Access Type

Open Access Dissertation

Date of Award

January 2011

Degree Type


Degree Name




First Advisor

Steven Cala


Cardiac CSQ (CSQ2) is a multifaceted protein, capable of binding significant quantities of Ca2+ and altering ryanodine receptor activity at the junctional sarcoplasmic reticulum (SR). Little is known about the trafficking of CSQ2 from its unknown site of biosynthesis, which appears to be of importance as its structure changes in a trafficking-dependent manner in various types of heart failure. Through the use of multiple antibodies specific to classic rough ER markers, and with the creation of CSQ-DsRed tetramer fusion protein, we were able to establish a juxtanuclear localization of rough ER in cardiomyocytes. Using fluorescence confocal microscopy, the translocon complex proteins TRAP-α and TRAM, along with the ribosomal protein S6, were all visualized and found to encapsulate both myonuclei. Additionally, time course studies of CSQ-DsRed, in conjunction with anti-DsRed immunostaining, highlighted a perinuclear rough ER site of biosynthesis for CSQ2. The fusion protein exhibited a tetramerization-dependent trafficking predicted to be very similar to CSQ2 polymerization-dependent trafficking with high and low secretory compartment Ca2+ concentrations leading to polymerization and monomerization, respectively.

Cardiac-specific C-terminal phosphorylation of CSQ2 serines was shown to effect CSQ2 trafficking according to mass spectrometric analysis of the protein's N-linked glycan. A Ser 378,382,386 Ala mutation (CSQ-nonPP) of these sites inhibited CSQ2 phosphorylation and led to an increased trafficking out of the ER as indicated by the increased mannose trimming of CSQ2 glycan. This effect was reversed with the phosphomimetic mutant Ser 378,382,386 Glu (CSQ-mimPP) which had glycoforms with mannose trimming nearly identical to that of CSQ-WT.

Pharmacological and molecular inhibition were used to establish the identity of CSQ2 kinase as protein kinase CK2. In vitro, inhibition of CSQ2 phosphorylation using cardiomyocyte and nonmuscle cell CSQ2 kinase sources was successfully carried out with three specific inhibitors of CK2: TBB, DMAT and TBCA. TBCA produced identical inhibition curves for both cellular kinase sources and commercial CK2α′ kinase, with increasing concentrations of drug. SiRNA knockdown of both CK2α and CK2α′ catalytic subunits in nonmuscle cells reduced CSQ2 kinase activity by nearly 2-fold according to similar in vitro CSQ2 phosphorylation assays.

Additionally, both ATP-binding site competitive inhibition and CK2 RNAi protein inhibition were used successfully in situ to suppress endogenous CSQ2 phosphorylation. TBCA used at 100 µM with CSQ-WT overexpressing cardiomyocytes and COS nonmuscle cells in culture led to an 82 and 66% decrease in CSQ2 phosphate incorporation, respectively, compared to control. Similarly, simultaneous knockdown of both CK2α and CK2α′ catalytic subunits in COS cells overexpressing adenoviral CSQ-WT, caused a near 2-fold decrease in CSQ2 phosphate incorporation, further supporting the identity of CSQ2 kinase as protein kinase CK2.

These studies present a global model for CSQ2 trafficking regulation in cardiomyocytes. As CSQ2 is translated at perinuclear rough ER, its C-terminus is exposed to the cytosolic protein kinase CK2 allowing for CSQ2 phosphorylation prior to translocation into the ER lumen. A combination of dephosphorylation and monomerization then promotes anterograde trafficking through the secretory system. CSQ2 subsequently becomes retained in junctional SR compartments due to increasing Ca2+ concentrations, which led to polymer formation. Following a decrease in Ca2+ levels, CSQ2 monomerizes to once again traffic anterogradely through the secretory system.