Different experimental approaches within the last two decades impressively document the fundamental role of TRPM7 for a vast variety of physiological processes including embryonic development, cell growth, proliferation and differentiation as well as adhesion and migration. In many cases, the impact of TRPM7 on these essential (cell) functions is mediated by regulation of various cell signaling pathways. However, with the exception of two papers [78, 79], all of the so far published work refers to the TRPM7 full-length protein, thus lacking differentiation between the action of the ion channel and the kinase domain.
Yet, to get a better understanding of the TRPM7 channel-kinase, it is more than important to discriminate between TRPM7-mediated ion influx and its phosphotransferase activity.
Given these aspects, overall aim of this work was to first confirm and expand the knowledge about TRPM7’s functional role in already identified signaling pathways in HAP1 cells and secondary to unravel the impact of the TRPM7 kinase domain on particular mediators of these signaling cascades.
To accomplish this goal, I will utilize two different HAP1 cell lines mutated for TRPM7: One lacks TRPM7 full-length protein expression (abbreviated TRPM7 KO), whereas the other mutated variant carries a point mutation within the kinase domain that leads to the loss of phosphotransferase activity (abbreviated TRPM7 KI). This enables me to at first decipher the role of full-length TRPM7 for distinct cellular signal transduction as well as gene transcription processes and secondly to relate TRPM7-directed actions directly to TRPM7`s kinase activity.
In particular, the following procedure is scheduled:
With use of DNA-based genetic sequencing, I will first verify the correct insertion of the two mutations (TRPM7 KO, TRPM7 KI) in the HAP1 cell line. These experiments will be completed by whole-cell patch clamp measurements: By a systematic comparison of currents typically displayed by TRPM7, the results not only allow confirming a successful deletion of TRPM7 but additionally enable me to detect current alterations which can be attributed to an inactivated TRPM7 kinase domain. In respect to TRPM7’s function in ion homeostasis, impact of TRPM7 respectively its kinase activity on intracellular ion concentrations will be reviewed using inductively coupled plasma mass spectrometry (ICP-MS).
In a next step, I plan to investigate the phosphorylation state of signaling molecules which already have been identified to be part of TRPM7-controlled cascades, such as Akt and GSK3β (PI3K/Akt pathway), ERK1/2 (MAPK/ERK pathway), STAT3 (JAK/STAT pathway) and SMAD2 (TGF-β /SMAD pathway). Phosphorylation of these target proteins will be determined using phospho-specific antibodies in Western Blot analysis. In the event of indeed discovering a TRPM7- respectively kinase activity-controlled phosphorylation of one of the examined signaling molecules, I
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next want to check for potential alterations of downstream effectors such as transcription factors.
Employment of a luciferase-based reporter gene assay will help to clarify domain-specific function of TRPM7 in transcriptional activity of potentially affected transcription factors. Furthermore, TRPM7 respectively kinase activity-dependent regulation of gene expression will subsequently be examined with the sensitive real-time quantitative polymerase chain reaction (RT-qPCR) technique which allows the simultaneous analysis of multiple genes. By directly comparing the results obtained for the TRPM7 KO and the KI clone, respectively, this strategy makes it possible to precisely differentiate between TRPM7 full-length protein and kinase-directed actions on distinct signaling molecules.
To finally verify results obtained by the genetic manipulations of TRPM7, I plan to selectively inhibit either TRPM7 or its kinase domain in primary cells. With the myeloid origin of the HAP1 cells in mind, I intend to utilize primary human neutrophils, which are developed from myeloid progenitor cells and can easily be isolated from blood samples. The results of such an experimental approach should permit the conclusion if identified target proteins of TRPM7 and/or its kinase domain in HAP1 cells are identical to the ones in primary cells.
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NH2 α-Kinase COOH
NH2 α-Kinase COOH
NH2 α-Kinase COOH
TRPM7 affected signaling pathways
Akt/GSK3β ERK1/2
STAT3 SMAD2
Downstream effectors
such as CREB, mTOR, NFκB, NFAT, MAPKAPKs
Target gene expression
Deployed TRPM7 modified HAP1 cell lines
HAP1 TRPM7 WT HAP1 TRPM7 KO HAP1 TRPM7 KI
X X
Figure 3.5 Scientific working strategy to study TRPM7 protein respectively TRPM7 kinase-specific function in cell signaling.
TRPM7 has been shown to modify various components of different cell signaling cascades such as the kinases Akt and ERK1/2 as well as the transcription factors STAT3 and SMAD2. However, whether the effect of TRPM7 is mediated by an ion flux through its channel pore and/or is catalyzed by a phosphorylation of target proteins by the fused kinase domain is currently not clear. To elucidate the exact role of the two TRPM7 domains in cell signaling, different HAP1 cell models modified for TRPM7 will be utilized and compared to a HAP1 TRPM7 wild-type (WT) model (black). First is the HAP1 TRPM7 knockout (KO) clone (red) lacking expression of the correct full-length protein, which enables one to study TRPM7-specific function(s) in general. Secondly, the HAP1 TRPM7 knock-in (KI) clone (blue), which carries a point mutation within the TRPM7 kinase domain inactivating its phosphotransferase activity, will be utilized to uncover the impact of the TRPM7 kinase unit.
Initially, each HAP1 cell model will be employed to assess the domain-specific effect of TRPM7 on the phosphorylation of Akt and GSK3β, ERK1/2, STAT3 and SMAD2. If indeed discovering a TRPM7 or kinase activity-dependent phosphorylation of these proteins, I will next focus on potential alterations regarding downstream effectors of the affected signaling pathway(s), such as further downstream located kinases or transcription factors. Finally, attempts will be made to depict the effect of TRPM7 and/or its kinase activity on specific target gene expression.
Akt – protein kinase B (PKB), COOH – C-terminus, CREB – cAMP response element-binding protein, ERK – extracellular signal-regulated kinase, GSK3β – glycogen synthase kinase 3 β, MAPKAPK – mitogen-activated protein kinase-activated protein kinase, mTOR – mechanistic target of rapamycin, NFAT – nuclear factor of activated T cells, NFκB – nuclear factor kappa-light-chain-enhancer of activated B cells, NH2 – N-terminus, SMAD – Mothers against decapentaplegic homolog, STAT – signal transducer and activator of transcription, TRPM – transient receptor potential, melastatin-like.
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