RICERCA ITALIANA     

Research program

Supramolecular complexes of sorcin in the generation and regulation of Calcium-dependent cellular functions

University Co-ordinator

Università degli Studi di PADOVA - CHIMICA BIOLOGICA - ()

Research Unit Leader

Maria Ruzzene

Description

The aim of the present research project is the investigation of the functional interactions between CK2 and sorcin, with special reference to the regulation of the cardiac ryanodine receptor Ryr2.
The major tasks for this Research Unit (R.U.) will be the following:

1. CHARACTERIZATION OF CK2/SORCIN INTERACTION
A. Characterization of CK2/sorcin interaction in vitro
In the laboratory of the applicant research group, sorcin has been found associated to the β regulatory subunit of CK2, by fishing cellular proteins on resin-immobilized CK2β (7). We plan to better characterize this association, first determining if only the free β subunit is able to bind sorcin, or the a subunit or CK2 holoenzyme are as well. Association could be related or not to the CK2 catalytic activity, depending on which subunit/domain will result to be involved in the interaction. The regions involved in the two proteins will be investigated exploiting mutants or deleted forms, already available in the laboratories, or designed on purpose, according to the first experimental data. We shall also consider the importance of Ca2+ in the regulation of the CK2/sorcin binding. It is worth to note that the interaction of sorcin with its other partner proteins requires a precise Ca2+ ion concentration, which promotes a sorcin conformational change thus exposing the hydrophobic regions involved in the protein/protein binding (16, 17). If also the CK2/sorcin association requires Ca2+, it will be physiologically relevant, considering that CK2, constitutively active, thus not responding to any cellular signaling, will be enclosed in a Ca2+ controlled pathway, not acting directly on its activity, but possibly regulating its association network. In this context, the evaluation of the catalytic activity of CK2 in the presence of sorcin will be particularly interesting, in the hypothesis that CK2 itself can be affected by the interaction.
B. Analysis of CK2/sorcin interaction in vivo
In cells expressing high levels of both CK2 and sorcin, by means of immunoprecipitation techniques, we shall investigate the in vivo association of the two proteins. If sorcin interacts with the only β subunit of CK2, it will be interesting to check if the association remove CK2β from the holoenzyme CK2 a2β2, allowing a pool of “free” a to act independently of β. This would depict a condition where, regardless the overexpression of CK2a, sorcin would ensure the presence of a not combined in the tetrameric form, thus altering the CK2 functions, considering that the physiological roles of CK2 a or a2β2 are probably distinct (1).
To investigate in more details the role of the different subunits of CK2 in the interaction/regulation of sorcin, we shall alter their expression in cells: to this purpose, we shall apply the RNA interference technique, by reducing with specific siRNA only one of the CK2 subunits, or we shall ectopically express only a or β subunit.
C. Analysis of the role of Ca2+ in the CK2/sorcin interaction in vivo
According to the results obtained from the in vitro analysis on this aspect, we shall investigate if Ca2+ alters the properties of the CK2/sorcin interaction in vivo. Experiments will be performed under conditions of cell stimulation inducing a [Ca2+] increase; on this regards, also the co/localization of the two proteins will be investigated, being Ca2+ known to promote sorcin translocation to the membranes. Moreover, we should take in mind that Ca2+ is an important signal in determining the cell choice between death and survival; it will be therefore evaluated in which measure sorcin and CK2, by means of their physical/function interactions, play their anti-apoptotic functions under different Ca2+ conditions. To these experiments, advantage will be taken by the use of already available (R.U. Colotti) sorcin mutants, which display an altered Ca2+ binding affinity (18).

