Bioinformatic search for potential phosphorylation sites of melusin – integrin b 1-binding protein

Phosphorylation is one of the most frequently occurring posttranslational modifications in proteins. It plays an essential role in transferring outside signals into a cell and regulates different cellular processes such as growth, metabolism, proliferation, motility and differentiation. Melusin is a stress response protein which strictly reacts to the threshold levels of mechanic stress and activates cardiomyocytes signaling pathways. The search for potential sites of melusin phosphorylation was performed using bioinformatic analysis of primary protein sequences. The comparative bioinformatic analysis of possible phosphorylation sites, evolutionary and structural motifs has identified Ser326,Ser329 and Ser334 as the most likely sites for phosphorylation of melusin by protein kinase CK2 in cardiamyocytes.


Introduction.
Melusin is a cytoplasmic protein with unique structural peculiarities synthesised in the skeletal tissues and cardiac muscle.The human melusin (integrin-b1-binding protein of 2-ITGB1BP2) is known to be one of so-called CHORD-proteins [1].It is a multidomain protein consisting of 347 amino acid residues with the N-terminal region represented by two cysteine/histidine rich domains (CHORD-domain) able to fix Zn 2+ ions (Fig 1).The C-terminal protein region contains so-called CS domain which is the binding site with the b1-integrin cytoplasmic domain.This domain was also discovered in a-crystalline and p23 and Stg1 proteins [2] belonging to the molecular chaperone family.The C-terminal end is rich in glutamatic and aspartic amino acids and represents a region of Ca 2+ ions fixation.Melusin interacts with the cytoplasmic domain of b1-integrine and is localized in the costameres participating in the molecular mechanism of sarcomere conjunction to the sarcolemma and intracellular matrix [1,3].Melusin is supposed to play a key role in the cardiac hypertrophy.
The experiments on the melusin gene inactivation in mice allowed assuming that this protein is not a critical one in the embryonic heart formation, sarcomeric organisation or heart functioning under normal conditions [2,4].The melusin removal breaks the hypertrophic response of the aortic ventricle and dramatically accelerates the transition to the cardiac dilatation [3,4].The diminishing of interaction forces between melusin, b1-integrine cytoplasmic domain and ILK kinase in the costameric junctions causes the cardiac dysfunction and dilated cardiomyopathy (DCM) development [5].At the same time by the melusin super expression the ventricles of transgenic mice hearts maintained the full contractile function in the case of long and constant overload [6].Such functional properties were connected with the protection against the apoptosis and lack of stromal tissue displacement.Melusin controls the hypertrophic heart reaction in response to the various stresses e.g.high pressure.The mechanical changes in the cardiomyocytes activate several cell signaling pathways: MAPK, JAK/STAT, PI3K/AKT, AKT/GSK3b [6][7][8].Melusin belongs to the proteins reacting acutely to the threshold levels of mechanical stress and activates AKT/GSK3b signaling pathways controlling the cardiomyocytes hypertrophy.This is a molecule with a unique ability to cause the compensatory hypertrophy and prevent the cardiac dilatation and cardiac failure [6].The study on a melusin role in the cardiomyocytes signaling pathways is of great theoretical and practical interest.
Our study is devoted to the search for potential sites of melusin phosphorylation using bioinformatic analysis of the protein sequence.The prediction is based on the analysis of sites availability on the linear molecule according to the hydrophobicity level with an assumption that the hydrophobic sequences are rather hidden inside the protein molecule than exhibited outside.As a result of performed analysis it was discovered that Ser 326 , Ser 329 and Ser 334 are the potential regions for phosphorylation by CK2 protein kinase.

