Angiotensin II signalling kicks out p27Kip1: casein kinase 2 augmentation of Cav1.2 L‐type Ca2+ channel activity in immature ventricular cardiomyocytes

Ca2+ influx via the L-type Ca2+ channel Cav1.2 triggers our heartbeat. Perinatally and particularly at birth, angiotensin II is produced to augment necessary physiological changes in blood flow such as pulmonary perfusion. In this issue of The Journal of Physiology Kashihara et al. now report that angiotensin II increases Cav1.2 channel activity via a novel pathway specific to immature but not adult ventricular cardiomyocytes (Kashihara et al. 2017). Using pharmacological and molecular approaches that elegantly complement each other, the authors convincingly show that angiotensin II regulates Cav1.2 via the AT1 receptor and its binding partner β-arrestin2 rather than the conventional AT1 receptor coupled Gq/11 signalling. β-Arrestin2 then stimulates a Src-family tyrosine kinase (SFK), which in turn phosphorylates the protein p27Kip1 (p27) on Tyr88. This phosphorylation prevents p27 from inhibiting the otherwise constitutively active casein kinase 2 (CK2). CK2 phosphorylates Thr1704 in the proximal half of the about 660 residue long C-terminus of Cav1.2. Nearby Arg1696 and Arg1697 electrostatically interact with Glu2103, Glu2106, and Asp2110 in the distal C-terminus, thereby reducing channel activity of Cav1.2 (Hulme et al. 2006). As the authors hypothesize, it thus appears likely that phosphorylation of Thr1704 acts by decreasing the interaction of the distal with the proximal C-terminus, thereby reducing this autoinhibitory interaction within Cav1.2. The role of p27 in this context is of particular interest. Originally identified as a cyclin-dependent kinase inhibitor (CDKi), p27 binds to cyclin–CDK2 subunit complexes. It inserts its 310-helix into the ATP binding pocket of the kinase and inhibits CDK2 activity, thereby blocking progression of mitotic cells through the G1-phase of the cell cycle (Russo et al. 1996). Thus, upregulation of p27 has been mainly associated with cell cycle inhibition and growth arrest of proliferating cells. Its function in tissues primarily composed of post-mitotic cells like the heart, where its expression is developmentally upregulated, has remained enigmatic and elusive. The report by Kashihara et al. now links p27 to regulation of Cav1.2 in cardiomyocytes (Kashihara et al. 2017). Unlike CDKs, which bind positive regulatory cyclin subunits required for kinase activity, catalytic CK2α/α’ subunits associate with β subunits that confer substrate specificity and target the CK2 holoenzyme to selected proteins (Montenarh & Goetz, 2013). Importantly, Kashihara et al. determined that CK2β binds to Cav1.2 and recruits p27 and CK2α’ to the Cav1.2 complex (Kashihara et al. 2017). Here the binding of p27 to both the CK2α’ and β subunits and blockage of the activation of the CK2 holoenzyme is akin to its regulation of cyclin–CDK2 complexes (Russo et al. 1996). Earlier work showed that CK2 itself can phosphorylate p27 (Montenarh & Goetz, 2013). Although this CK2-mediated modification apparently altered the secondary structure of p27, the biological significance of these findings was at the time unclear. Later work indicated that AngII induces the proteasomal degradation of p27 through CK2α’-dependent phosphorylation of p27 on Ser83 and Thr187 (Hauck et al. 2008). However, these studies did not resolve what leads to CK2α’ activation. Kashihara et al. take these earlier observations several steps further (Kashihara et al. 2017). Their findings indicate that AngII signalling activates an SFK that phosphorylates Tyr88 of p27, reducing its CK2α’β inhibitory effect. This suggests that AT1 receptor signalling triggers CK2α’β activity towards the channel via SFK-mediated alteration of the p27 interaction with CK2α’β. Indeed, earlier studies have shown that the non-receptor tyrosine kinases Lyn, Abl, and Src regulate p27 stability via phosphorylation of Tyr74 and Tyr 88 (reviewed in Kaldis, 2007). Phosphorylation of Tyr88 on p27 hinders insertion of its 310-helix into the ATP binding site of CDK2 and thereby impairs the CDK2 inhibitory action of p27 10-fold (Kaldis, 2007). Although pTyr88-p27 initially remains bound to cyclin–CDK2 complexes, the intermolecular interaction with both cyclin and CDK2 subunits facilitates CDK2-mediated phosphorylation of p27 on Thr187, which in turn promotes ubiquitin-mediated degradation of p27 (Kaldis, 2007). Angiotensin II stimulates CK2α’ phosphorylation of p27 on Ser83 and Thr187 and thereby antagonizes p27 binding to and inhibition of CK2α' (Hauck et al. 2008). This positive feedback mechanism is analogous to the above feedback loop arising from CDK2 interaction with pTyr88–p27. Thus regulation of p27 stability is not restricted to mitotic cells and the promotion of growth through cellular proliferation. Rather p27–CK2α’ interaction in differentiated but immature cardiomyocytes is regulated by AngII and CK2-mediated inactivation of p27 is clearly critical for cardiac hypertrophic growth in the neonate. Importantly, expression of CK2α’ subunits decreases during the postnatal period during which p27 expression in cardiomyocytes is known to increase. The findings of Kashihara et al. now indicate that the positive inotropic effect that AngII stimulated AT1 receptors has on immature heart results from β-arrestin2 and CK2α’β activation of Cav1.2 channels in the absence of high p27 levels and points towards the importance of the balance of p27 levels relative to CK2α’ expression and the pre-assembly of a p27–CK2–Cav1.2 complex (Kashihara et al. 2017). One open question is how β-arrestin2 induces Src activation. Part of this mechanism could be to move Src close to a signalling complex that could be formed between the AT1 receptor and Cav1.2, which would be analogous to the β2 adrenergic receptor–Gs–adenylyl cyclase–PKA–Cav1.2 complex (Patriarchi et al. 2016). In fact, β2 adrenergic receptor and AT1 receptor signalling might very well intersect because CK2 typically phosphorylates serine and threonine residues near a negatively charged residue, which is created by PKA-mediated phosphorylation of Ser1700 during β2 adrenergic receptor signalling (Patriarchi et al. 2016). Perhaps requirement for the activation of two different signalling cascades could explain the slow time course of the AT1 receptor–Thr1704 signalling, which requires several hours for optimal phosphorylation. None declared.