Investigation into AMPK phosphatases and identification and characterisation of the interaction between AMPK and PAK1

Abstract

Abstract AMP-activated protein kinase (AMPK) plays important roles in the regulation of energy homeostasis and has been implicated in a number of diseases including diabetes and cancer. Obtaining a full picture of AMPK signalling is important for the understanding of these disease states and the development of novel therapies. AMPK is activated by phosphorylation of T-172 within the α catalytic subunit. While a number of AMPK upstream kinases have been identified, identification of the AMPK phosphatase has proved elusive. In the first part of this thesis, the identity of the AMPK phosphatase(s) was investigated. The data presented shows that treatment of mammalian cells with the protein phosphatase (PPP) family inhibitor Calyculin-A caused marked increases in AMPK activity. These results indicate that one or more of the AMPK phosphatases belong to the PPP family, with the concentration profile pointing towards PP1 involvement. However, using siRNA based techniques; it was not possible to pinpoint the specific phosphatase(s) that dephosphorylates AMPK. Our results, combined with recently published studies, suggest that regulation of AMPK dephosphorylation is a complex process with involvement of different protein phosphatases depending on cell-type and/or conditions of cell stress. Next, quantitative mass spectrometry techniques were utilised in order to identify AMPK interacting proteins. From the list of candidate interactors, the interaction between AMPK and the PAK1 signalling complex was characterised. AMPK directly phosphorylated PAK1 on Ser-21 leading to its activation. Activation of AMPK in mammalian cells led to increased activity and phosphorylation of endogenous PAK1. PAK1 activity/phosphorylation was reduced in cortical neurons derived from AMPK α1 knockout mice compared to wild-type neurons. Furthermore, activation of AMPK with A769662 in melanoma cell-lines significantly increased cell invasion and this effect was abolished by the PAK1 inhibitor, IPA3. These results show that PAK1 is an AMPK substrate and suggest that at least part of the effects of AMPK on axonogenesis and tumour formation is mediated through PAK1 signalling. In addition to the effects of AMPK on PAK1, PAK1 was shown to directly phosphorylate the AMPK α1 subunit at Thr-377 and Ser-403/405. This phosphorylation was able to further increase the activity of CaMKKβ phosphorylated AMPK. This finding adds a novel layer of control to the regulation of AMPK.