2. ANALYSIS OF SORCIN PHOSPHORYLATION BY CK2
A. Analysis of sorcin phosphorylation by CK2 in vitro
The presence of consensus sites for CK2 (10) in the sorcin sequence allows to postulate that CK2 can also phosphorylate sorcin; in particular, Ser80 and Thr155 of human sequence are predicted sites for CK2 (and are conserved in other species). By means of in vitro enzymatic assays with radioactive ATP, the actual phosphorylation of sorcin by CK2 will be evaluated. Reaction will be biochemically characterized, determining the phosphorylation stoichiometry, kinetic parameters, and the optimal conditions, for example as for Ca2+ ions requirement. The possibility will be assessed that different CK2 isoforms (monomeric or tetrameric) display different phosphorylation efficiency towards sorcin. It has to be considered that CK2 is normally present in the cells as a tetrameric holoenzyme, constituted of two catalytic and two regulatory subunits, but the monomeric catalytic subunit is also active in vitro, and displays a substrate specificity in some cases distinct from that of the holoenzyme (1). This could be of great relevance in the MDR context, where both CK2 and sorcin are involved, but in the case of CK2 the only catalytic subunit has been found overexpressed in MDR cell lines (15), where, therefore, it should be present in vivo not associated to the regulatory subunits.
We shall then determine which aminoacids are phosphorylated in sorcin by CK2, trying to indentify the actual target sites, by means of mass spectrometry analysis of the protein phosphorylated in vitro. The exact identification of the phosphorylatyed sites could reveal particularly useful to design and produce phospho-specific antibodies, to be subsequently used for in vivo analysis of the sorcin CK2-dependent phosphorylation state.
B. Analysis of sorcin phosphorylation by CK2 in vivo
If the in vitro study gives positive indications on the phosphorylation of sorcin by CK2, this aspect will be investigated in vivo; we shall immunoprecipitate sorcin endogenously or ectopically expressed in cells, and check its phosphorylation degree, exploiting radioactive isotopic techniques, or phospho-specific antibodies, produced on this purpose on the basis of in vitro data. A great help in this analysis will come from the availability, in the applicant laboratory, of a wide panel of very specific and powerful CK2 inhibitors (6), which allow to quantify the CK2 contribution in the sorcin phosphorylation in vivo; in case, we shall then produce and express suitable mutants of the putative CK2 target sites. Cellular conditions will be varied, as far as the Ca2+ concentration is concerned, considering that Ca2+-induced sorcin translocation can possibly affect its phosphorylation as well.

3. EFFECTS OF CK2 ON SORCIN FUNCTIONS WITH SPECIAL REFERENCE TO ITS INTERACTION WITH Ryr2
First, in collaboration with R.U. Colotti, we shall evaluate if sorcin phosphorylation by CK2, and/or the interaction of the two proteins, cause a change in sorcin affinity for Ca2+, with obvious consequences in its ability to interact with other proteins. In fact, sorcin is known to form Ca2+-dependent complexes with different partners; among them, the ryanodine receptor, and in particular the cardiac isoform Ryr2 (18) is considered with special attention in this project. It is conceivable that CK2 controls this function of sorcin, either indirectly, by altering sorcin affinity for Ca2+, or, more directly, by sorcin association/phosphorylation. We therefore plan to analyze if CK2 participates to the sorcin/Ryr2 complex, or if the sorcin partners are mutually exclusive. The study will be performed in vitro, with recombinant proteins, and in vivo, by co-precipitation experiments from cell lysates. Even in the case CK2 is not found in the complex with sorcin and Ryr2, its involvement as a regulating factor is still possible, due to phosphorylation; we shall therefore evaluate if sorcin phosphorylated by CK2 displays a different affinity for Ryr2. The previous identification of the precise CK2 target sites will allow the generation of phospho-mimetic sorcin mutants, where Ser/Thr residues will be replaced by Asp. This strategy (in collaboration with R.U. Colotti) will allow to overcome a possible under-stoichiometric in vitro phosphorylation of sorcin, thus generating a homogenous molecule population, useful to better understand the functional role of sorcin phosphorylation.
Interesting data could come from the comparison of sorcin functionality under different experimental conditions, namely treatment of cells with specific siRNA (RNA interference technique) for reducing the CK2 amount (single of both subunits), or with specific CK2 inhibitors, for reducing its catalytic activity without altering its amount; if these two different approaches for interfering with CK2 action will give different effects on sorcin, a scenery could be depicted where not the activity, but the presence of CK2 as interacting protein, would be relevant in the modulation of sorcin functions.
As far as the role of CK2 in regulating the sorcin/Ryr2 complex formation is concerned, a further possibility will be taken into account, that CK2 phosphorylates thus directly regulates, Ryr2 itself. This part of the project will be detailed in the next paragraph.

