Cotranslational cis -phosphorylation of the COOH-terminal tail is a key priming step in the maturation of cAMP-dependent protein kinase

Abstract

cAMP-dependent protein kinase A (PKA), ubiquitously expressed in mammalian cells, regulates a plethora of cellular processes through its ability to phosphorylate many protein substrates, including transcription factors, ion channels, apoptotic proteins, transporters, and metabolic enzymes. The PKA catalytic subunit has two phosphorylation sites, a well-studied site in the activation loop (Thr 197 ) and another site in the C-terminal tail (Ser 338 ) for which the role of phosphorylation is unknown. We show here, using in vitro studies and experiments with S49 lymphoma cells, that cis-autophosphorylation of Ser 338 occurs cotranslationally, when PKA is associated with ribosomes and precedes posttranslational phosphorylation of the activation loop Thr 197 . Ser 338 phoshorylation is not required for PKA activity or formation of the holoenzyme complex; however, it is critical for processing and maturation of PKA, and it is a prerequisite for phosphorylation of Thr 197 . After Thr 197 and Ser 338 are phosphorylated, both sites are remarkably resistant to phosphatases. Phosphatase resistance of the activation loop, a unique feature of both PKA and PKG, reflects the distinct way that signal transduction dynamics are controlled by cyclic nucleotide-dependent PKs.

Mechanisms of cyclic AMP/protein kinase A- and glucocorticoid-mediated apoptosis using S49 lymphoma cells as a model system

“…PKA-Cα is phosphorylated at both Ser 338 and Thr 197 in WT cells, but neither site is phosphorylated in kin -cells ( Fig. 2A), suggesting that both its cotranslational and posttranslational processing steps are compromised in kin -cells, results consistent with earlier reports (17,20). Imaging of PKA-Cα by immunofluorescence microscopy of WT and kin -cells provided complementary results: PKA-Cα localizes to perinuclear (likely Golgi) regions in WT cells (Fig.…”

“…As shown in Fig. 1A, PKA-Cα is soluble and phosphorylated in WT and Cos7 cells but insoluble and not phosphorylated in kin -cells, consistent with earlier results (19,20). We also detected two PKA-C isoforms based on the phosphoantibody blots, both of whose isoforms are absent in kin - (Fig.…”

“…1 and 2) (17). The lack of soluble PKA-Cα protein derives from a loss in Ser 338 phosphorylation, which is essential for maturation of PKA-Cα and phosphorylation of Thr 197 (20). Cellular expression of PKA-RIα depends in part on the ubiquitin ligase Praja (32).…”

“…In kincells, expression of the mRNA transcript of the PKA-Cα subunit is normal, but there is no soluble PKA-Cα protein (17)(18)(19). We have previously shown that cis-autophosphorylation of Ser 338 ribosome-associated PKA-Cα occurs cotranslationally, but posttranslational phosphorylation of the activation loop Thr 197 is mediated in trans; absence of phosphorylation of Ser 338 leads to the accumulation of insoluble, inactive PKA-Cα (20). Ser 338 is thus critical for processing and maturation of PKA-Cα and is a prerequisite for phosphorylation of Thr 197 (20).…”

“…We have previously shown that cis-autophosphorylation of Ser 338 ribosome-associated PKA-Cα occurs cotranslationally, but posttranslational phosphorylation of the activation loop Thr 197 is mediated in trans; absence of phosphorylation of Ser 338 leads to the accumulation of insoluble, inactive PKA-Cα (20). Ser 338 is thus critical for processing and maturation of PKA-Cα and is a prerequisite for phosphorylation of Thr 197 (20). However, the precise mechanism by which kin -cells are able to escape cAMP/PKA-mediated apoptosis is not known, and understanding is limited regarding the mechanisms that may link cAMP-and glucocorticoid-promoted cell killing (21,22).…”