Functional Interaction between Casein Kinase 1 and HAMLET

Abstract

Current cancer treatments are associated with high mortality because anticancer drugs are highly toxic and have numerous side effects. Human α-lactalbumin Made Lethal to Tumour cells (HAMLET) is protein-lipid complex that kills tumor cells and spares healthy differentiated cells. HAMLET interacts with a broad range of cellular targets. Our group has shown that the cell death induced by HAMLET can be correlated with a caspase independent pathway, a transcriptional response characterized by the activation of ion fluxes and p38 MAPK signalling pathway. There are still many cellular events in response to HAMLET that need to be address. HAMLET is isolated from a casein fraction of human milk and Casein Kinase 1 (CK1) has been shown to phosphorylate several proteins contained in casein. Furthermore CK1 has been showed to phosphorylate the partially unfolded state of α-lactalbumin compared to its native state. For this reason we wanted to investigate if there was any functional interaction between HAMLET and CK1. Here we showed by a protoarray, confocal imaging, and co-IP assays that HAMLET interacts with CK1 isoforms. Treatment of cancer cells with HAMLET triggered a marked change in distribution of CK1 isoforms. CK1δ was found to accumulate in a network of filaments corresponding to the cytoskeleton, as confirmed by CK1δ colocalization with α-actinin1. This was explained by HAMLET inhibition of CK1δ dependent phosphorylation of Tau. CK1α and CK1ε accumulated in the perinuclear region of the cell. The perinuclear compartment was identified as the endoplasmic reticulum (ER) as confirmed by CK1α colocalization with the ER marker, calnexin. HAMLET triggered a marked ER stress response by activation of all the three main branches of the Unfolded Protein Response (UPR): activation of the inositol requiring protein 1 (IRE1), the protein kinase RNA-like endoplasmic reticulum kinase (PERK) and the activating transcription factor 6 (ATF6). Involvement of CK1 in the ER stress response triggered by HAMLET still needs to be clarified. Furthermore, inhibition of CK1 isoforms with the CK1 specific inhibitor IC261 caused partial inhibition in HAMLET induced CK1 distribution and caused synergistic cell death. Finally, ion fluxes which are essential for HAMLET-induced tumor cell death partially prevented CK1 change in distribution.

Popular Abstract

Casein Kinase 1 and HAMLET functional interaction in Cancer Cells

Casein Kinase 1 (CK1) are enzymes that regulate many signaling pathways in cells. A malfunction in CK1 activity has been proved to lead to cancer development. HAMLET is a potential drug that could be used for the treatment of cancer, because it selectively kills tumor cells and it spares the healthy ones. For this reason we wanted to investigate if there was any interaction between CK1 and HAMLET, and if this interaction existed, how does it contributes to the tumor cell death triggered by HAMLET.

Cell death under different conditions was studied with ATP lite and Presto Blue assays. HAMLET and CK1 interaction was studied with a Protoarray, Co-immunoprecipitation assay, immunohistochemistry and confocal imaging. Immunohistochemistry and confocal imaging were also performed to observe CK1 distribution in response to HAMLET. Phosphorylation levels of CK1 targets after treatment of tumor cells with HAMLET were quantified with a phosphoarray. Finally to see if HAMLET triggered an Endoplasmic Reticulum (ER) stress response in tumor cells which can lead to cell death, techniques like transcriptomics, Western Blot, and RT-PCR were done to analyze different genes and proteins that are known to be involved in the ER stress response.

Several CK1 isoforms were found to interact directly with HAMLET. HAMLET dramatically changed the localization of CK1 in cells. CK1δ was found to accumulate in the cytoskeleton. This was explained by HAMLET inhibition of CK1δ dependent phosphorylation of the microtubules associated protein Tau. On the other hand, CK1α and CK1ε accumulated in a cellular organelle, the endoplasmic reticulum (ER), which has a fundamental role in protein folding. HAMLET triggered a marked ER stress response through the activation of different proteins that have key roles in trying to recover the normal functions of the ER. Involvement of CK1 in the ER stress response triggered by HAMLET still needs to be clarified. Furthermore, inhibition of CK1 isoforms with the CK1 specific inhibitor IC261 caused a partial inhibition in HAMLET induced CK1 change in distribution and caused synergistic cell death. Finally, ion fluxes, which are essential for HAMLET-induced tumor cell death, partially prevented CK1 change in distribution.

The results of this study are a good approach to understand many of the cellular events that are involved in HAMLET’s tumouricidal effect. In addition it suggests that drugs directed against CK1 family members could be used for the treatment of cancer.




Advisor: Prof. Catharina Svanborg
Master´s Degree Project in Biology, 45 credits
Department of Biology, Lund University

• author: Duque, Luisana
• supervisor: Catharina Svanborg
• organization: Degree Projects in Biology
• course: BION01 20142
• year: 2015
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• language: English
• id: 8052647
• date added to LUP: 2015-10-07 11:05:24
• date last changed: 2015-10-07 11:05:24