4. ANALYSIS OF RyR2 PHOSPHORYLATION BY CK2
The cardiac isoform of the ryanodine receptor, Ryr2, presents several sites in its sequence, with an optimal prediction of phophorylation by CK2 (10). We therefore intend to perform a study, in close collaboration with R.U. Sorrentino, to investigate the actual phosphorylation of Ryr2 by CK2, and in case to characterize it from a biochemical and functional point of view. First experiments will be in vitro kinase assays, in the presence of radioactive ATP, with recombinant CK2 and microsomal fractions containing Ryr2, whose phosphorylation will be visualized after SDS-PAGE and western blot. Then, fragments of Ryr2, corresponding to different regions of the whole receptor, will be expressed as recombinant proteins by R.U. Sorrentino, and used as potential substrates for CK2 in vitro. This approach will be useful to have a first idea on the actual location of CK2 sites in Ryr2, then this aspect will be further deepened, by means of mass spectrometry analysis, for the exact identification of the target sites. In case of positive results, we plan to produce phospho-specific antibodies, to confirm also in vivo the occurrence of these CK2-dependent phosphorylation sites.
In order to disclose a possible regulatory function of CK2 on Ryr2 activity, in collaboration with R.U. Sorrentino we plan to use a panel of different specific CK2 inhibitors (6) for treatment of cultured cardiomyocites, where to measure the Ca2+ release properties of Ryr2 in comparison with not-treated cells.
In the context of Ryr2 phosphorylation, special attention will be given to the connection between CK2-dependent phosphorylation and other phosphorylations already known for Ryr2, catalyzed by different kinases and related to pathological conditions. In fact, hyperphosphorylation of Ryr2 seems to be associated to the pathogenesis of heart failure, and protein kinase A (PKA) and Ca-Calmodulin dependent kinase II (CaM KII) are the two enzymes known so far as involved in this process; however, the phosphorylation sites identified as the targets of PKA and CaMKII (Ser2808 and Ser2815, respectively) are probably not exclusive (for examples, also Ser2030 has been reported as site for PKA), and also their relevance when hyperphosphorylated under pathological conditions is still debated (11, 12, 19). We can imagine, therefore, that a coordinated action of more than one enzyme should occur to produce a final effect; in this project the possibility will be considered that also CK2 is involved. We shall perform experiments of phosphorylation by CK2 of Ryr2 previously phosphorylated by other kinases: in vitro, we shall use PKA and CaMKII (commercially available as active enzymes); in vivo, we shall exploit β-adrenergic stimulation of cardiomyocites and we shall evaluate if significant changes in Ryr2 activity are induced, under these conditions, by pharmacological inhibition of CK2.
As described so far, the study will be mainly performed on the cardiac isoform Ryr2, but in case of interesting results, and given the high homology of other members of the Ryr family, we shall later evaluate if other Ryr isoforms are phosphorylated and regulated by CK2.

5. ROLE OF CK2/SORCIN CONNECTION IN THE MDR PHENOTYPE
Since both proteins, CK2 and sorcin, display an anti-apototic role, we plan to elucidate at which extent their connection regulate this function. We shall first investigate whether the MDR cell lines that overexpress CK2 display also high levels of sorcin; this analysis will be performed mainly by western blot techniques, exploiting pair cell lines (sensitive/MDR) already available in the applicant laboratory. However, it has also to be considered that CK2 is always very highly expressed in tumor cells, even when the MDR cell variant does not display a higher level compared to the parent line; we can therefore suppose that sorcin, in cancer cells, can always find a CK2 amount sufficient for its phosphorylation/association. In cell lines overexpressing sorcin, the specific activity of CK2 will be calculated, in the hypothesis that a cross-control can occur, where also sorcin affects CK2. CK2 activity will be measured in lysates from cells overexpressing sorcin, by means of CK2-specific peptide substrates, or analysis of in vivo phosphorylation of CK2 substrates (by means of 32Pi cell loading or western blot with phospho-specific antibodies); the results will be compared with those obtained from cells not overexpressing sorcin.
To estimate the effects of CK2/sorcin connection in term of ability to induce a MDR-like phenotype, we could overexpress CK2 (a, β or both) and sorcin, separately or together, in normally sensitive (not MDR) cells, and evaluate a possible synergistic effect of the two proteins in determining an increased resistance to apoptosis. This would suggest that sorcin is one of the targets that CK2 exploits to exert its anti-apoptotic role, and that sorcin functions are assisted by CK2. On the other hand, by applying RNA interference techniques, we could reduce CK2 expression in cells overexpressing sorcin: by comparing the residual resistance degree with data obtained from cells not overexpressing sorcin, we could gain information on the actual requirement of CK2 for the anti-apoptotic function of sorcin.