Comparison of the Effects of PRKAR1A and PRKAR2B Depletion on Signaling Pathways, Cell Growth, and Cell Cycle Control of Adrenocortical Cells

Abstract

The cyclic AMP/protein kinase A signaling cascade is one of the main pathways involved in the pathogenesis of adrenocortical tumors. The PKA R1A and R2B proteins are the most abundant regulatory subunits in endocrine tissues. Inactivating mutations of PRKAR1A are associated with Carney complex and a subset of sporadic tumors and the abundance of R2B protein is low in a subset of secreting adrenocortical adenomas. We previously showed that PRKAR1A and PRKAR2B inactivation have anti-apoptotic effects on the adrenocortical carcinoma cell line H295R. The aim of this study was to compare the effects of PRKAR1A and PRKAR2B depletion on cell proliferation, apoptosis, cell signaling pathways, and cell cycle regulation. We found that PRKAR2B depletion is compensated by an upregulation in the abundance of R1A protein, whereas PRKAR1A depletion has no effect on the production of R2B. The depletion of either PRKAR1A or PRKAR2B promotes the expression of Bcl-xL and resistance to apoptosis; and is associated with a high percentage of cells in S and G2 phase, activates PKA and MEK/ERK pathways, and impairs the expression of IkB leading to activate the NF-κB pathway. Nonetheless, we observed differences in the regulation of cyclins. The depletion of PRKAR1A leads to the accumulation of cyclin D1 and p27kip, whereas the depletion of PRKAR2B promotes the accumulation of cyclin A, B, cdk1, cdc2, and p21Cip. In conclusion, although the depletion of PRKAR1A and PRKAR2B in adrenocortical cells has similar effects on cell proliferation and apoptosis; loss of these PKA subunits differentially affects cyclin expression.

Hypoxia and the hypoxia inducible factor 1α activate protein kinase A by repressing RII beta subunit transcription

“…In contrast to many other protein kinases which are regulated by the turnover of an activation loop phosphate, PKA is regulated by the composition of its holoenzyme structure, which determines its activation potential by cAMP [57]. As such, alterations in the structure of the holoenzyme complex make it susceptible to deregulation of kinase activity [48,49]. As we found no significant alterations in intracellular cAMP levels, we propose that the suppression of PRKAR2B expression is the sole trigger of overactive PKA under these conditions.…”

“…PKA is the major kinase phosphorylating CREB [48,49]. Both HIF-1α overexpression and hypoxic incubation significantly increased PKA activity (Fig.…”

“…4c-e), demonstrating that hypoxia and consequently HIF-1α requires a catalytically active PKA to mediate its effects. PKA activation first occurs following an increase in intracellular cAMP concentrations [48,49]. However, neither HIF-1α inhibition with RNA interference nor hypoxic incubation affected basal and forskolin-induced intracellular cAMP levels (Fig.…”

“…PKA activity can be dysregulated as a result of altered subunit expression patterns activity [48,49]. As a transcriptional regulator, we therefore speculated that HIF-1α may induce basal PKA activity by altering the balance of the expression of the different PKA subunits.…”

Expression of protein kinase A regulatory subunits in benign and malignant human thyroid tissues: A systematic review

“…In addition, the depletion of R1A or R2B enhances the resistance to apoptosis and induces the expression of Bcl-xL. In addition, a loss in R1A and R2B subunit expression results in the accumulation of cyclin D1 and p27kip for R1A, and cyclins A, B, cdk1, cdc2, and p21Cip for R2B (14).…”