Materials and Methods. Phylogenetic analysis.
The primary sequence comparison was carried out applying the program CLUSTAL W [9].The protein sequences of human melusin 1 and 2 were received from NCBI and SwissProt databases and used to search out the appropriate proteins from the other eukaryotes through the instrumentality of BLAST 2.0 on the BLAST NCBI.
Search for the phosphorylation sites.The search for potential phosphorylation regions was carried out applying the following online resources programs: ScanSite [10], NetPhos 2.0 [11], and KinasePhos 2.0 [12].These programs, based on protein primary sequences, enable the prediction of the whole motifs for various known protein kinases applying the selective matrix, calculated as a result of protein libraries screening.They are also based on the availability analysis of sites on the protein molecule.
The protein secondary structure prediction.The protein secondary structure was predicted applying the program GORNIER4 [13] and PHD web server [14].
The protein 3D structure construction.3D structure of melusin was simulated on the basis of CHORD containing protein-1 (2YRT) structure [15] applying the web server of the program 3D-JIGSAW (version 2.0) www.bmm.icnet.uk[16,17].The appropriate amino acid exposition was determined by the 3D-structure applying the program SWISS PDB Viewer.
Results and discussion.We performed the computer analysis of human melusin structure in order to reveal the potential phosphorylation sites.Special attention was paid to the Ser/Thr protein kinases participating in PI3K-AKT, AKT/GSK3b and other signaling pathways activated under conditions of various stresses and resulting in the mechanical changes in the cardiomyocytes.Using the program NetPhos 2.0 we discovered seven potential on-serine and one on-threonine phosphorylation sites in melusin, selecting these sites with prediction probability of 0.8 and higher, and considering the coincidence with the phosphorylation sites predicted by other programs: ScanSite and KinasePhose 2.0 (see the table).The most probable phosphorylation sites for protein kinase CK2 (S 326 , S 329 , S 334 ) were selected as a result of comparative analysis.In order to ascertain how conservative these It was found out that tree mentioned sites (S 326 , S 329 , S 334 ) are conservative for melusins of all the investigated eukaryotes (Fig. 2).Fig. 2 shows that the phosphorylation sites and surrounding amino acid residues did not change in the process of evolution which indicates the importance of these amino acids phosphorylation.
For the effective phosphorylation the aminoacids have to be available for protein kinases i.e. to be located on the protein surface.We built the models of secondary structures and performed 3D modelling for human melusin and other 12 known proteins.The analysis of predicted secondary structures for all studied proteins and corresponding 3D structures indicates that all predicted amino acids are located on the surface of the protein molecule.This is an essential confirmation that exactly S 326 , S 329 , S 334 are the potential phosphorylation sites for CK2 protein kinase characterized by a high activity in the heart [18].
The protein phosphorylation as an important mechanism of post-translational modification has a significant influence on the cellular processes e.g.metabolism, differentiation, membrane transport and cell signaling pathways [19].
It is known that the C-domain of melusin is structurally similar to the C-domain of calsequestrin    [20], which contains the phosphorylation sites for CK2 protein kinase [21] and fixes Ca 2+ ions like melusin.The interaction of melusin with b1-integrine cytoplasmic domain is regulated by Ca 2+ ions presence or absence.The attenuation of interaction between melusin, b1-integrine cytoplasmic domain and ILK in the costameres results in the disfunction and dilatation in the cardiac muscle [5].Melusin is known to participate in the Ca 2+ homeostasis [22], namely, in the Ca 2+ ions concentration decrease in the sarcoplasmic reticulum of cardiomyocytes.This leads to an incorrect protein folding in response to a stress causing the cardiomyocytes apoptosis and heart failure as a consequence.We suppose that precisely the phosphorylated melusin forms can perform the function similar to that of calsequestrin i.e. to bind incorrectly folded proteins and transfer them through endoplasmic reticulum to the Golgi apparatus, ensuring the complicated cellular transport and thus participating in the cell signaling pathways.At the same time, the CS-domain and C-terminal end in melusin are also a signature for S100 proteins fixation involved in the cell Ca-regulatory function [2,23].The melusin phosphorylation seems to result in the post-translational change of the protein structure, whereby it binds S100 proteins.

Fig. 3 .
Fig. 3. Prediction of secondary structure of human melusin ( eexposed regions) (a) and predicted 3D structure of human melusin (b) Table of melusin phosphorylation sites