The Role of PRAJA2 in TSH- or Isoproterenol- Stimulated Lipolysis in Human Adipocytes

Abstract

Thyrotropin (TSH) binds to TSH receptors on thyrocytes to regulate development and growth of the thyroid gland, and to stimulate thyroid hormone production. Thyrotropin has also been shown to act in an extra-thyroidal fashion, and to engage TSH receptors on adipocytes to induce lipolysis, similar to the response seen by stimulation with β-adrenergic receptor agonists (i.e. isoproterenol). In both cell types, cAMP-dependent kinase (PKA) is activated. Recently, PRAJA2, a novel AKAP and E3 ubiquitin ligase that targets the regulatory subunits of PKA was identified. The ubiquitin-dependent proteasomal degradation of the PKA regulatory subunits, due to PKA- phosphorylated PRAJA2, prolongs the catalytic activity of PKA, as shown in neuroblastoma cells by Lignitto et al., 2011. In adipocytes, stimulated PKA activity is required for lipolysis. Additionally, PRAJA2 has been described to have increased expression in TSH-responsive, differentiated thyroid cancer cells when compared to anaplastic thyroid tumor (Cantara et al., 2012) The aim of this study was to characterize PRAJA2 and its potential influence on adipocyte lipolysis. These data confirm that TSH and isoproterenol stimulate lipolysis in primary human differentiated adipocytes. PRAJA2 is expressed at the mRNA and protein level in differentiated adipocytes, with no change following stimulation with TSH or isoproterenol. Stimulation with isoproterenol, but not TSH, increases PKA-dependent phosphorylation of a 122kDa (potentially PRAJA2) and 69kDa protein identified in PRAJA2 immunoprecipitates. These proteins may prove important for lipolytic signaling or other PRAJA2-dependent process in adipocytes. Experimentation was unable to identify interactions between PRAJA2 and PKAR2 in differentiated adipocytes; however further investigations are required before discounting this interaction. An attempt was made to knockdown PRAJA2 in this model, and measure effects on lipolytic response; however, this was unsuccessful. Taken together, PRAJA2 appears to be phosphorylated following β-adrenergic stimulation in human adipocytes; however, further studies are needed to delineate the specific role of PRAJA2 in this human differentiated adipocyte model.