Signalling mechanisms of the tyrosine phosphatase SHP2 upstream of Ras
Normal cell regulation is essential for healthy cell function, including cell growth, differentiation, migration, and apoptosis. Dysregulation of intracellular signalling pathways, including Ras – mitogen activated protein kinase (MAPK), disrupts normal cell functioning and can lead to various disorders, including cancer and developmental pathologies. SHP2 regulates multiple signalling cascades, including its crucial role in the complete activation of RasMAPK in KRas driven cancers. SHP2 is also the most commonly mutated protein in RASopathies such as Noonan syndrome. SHP2 is therefore an important therapeutic target, with allosteric inhibitors ‘locking’ SHP2 in its autoinhibited state currently in clinical trials. However, the efficacy of such allosteric inhibitors is significantly reduced by strongly activating mutations, which account for 50% of SHP2 variants detected in human cancers. Improved understanding of SHP2 function in intracellular signalling is needed to identify novel therapeutic approaches targeting SHP2, without the previously observed toxicity. Despite the well-described structure and mode of SHP2 autoinhibition, its precise signalling mechanisms remain to be elucidated. SHP2 is a cysteine-based non-receptor protein tyrosine phosphatase (PTP). It is susceptible to redox regulation; although canonically it is thought to be inactivated by oxidation, our lab has identified the capacity for specific redox-regulated interactions with PTPs, at least in vitro. To better understand SHP2 signalling, I focused on identifying substrates and potential redox-switchable interactions. Using a combination of protein pull downs, western blots, and mass spectrometry (MS). DLG5, a scaffolding protein, and RASAL2, a putative RasGAP, were identified as novel redox-regulated SHP2 interactors. I have shown that DLG5 is a likely SHP2 substrate, and mapped the interaction between the SHP2 PTP domain and the C-terminus SH3-GUK domains of DLG5 using transiently expressed DLG5 truncated constructs. The RASAL2 N-terminal C2 domain appears to be key in binding directly to the SHP2 PTP, according to interaction mapping in vitro. Both interactors were also found to localize in close proximity to SHP2 in cells. Focusing on RASAL2, since it is linked to Ras regulation via its conserved GAP domain, I identified a synergistic role between SHP2 and RASAL2, where RASAL2 counterintuitively positively regulates signalling, including the MAPK/ERK pathway, and further tested this in different KRas mutant settings. This positive regulation occurs upstream from MEK and not as a result of negative feedback regulation of the RasMAPK pathway downstream from ERK. RASAL2 knockdown (KD) leads to a decrease in phosphorylation and total protein levels of SHP2 and FAK, as well as extensive transcriptional changes in SHP2-dependent and independent manners, including regulation of receptor tyrosine kinase signalling.
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