DISSECTING THE FUNCTION AND INTERACTOME OF PLEKHS1 IN PROSTATE TUMORIGENESIS
University of Cambridge
Postgraduate School of Life Sciences (PSLS)
Doctor of Philosophy (PhD)
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Jung, P. (2021). DISSECTING THE FUNCTION AND INTERACTOME OF PLEKHS1 IN PROSTATE TUMORIGENESIS (Doctoral thesis). https://doi.org/10.17863/CAM.63289
Phosphatidylinositol(3,4,5)trisphosphate (PI(3,4,5)P3 or PIP3) is the lipid product and key signalling output of Class I phosphoinositide 3-kinases (PI3Ks) and acts as a coordinator of a huge network that controls cellular processes such as metabolism, cell growth and movement. Mutations that augment the activity of this network are amongst the most common drivers of tumour progression. Upon binding of many ligands to their receptors, Class I PI3Ks are activated and 3-phosphorylate the ubiquitous phospholipid PI(4,5)P2 to yield PIP3 in the plasma membrane. This leads to the local accumulation of a variety of PIP3-sensor proteins, typically hosting PIP3-binding pleckstrin homology (PH) domains. These PIP3-sensors then propagate the PI3K signal via a multitude of mechanisms to regulate keystone effectors like Akt, mTOR and PRex proteins that shape cell behaviour on many levels. Loss of the tumour-suppressor and PIP3-3-phosphatase PTEN, leads to constitutive activation of the PIP3-network and is a very common event in many human tumours, including prostate cancer. Mouse models in which Pten and/or Class I PI3K signalling can be conditionally deleted in the prostate have been developed and used to show that unrestrained Class IA PI3K signalling can drive over growth of tissues and eventually cancer. The PI3K signalling pathway has been studied intensively for many years, however, due to a variety of intractable problems, the endogenous proximal upstream regulators and downstream effectors of Class IA PI3Ks are still largely unknown in either health or disease. To identify the potential interactors, that could include both activators and targets, of Class IA PI3Ks in healthy and Pten-/- mouse prostate tissue, we engineered mouse strains in which selected subunits of their Class IA PI3Ks could be specifically biotinylated in vivo (AviTag technology) and hence efficiently purified. Using this methodology, we have found that PLEKHS1 was specifically recovered with Class IA PI3Ks and that association was dramatically increased in Pten -/- prostate. To further investigate the function and interactions of PLEKHS1 in vivo, we generated two mouse models: a Plekhs1-null line (Plekhs1-/-) and a strain in which endogenous Plekhs1 was AviTagged(Plekhs1Avi/Avi). We also performed experiments with cells transfected with Plekhs1 constructs and with recombinant Plekhs1 in vitro to dissect its structure/functions. The transfection studies confirmed that PLEKHS1-PI3K interaction is dependent on PLEKHS1 phosphorylation and suggest PLEKHS1 can activate Class IA PI3K signalling. In vitro studies performed by Dr Tamara Chessa showed the isolated PH domain of PLEKHS1 interacted selectively with PI(3,4)P2 and PIP3 and suggest that PLEKHS1 can also function downstream of Class I PI3Ks. Using Plekhs1Avi/Avi mice we were able to efficiently recover PLEKHS1 from healthy and Pten -/- prostates and have identified a set of proteins that can interact selectively with PLEKHS1 in vivo. We observed increased interaction with Class IA PI3Ks, confirming the above results, and phosphorylation of PLEKHS1, in Pten -/- tissue. Importantly, the absence of PLEKHS1 substantially reversed the impact of loss of PTEN on the prostate weight, phosphorylation of Akt and levels of PIP3, with no or small effects on healthy prostate. Collectively, these data suggest that PLEKHS1is an unexpectedly important activator, and effector, of Class IA PI3K signalling in Pten -/- mouse prostate.
PTEN, Plekhs1, Prostate, PI3K, Cancer
This project has received funding from the European Union‘s Horizon 2020 research and Innovation programme under the Marie Skłodowska-Curie grant agreement No 675392.
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This record's DOI: https://doi.org/10.17863/CAM.63289
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