## 4 Discussion
Based on our binding assays, in which we narrowed the PTEN binding site down to residues 239-278, it is also possible that hSMN S290 is the target site for PTEN. S290 localizes to the sequence encoded by exon 7. Deletion of exons 6 or 7 decreased half-life of SMN by approximately half, indicating an important role of those sequences for regulation of SMN stability [26]. Moreover, this region is involved in self-association of SMN, direct binding of Gemini3, and indirect interaction with Gemini4 as well as the zinc finger protein ZPR1 [2; 13; 15; 71]. Focusing on defined C-terminal functions of SMN, we found that SMN oligomerization was impaired in the phospho-mimetic hSMN S290D. Furthermore, hSMN S290D failed in binding to and co-localization with ZPR1. Of note, hSMN S290D shows several impairments in SMN functions, which are not prominent in hSMN S290A. However, this does not necessarily imply that the non-phosphorylated hSMN S290A shows completely adverse effects. Therefore, we suppose that regulation of SMN stability is not restricted to the phosphorylation state at S290 only. A first antisense oligonucleotide (ASO)-based therapy (Nusinersen/Spinraza(tm)) has been approved in 2016 by the FDA and in 2017 by European Medicines Agency (EMA). However, it is currently unclear to what extent possible non-motoneuron phenotypes might emerge in treated severely affected patients and whether all patients can be successfully treated with this approach since about 50% of the SMA type I patients are non-responders [6; 7; 72]. Treatment possibilities have recently been extended by approval of an AAV9-based gene replacement therapy onasemnogene abeparvovec-xioi (Zolgensma(tm)) [8] and the systemically administered small compound Risdiplam (Evrysdi(tm)). This changing therapeutic landscape has identified the need for novel combinatorial treatments that consist of SMN-independent and -dependent treatment strategies that can support or augment the benefits of the currently approved SMN gene therapies [73]. The identification of SMN phosphatases and kinases is of great importance for the development of such new strategies that could stabilize the SMN protein and maximize its functional benefits. In summary, our data provide novel insights into post-translational modification of the SMN protein, which is of importance for putative future combinatorial SMA therapies comprising expressional increase and stabilization of SMN. Although many kinases display pleiotropic effects, novel approaches aim to target specifically the substrate-kinase interface [74]. Inhibition of the kinase responsible for phosphorylation of serine residues at the C-terminus could be such a putative strategy. **Author Contributions:** [PERSON] and [PERSON] designed the project and planned the experiments. [PERSON], [PERSON], [PERSON], [PERSON], [PERSON], I.-M.W., [PERSON], [PERSON], [PERSON], [PERSON], M.B., [PERSON] and [PERSON] performed the experiments and analyzed the data. [PERSON] and A.N. generated and analyzed the mass spectrometry data. [PERSON] and [PERSON] wrote the manuscript with the contribution of all co-authors. All authors have read and agreed to the published version of the manuscript. **Funding:** This project was funded by The SMA Trust (now SMA UK) (to M.B. ), by the Deutsche Muskelstiftung (DMS)/Philipp & