cAMP signaling regulates histone H3 phosphorylation and mitotic entry through a disruption of G2 progression.

cAMP signaling is known to have significant effects on cell growth, either inhibitory or stimulatory depending on the cell type. Study of cAMP-induced growth inhibition in mammalian somatic cells has focused mainly on the combined role of protein kinase A (PKA) and mitogen-activated protein (MAP) kinases in regulation of progression through the G1 phase of the cell cycle. Here we show that cAMP signaling regulates histone H3 phosphorylation in a cell cycle-dependent fashion, increasing it in quiescent cells but dramatically reducing it in cycling cells. The latter is due to a rapid and dramatic loss of mitotic histone H3 phosphorylation caused by a disruption in G2 progression, as evidenced by the inhibition of mitotic entry and decreased activity of the CyclinB/Cdk1 kinase. The inhibition of G2 progression induced through cAMP signaling is dependent on expression of the catalytic subunit of PKA and is highly sensitive to intracellular cAMP concentration. The mechanism by which G2 progression is inhibited is independent of both DNA damage and MAP kinase signaling. Our results suggest that cAMP signaling activates a G2 checkpoint by a unique mechanism and provide new insight into normal cellular regulation of G2 progression.

Distinct phosphorylation sites on the ß(2)-adrenergic receptor establish a barcode that encodes differential functions of ß-arrestin.

Phosphorylation of G protein-coupled receptors (GPCRs, which are also known as seven-transmembrane spanning receptors) by GPCR kinases (GRKs) plays essential roles in the regulation of receptor function by promoting interactions of the receptors with ß-arrestins. These multifunctional adaptor proteins desensitize GPCRs, by reducing receptor coupling to G proteins and facilitating receptor internalization, and mediate GPCR signaling through ß-arrestin-specific pathways. Detailed mapping of the phosphorylation sites on GPCRs targeted by individual GRKs and an understanding of how these sites regulate the specific functional consequences of ß-arrestin engagement may aid in the discovery of therapeutic agents targeting individual ß-arrestin functions. The ß(2)-adrenergic receptor (ß(2)AR) has many serine and threonine residues in the carboxyl-terminal tail and the intracellular loops, which are potential sites of phosphorylation. We monitored the phosphorylation of the ß(2)AR at specific sites upon stimulation with an agonist that promotes signaling by both G protein-mediated and ß-arrestin-mediated pathways or with a biased ligand that promotes signaling only through ß-arrestin-mediated events in the presence of the full complement of GRKs or when either GRK2 or GRK6 was depleted. We correlated the specific and distinct patterns of receptor phosphorylation by individual GRKs with the functions of ß-arrestins and propose that the distinct phosphorylation patterns established by different GRKs establish a "barcode" that imparts distinct conformations to the recruited ß-arrestin, thus regulating its functional activities.