BAG-1M regulates keratin-associated Hsp70 chaperoning of aPKC in intestinal cells under inflammatory signaling

Abstract

Atypical PKC (i/l and f; hereafter referred to as aPKC) is a key player in the acquisition of epithelial polarity and participates in other signaling cascades including the control of NF-kB signaling. This kinase is post-translationally regulated through Hsp70-mediated refolding. Previous work has shown that such a chaperoning activity is specifically localized to keratin intermediate filaments. Our work was performed with the goal of identifying the molecule(s) that block Hsp70 activity on keratin filaments during inflammation. A transcriptional screen allowed us to focus on BAG-1, a multifunctional protein that assists Hsp70 in nucleotide exchange but also blocks its activity at higher concentrations. We found the BAG-1 isoform BAG-1M upregulated threefold in human Caco-2 cells following stimulation with tumor necrosis factor receptor a (TNFa) to induce a pro-inflammatory response, and up to sixfold in mouse enterocytes following treatment with dextran sodium sulfate (DSS) to induce colitis. BAG-1M, but no other isoform, was found to copurify with intermediate filaments and block Hsp70 activity in the keratin fraction but not in the soluble fraction within the range of concentrations found in epithelial cells cultured under control and inflammation conditions. Constitutive expression of BAG-1M decreased levels of phosphorylated aPKC. By contrast, knockdown of BAG-1, blocked the TNFa-induced decrease of phosphorylated aPKC. We conclude that BAG-1M mediates Hsp70 inhibition downstream of NF-kB.

Multiple roles for keratin intermediate filaments in the regulation of epithelial barrier function and apico-basal polarity

“…Likewise some members of the Hsp40 family bind stably to the C-terminal region of K18 46 . Small cochaperones, such as Bag1 also bind to the IF scaffold under pro-inflammatory upregulation 53 . Filensin IF bind α-crystallin 54 .…”

Functional Analysis of Keratin-Associated Proteins in Intestinal Epithelia

“…On the other hand, there is no specificity for luciferase refolding in any of the fractions. Soluble or keratin-associated chaperones seem to be equally effective (Mashukova et al, 2014). …”

“…1B). Furthermore, the levels of IF-bound Bag-1M were dependent on the total cellular Bag-1M (Mashukova et al, 2014). These data suggest that attached Bag-1M is in equilibrium with soluble Bag-1M.…”

“…We found that Bag-1M is necessary for Hsp70 activity at basal concentrations (around 1 μM) but inhibits chaperoning activity in kP at concentrations usually found in epithelial cells under inflammatory stimulation (around 6 μM). Importantly, the inhibitory concentration is higher for soluble, cytosolic Hsp70 which showed another aspect of specificity for chaperones attached to the keratin scaffold (Mashukova et al, 2014). Moreover, these results suggested that the Hsp70 keratin scaffold may not be functional in cells expressing high levels of Bag-1M, such as some carcinomas (Maki et al, 2007).…”

The Dynamic Duo of Small Heat Proteins and IFs Maintain Cell Homeostasis, Resist Cellular Stress and Enable Evolution in Cells and Tissues

“…Therefore, we suggest this is a reasonable expectation of the association with IFs. For example, the association of HSP70 with keratin fi laments is required for maintaining aPKC activity (Mashukova et al 2009(Mashukova et al , 2014 and HSP27 regulates the activity of cdk5 and the subsequent phosphorylation of neurofi laments (Holmgren et al 2013 ). In the epidermis, the regulation of fi laggrin expression by HSP27 involves phosphorylation and transition to the keratin fi lament network (Jonak et al 2011 ;Robitaille et al 2010 ).…”