Biochemical investigation of phosphodiesterase type IV post-translational modification, cellular localisation and interaction with associated binding proteins.
PhD thesis, University of Glasgow.
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cAMP is a secondary messenger that is involved in a variety of signalling pathways through its effectors including EPAC, PKA and ion channels. cAMP signalling regulates processes such as memory, muscle contraction and inflammatory responses. PDE enzymes offer a mechanism to negatively regulate elevated cAMP levels elicited by activators of adenylyl cyclase. Studies have shown that cAMP signalling is compartmentalised through binding of PDEs to A-kinase anchoring proteins (AKAPs) that scaffold PKA regulatory subunits.
In this study post-translational-modification of PDE4 isoforms is investigated. SUMOylation is a relatively newly identified post-translational modification that is known to regulate the structure and function of its substrates. PDE4 isoforms of the PDE4A and 4D subfamilies are SUMOylated by an E3 ligase, PIASy. SUMOylation alters the rolipram sensitivity and potentiates the PKA mediated activation of the isoforms whilst it confers protection from ERK-mediated inhibition of PDE4 activity. SUMOylation alters the association of PDE4 isoforms with binding partners like β-Arrestin, AKAP18 δ and UBC9.
Rolipram is an archetypal PDE4 specific inhibitor. In this study it is shown that in cells expressing a GFP tagged form of PDE4A4 undergoes redistribution into accretion foci upon chronic treatment with rolipram. Data suggests that foci formation requires protein turnover and is regulated by signalling pathways such as PI3 kinase pathway, p38 MAP kinase pathway and PKC pathways. Further, the Immunomodulatory drug Thalidomide® also inhibits foci formation.
PDE4 isoforms have isoforms specific N-terminal regions, which play a crucial role in sub-cellular localisation and protein-protein interactions. It is shown here that PDE4D5 interacts with a novel RhoGAP called ARHGAP21 which has been previously reported to bind β-arrestins. This interaction is independent of GAP activity of ARHGAP as well as PDE4 activity. Previous reports have indicated a role of β-Arrestin, PDE4 and ARHGAP21 in regulation of actin cytoskeleton dynamics. Hence complex β-Arrestin-PDE4-ARHGAP21 may play a crucial role in regulating actin dynamics.
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