Regulation of Phosphorylation of PPARγ by CDK5 in Obesity

Abstract

PPAR? is the receptor for thiazolidinedione (TZD) anti-diabetes drugs, and a key regulator of adipose differentiation and insulin sensitivity. Increased heart failure associated with specific TZDs has reduced their clinical use. However recent work suggested that phosphorylation of PPAR? at Ser273 modified its response to TZDs, shifting the risk-benefit ratio of these drugs. Cyclin dependent kinase-5 (CDK5) which requires a non-cyclin partner (p35) for activity was proposed to phosphorylate Ser273, while obesity promoted cleavage of p35 to p25 thereby enhancing CDK5 phosphorylation of PPAR? (Choi et al 2010). However the sequence surrounding Ser273 is not a CDK5 consensus, therefore I have analysed PPAR? phosphorylation more carefully. I mutated Ser273 on PPAR? to alanine and compared phosphorylation of mutant and wild-type by multiple protein kinases. I raised a phosphospecific antibody to Ser273 of PPAR? and used this to monitor phosphorylation of PPAR? isoforms. P35-CDK5 and p25-CDK5 phosphorylate wild-type and S273A PPAR? to similar levels in vitro. The phosphospecific Ser273 antibody did not detect phosphorylation of endogenous PPAR? in adipose tissue of rats (with or without obesity). I could not detect phosphorylation of Ser273 on recombinant PPAR? when over-expressed in HEK293 or 3t3-L1 cells (± differentiation). Finally, phospho-Ser273 was not present on PPAR? immunoprecipitated from adipose tissue (obese and non-obese), or from cell lines, as assessed by phosphopeptide analysis or immunoblot. I have tried multiple approaches to validate that CDK5 phosphorylates Ser273 of PPAR? but our data questions the clinical potential of targeting this modification.

Date of Award	2014
Original language	English
Awarding Institution	University of Dundee
Sponsors	Diabetes UK
Supervisor	Calum Sutherland (Supervisor)