1Universitätsklinikum Heidelberg Klinik für Innere Med. III, Kardiologie, Angiologie u. Pneumologie Heidelberg, Deutschland
Heart failure is still the number one cause for hospital admission in Germany as well as a leading factor of morbidity and mortality worldwide. Thus, the underlying molecular pathways remain to be better understood in order to identify potential targets for treatment.
An important regulatory mechanism in cells is phosphorylation. Striatin interacting phosphatases and kinases complexes (STRIPAK) are an emerging group of multiprotein complexes responsible for integrating different cellular pathways and altering phosphorylation patterns in the cell. Despite their potential importance in cellular signalling STRIPAK complexes have not been studied in cardiac tissue to date. We here show that several STRIPAK members such as SLMAP, Striatin, Striatin 3, STRIP1, STRIP2, CCM3, MST3 and mammalian STE20-like kinase 4 (MST4) are abundantly expressed in rodent hearts as shown in multi tissue immunoblot analyses.
Furthermore, we could demonstrate the existence of STRIPAK complexes including MST4 in cardiomyocytes through Co-immunoprecipitation experiments using neonatal rat ventricular cardiomyocytes (NRVCM) as well as heart tissue of adult rats. Core member of STRIPAK complexes are proteins from the striatin family. Fishing with MST4, we could co-immunoprecipitate both Striatin and the related Striatin 3. Further STRIPAK members identified were STRIP1, STRIP2 and SLMAP. We could also demonstrate these interactions for the MST4 sister kinase MST3, including MST3-Striatin and MST3-SLAMP. Interestingly, adenoviral MST4 overexpression in NRVCM leads to a decrease in MST3 expression and overexpression of MST3 reduces MST4 expression in return.
Another intra-STRIPAK regulation could be observed involving protein phosphatase 2A. This phosphatase consists of a core enzyme made up of a scaffolding subunit A and a catalytic subunit C. The role of the regulatory subunit B can be taken on by different proteins as well as protein complexes such as STRIPAK. In cardiomyocytes, previously described PP2A targets include major proteins such as Troponins, L-type calcium channel, protein kinase B, connexin 43 and phopholamban. Using a PP2A-activator, we could demonstrate an impairment of MST4 activity indicated by less MST4 phosphorylation at T178. This phospho-site is essential for the formation of MST4-dimers and its ability to phosphorylate. In line with this, PP2A inhibition using a small molecule results in increased MST4 activity.
The different regulatory subunits B of PP2A are important for its targeting to different cellular components. Against this background we performed further interaction analyses showing co-immunopreciptation of MST4 with Serca2A, alpha-Actinin 1 and beta-Catenin. This seems particularly interesting considering that we also observed an improvement in contractility and relaxation in isolated adult rat ventricular cardiomyocytes upon MST4 overexpression.
Taken together, this is the first demonstration of the existence of functioning STRIPAK complexes including MST4 in the heart. MST4 is being regulated by other STRIPAK members such as PP2A and MST3 and we also found different interaction partners hinting at a targeting function of STRIPAK complexes for MST4 that has previously been described for PP2A.