The BAG-1 isoform BAG-1M regulates keratin-associated Hsp70 chaperoning of aPKC in intestinal cells during activation of inflammatory signaling

Atypical PKC (iota/lambda and zeta; 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-kappa B 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 alpha (TNF alpha) 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 co-purify 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 TNF alpha-induced decrease of phosphorylated aPKC. We conclude that BAG-1M mediates Hsp70 inhibition downstream of NF-kappa B.