Exploring the nuclear translocation of TPL2 and its interaction with MEK1

Shi, Xiaofeng (2026) Exploring the nuclear translocation of TPL2 and its interaction with MEK1. MSc(R) thesis, University of Glasgow.

Abstract

The mitogen-activated protein kinase (MAPK) signalling pathway is a conserved pathway involved in cell proliferation, survival, and therapeutic resistance. TPL2 (also known as MAP3K8) functions as a serine/threonine kinase upstream of the MAPK cascade. By activating MEK1/2 and ERK1/2, it triggers the production of pro-inflammatory cytokines such as TNF-α and IL-6, thereby regulating inflammatory immune responses and transmitting cellular signals. Additionally, TPL2 modulates cell proliferation and survival. Collins et al, (2019) previously demonstrated that TPL2 shuttles between the nucleus and cytoplasm via a CRM1-dependent nuclear export mechanism mediated by its NES motif, confirming its transport into the nucleus followed by rapid degradation by the proteasome. However, the regulatory mechanisms governing its nuclear transport and the significance of its nuclear-cytoplasmic shuttling as an upstream scaffold protein in signal transduction remain unclear.

This study revealed TPL2’s role in subcellular signalling organization by analysing the structural components guiding its nuclear entry and its ability to interact with MEK1. This study constructed a TPL2 kinase-inactive mutant (TPL2D270A) and MEK1 mutants with nuclear import defects (T218A/T222A/T226A) through site-directed mutagenesis to investigate their intracellular dynamics and evaluate their colocalization patterns with two TPL2 structural mutants: a C-terminal truncation mutant (TPL2ΔC, lacking residues 397-467 that include a putative degradation motif), and a NES deletion mutant (TPL2ΔNES, lacking the nuclear export signal), co-expressed in HEK293 cells. The behavioural characteristics of the mutant proteins were assessed using subcellular fractionation combined with immunoprecipitation, Western blotting, and confocal imaging techniques.

This study shows that the deletion of the C-terminal region can stabilize TPL2 and increase ERK phosphorylation. The kinase activity of TPL2 contributes to the stabilization of MEK1, and removal of the inhibitory C-terminal domain further enhances this effect. These findings are consistent with previous reports suggesting that the C-terminal region functions as a negative regulatory domain (Gantke et al.,2011). Additionally, this study observed that the activation and stable expression of MEK1 the kinase activity of TPL2. More intriguingly, TPL2 promotes nuclear accumulation of MEK1 mutants with nuclear import defects. These results indicate that TPL2 may assist MEK1 nuclear import even when MEK1’s own import signal is defective, suggesting that TPL2 contributes to MEK1 subcellular localization through a non-catalytic, potentially scaffold-like mechanism rather than solely through kinase-dependent signalling, as observed in the co-expression experiments with MEK1 mutants and TPL2 mutants.

In summary, this study reveals that TPL2 contributes to the subcellular localization and stabilization of MEK1 through both its kinase activity and structural domains. Specifically, co-expression experiments using MEK1 mutants with impaired nuclear import signals showed that TPL2 facilitates MEK1 nuclear accumulation, even in the absence of canonical import motifs. This suggests that TPL2 may function as a scaffold-like carrier in addition to its catalytic role. These findings highlight a previously unrecognized bidirectional interaction between MEK1 and TPL2, where TPL2 regulates MEK1 localization, and MEK1 mutants, in turn, reveal regulatory features of TPL2 localization. This expands our understanding of MAPK pathway regulation through subcellular localization dynamics and suggests new potential targets for therapeutic intervention. However, several limitations should be acknowledged. First, this study was mainly performed using overexpression systems in HEK293 cells, which may not fully reflect endogenous physiological conditions. Second, the study relied primarily on static imaging and biochemical assays, and therefore could not directly assess the real-time dynamics of TPL2-MEK1 trafficking. Third, although the findings support a scaffold-like role for TPL2, the precise mechanism underlying TPL2-mediated nuclear transport remains unclear. In addition, ubiquitination assays and proteasome inhibition experiments were not performed in this study, which limits mechanistic understanding of TPL2 stability and degradation. Future studies using endogenous models, live-cell imaging, and transport-related mechanistic assays will be required to further validate these observations.

• Item Type: Thesis (MSc(R))
• Qualification Level: Masters
• Subjects: Q Science > QH Natural history > QH301 Biology; Q Science > QH Natural history > QH426 Genetics
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
• Supervisor's Name: Carmody, Dr. Ruaidhri
• Date of Award: 2026
• Depositing User: Theses Team
• Unique ID: glathesis:2026-86184
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 14 Aug 2026 10:21
• Last Modified: 14 Aug 2026 10:22
• URI: https://theses.gla.ac.uk/id/eprint/86184