The cytosolic protein kinase CK2 phosphorylates cardiac calsequestrin in intact cells

Abstract

The luminal SR protein CSQ2 contains phosphate on roughly half of the serines found in its C-terminus. The sequence around phosphorylation sites in CSQ2 suggest that the in vivo kinase is protein kinase CK2, even though this enzyme is thought to be present only in the cytoplasm and nucleus. To test whether CSQ2 kinase is CK2, we combined approaches that reduced CK2 activity and CSQ2 phosphorylation in intact cells. Tetrabromocinnamic acid, a specific inhibitor of CK2, inhibited both the CSQ2 kinase and CK2 in parallel across a range of concentrations. In intact primary adult rat cardiomyocytes and COS cells, 24 h of drug treatment reduced phosphorylation of overexpressed CSQ2 by 75%. Down-regulation of CK2α subunits in COS cells using siRNA, produced a 90% decrease in CK2α protein levels, and CK2-silenced COS cells exhibited a twofold reduction in CSQ2 kinase activity. Phosphorylation of CSQ2 overexpressed in CK2-silenced cells was also reduced by a factor of two. These data suggested that CSQ2 in intact cells is phosphorylated by CK2, a cytosolic kinase. When phosphorylation site mutants were analyzed in COS cells, the characteristic rough endoplasmic reticulum form of the CSQ2 glycan (GlcNAc2Man9,8) underwent phosphorylation site dependent processing such that CSQ2-nonPP (Ser to Ala mutant) and CSQ2-mimPP (Ser to Glu mutant) produced apparent lower and greater levels of ER retention, respectively. Taken together, these data suggest CK2 can phosphorylate CSQ2 co-translationally at biosynthetic sites in rough ER, a process that may result in changes in its subsequent trafficking through the secretory pathway.

Basal and β-adrenergic regulation of the cardiac calcium channel Ca V 1.2 requires phosphorylation of serine 1700

“…One plausible reason for the difference in results is the possibility that exogenously expressed Ca V 1.2 channels do not achieve the correct subcellular location or the correct protein composition and stoichiometry of the Ca V 1.2 signaling complex. T1704 is predicted to be a substrate for casein kinase II (13,27), which is constitutively active in cardiac myocytes (34,35). Thus, basal phosphorylation of this site may be high and not strongly regulated by Iso.…”

Transitions of protein traffic from cardiac ER to junctional SR

“…The results of these studies serve as a basis for understanding the cellular physiology that accompanies co-translational and posttranslational features of jSR proteins. Biochemical changes that occur within early subdomains of the cardiac secretory pathway include: trimming of the CSQ2 glycan in heart to very low levels of mannose (Man1-5) [34], replacement of CSQ2 in normal heart tissue with less trimmed CSQ2 glycoproteins in failed canine tissue [35, 36], regulation by TRD glycosylation of its breakdown by the proteasome [37], and phosphorylation of CSQ2 by protein kinase CK2 which is thought to occur in rough ER [23, 38]. …”

Calsequestrin Accumulation in Rough Endoplasmic Reticulum Promotes Perinuclear Ca2+ Release

“…The second possible mechanism is that the ER form of CSQ2 in the heart is more fully phosphorylated, whereas junctional SR forms of CSQ2 are relatively dephosphorylated (38). The result of increased level of CSQ2 phosphorylation is thought to be increased rough ER retention (15,39). This mechanism is predicted to underlie increased levels of perinuclear CSQ2 in tissue from tachycardia-induced canine heart failure (16).…”