The Role of CK1 in Necroptosis and Confirmation of Amyloid-Like Fibers in Necroptosis Using 2D SDD-AGE




Addams, Sarah Elizabeth

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Necroptosis is a regulated necrotic cell death pathway, mediated by a supermolecular complex called the necrosome, which contains receptor-interacting protein kinase 1 and 3 (RIPK1, RIPK3) and mixed-lineage kinase domain-like protein (MLKL). Phosphorylation of human RIPK3 at serine 227 (S227) has been shown to be required for downstream MLKL binding and necroptosis progression. Tandem immunoprecipitation of RIPK3 reveals that casein kinase 1 (CK1) family proteins associate with the necrosome upon necroptosis induction, and this interaction depends on the kinase activity of RIPK3. In addition, CK1 proteins colocalize with RIPK3 puncta during necroptosis. Importantly, CK1 proteins directly phosphorylate RIPK3 at S227 in vitro and in vivo. Loss of CK1 proteins abolishes S227 phosphorylation and blocks necroptosis. Furthermore, a RIPK3 mutant with mutations in the CK1 recognition motif fails to be phosphorylated at S227, does not bind or phosphorylate MLKL, and is unable to activate necroptosis. These results strongly suggest that CK1 proteins are necrosome components which are responsible for RIPK3-S227 phosphorylation. During necroptosis, RIPK1 and RIPK3 are known to form an amyloid fiber. During our lab's investigation into the necroptosis signaling pathway, an amyloid fiber containing MLKL was observed. Based on size, the MLKL fiber appeared to represent a previously undescribed complex. However, the possibility this was the result of either degradation or dissociation of some of the proteins during electrophoresis could not be ruled out. To address this, I developed a protocol based on semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) which allows detection of the SDS-resistant amyloid-like fibers in the cell extracts without purification. Performing second dimension of SDD-AGE determined that the size heterogeneity previously noted was due to a distinct amyloid species. This method allows fast, qualitative confirmation that the amyloid or amyloid-like fibers are not partially dissociating during the SDD-AGE process, and is not limited to use in the necroptosis field.

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