Ser-2030, but not Ser-2808, is the major phosphorylation site in cardiac ryanodine receptors responding to protein kinase A activation upon β-adrenergic stimulation in normal and failing hearts

Abstract

We have recently shown that RyR2 (cardiac ryanodine receptor) is phosphorylated by PKA (protein kinase A/cAMP-dependent protein kinase) at two major sites, Ser-2030 and Ser-2808. In the present study, we examined the properties and physiological relevance of phosphorylation of these two sites. Using site- and phospho-specific antibodies, we demonstrated that Ser-2030 of both recombinant and native RyR2 from a number of species was phosphorylated by PKA, indicating that Ser-2030 is a highly conserved PKA site. Furthermore, we found that the phosphorylation of Ser-2030 responded to isoproterenol (isoprenaline) stimulation in rat cardiac myocytes in a concentration- and time-dependent manner, whereas Ser-2808 was already substantially phosphorylated before beta-adrenergic stimulation, and the extent of the increase in Ser-2808 phosphorylation after beta-adrenergic stimulation was much less than that for Ser-2030. Interestingly, the isoproterenol-induced phosphorylation of Ser-2030, but not of Ser-2808, was markedly inhibited by PKI, a specific inhibitor of PKA. The basal phosphorylation of Ser-2808 was also insensitive to PKA inhibition. Moreover, Ser-2808, but not Ser-2030, was stoichiometrically phosphorylated by PKG (protein kinase G). In addition, we found no significant phosphorylation of RyR2 at the Ser-2030 PKA site in failing rat hearts. Importantly, isoproterenol stimulation markedly increased the phosphorylation of Ser-2030, but not of Ser-2808, in failing rat hearts. Taken together, these observations indicate that Ser-2030, but not Ser-2808, is the major PKA phosphorylation site in RyR2 responding to PKA activation upon beta-adrenergic stimulation in both normal and failing hearts, and that RyR2 is not hyperphosphorylated by PKA in heart failure. Our results also suggest that phosphorylation of RyR2 at Ser-2030 may be an important event associated with altered Ca2+ handling and cardiac arrhythmia that is commonly observed in heart failure upon beta-adrenergic stimulation.

Cardiac ryanodine receptor phosphorylation: target sites and functional consequences

“…In the latest issue of the Biochemical Journal, Xiao et al [1] provide further compelling evidence that Ser-2030 in RyR2 in mouse, rat, rabbit and human can be phosphorylated by PKA. They show that Ser-2030 phosphorylation is extremely low before PKA activation, but increases rapidly upon PKA activation (and Ser-2030 is a poor substrate for CaMKII or PKG).…”

“…So, even if they had somehow failed to detect phosphorylation, there was still no detectable channel activation. Nevertheless, the compelling data from Xiao et al [1,17] makes it inappropriate to dismiss the possibility that PKA-dependent RyR2 phosphorylation can occur at Ser-2030. If these laboratory groups swapped reagents and conditions, perhaps these differences could be reconciled.…”

“…As attractive as this working hypothesis is, the attention it has received and its extensive testing by this group, it remains controversial. This is because numerous laboratory groups have found results that do not support different aspects of this hypothesis [1,[13][14][15][16][17]. These include findings that RyR2 is not hyperphosphorylated by PKA in HF, that PKA-dependent phosphorylation does not cause FKBP12.6 release, that PKA activation does not increase basal RyR2 activation, that there are additional PKA sites on RyR2 and that Ser-2809 can also be phosphorylated by CaMKII or PKG (protein kinase G).…”

“…Xiao et al [1] show in this issue of the Biochemical Journal that PKA (protein kinase A)-dependent phosphorylation of the cardiac ryanodine receptor (RyR2) can occur at Ser-2030, and that this is dynamically regulated by β-adrenergic agonists in isolated myocytes. During normal cardiac EC (excitation-contraction) coupling a relatively small amount of Ca 2+ enters the cell via the Ca 2+ current (I Ca ) and this triggers the release of a larger amount of Ca 2+ from the SR (sarcoplasmic reticulum) via RyR2 in a process called Ca 2+ -induced Ca 2+ release [2].…”

Effects of therapy using the Celacade system on structural and functional cardiac remodelling in rats following myocardial infarction

“…The molecular mechanisms behind this reduction of diastolic cardiac muscle fluctuation were not revealed by the present study. Considering the important role of RyR function in SL fluctuation, the effect of Celacade on SL fluctuation in MI trabeculas might involve a regulatory step of the RyR functional pathway, possibly including RyR phosphorylation by either protein kinase A or Ca 2+ -calmodulin-dependent protein kinase II (2,(15)(16)(17), or alteration of stoichiometry of the FK506 binding protein 12.6 to the channel (18), and increased oxidative stress in the MI heart (19).…”