validated binding of 29 peptides using isothermal titration calorimetry. Of the 29 peptides, 19 were entirely novel, and all had the canonical TQT motif anchor. A striking observation is that numerous peptides containing the TQT anchor do not bind LC8, indicating that residues outside of the anchor facilitate LC8 interactions. Using both LC8-binding and nonbinding peptides containing the motif anchor, we developed the "LC8Pred" algorithm that identifies critical residues flanking the anchor and parses random sequences to predict LC8-binding motifs with ∼78% accuracy. Our findings significantly expand the scope of the LC8 hub interactome.
Highlights
Most proteins interact with few partners, but a class of proteins referred to as hubs interact with a large number of partners in complex protein–protein interaction networks (Jeong et al, 2001, 2000)
LC8-GFP is present throughout the cytoplasm, within the cell nucleus, and enriched in patch-like structures at the cell cortex located in the vicinity of focal adhesions (Fig 2A)
The mitotic LC8-GFP localization is consistent with LC8 being part of the cytoplasmic dynein complex (Cianfrocco et al, 2015), the nuclear localization and cortical accumulations in interphase cells are not observed for the other core subunits of cytoplasmic dynein, such as the heavy, intermediate, or light intermediate chain (IC), which were previously tagged with GFP and detected in a similar manner (Splinter et al, 2012)
Summary
Most proteins interact with few partners, but a class of proteins referred to as hubs interact with a large number of partners in complex protein–protein interaction networks (Jeong et al, 2001, 2000). Static hubs bind a large number of partners simultaneously at different sites, for example, BRCA2 (Komurov & White, 2007). Dynamic hubs bind multiple partners that compete for the same site (Wu et al, 2009; Patil et al, 2010). Well-known examples of dynamic hubs include calmodulin and 14-3-3 proteins (Aitken, 2006; Uchikoga et al, 2016; Uhart et al, 2016). A more recently discovered member of dynamic hub proteins is the dynein light chain LC8 (Barbar, 2008)
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