Calcium/Calmodulin-dependent protein Kinase II (CaMKII) and Protein Kinase A (PKA) are both pivotal signal transducers in the heart. Over the last decade, it became clear that CaMKII and PKA are ‘opposing’ kinases. CaMKII mostly mediates maladaptive signaling, whereas PKA is mostly implicated in the maintenance of cardiac function. It is, however, currently unknown how during sustained β-adrenergic stress the balance shifts from (adaptive) PKA-signaling towards (maladaptive) CaMKII-signaling. PKA can be activated by binding of cAMP to its regulatory subunits, but also by reactive oxygen species (ROS)-dependent dimerization of the R1α regulatory subunits, both of which release PKA’s catalytic subunits. In effect, this activates PKA and allows it to phosphorylate its targets.  Using a SILAC-mass spectrometry screen of wildtype vs. cardiomyocyte-specific CaMKIIγ/δ double knockout (DKO) mouse hearts, we have identified Serine 77 of the regulatory subunit of PKA, R1α, as a yet unrecognized phosphorylation target of CaMKII. Subsequently, we could validate R1α as a direct phosphorylation target in CaMKII kinase assays. We further investigated R1α phosphorylation at Serine 77, and found that R1α phosphorylation was almost completely lost in the hearts of CaMKIIγ/δ DKO mice. Adenoviral overexpression of constitutively active CaMKII in neonatal rat cardiomyocytes increases R1α Serine 77 phosphorylation, diminishes R1α dimerization, and subsequently ROS-dependent activation of PKA. This would mean that decreased R1α Serine 77 phosphorylation corresponds with an increase in PKA activity. Indeed, we found PKA-dependent phosphorylation of PLN at S16 markedly increased in CaMKIIγ/δ DKO mice, indicative of increased PKA activity. To be able to investigate the in vivo importance of R1α Serine 77 phosphorylation, we used CRISPR/Cas9 to knock in a point mutation in R1α, which changes Serine 77 to a phospho-resistant Alanine. We have confirmed correct targeting using restriction digestion and Sanger sequencing and are currently breeding these R1α-S77A mice for homozygosity and cardiac phenotyping at baseline and after pressure overload of the heart.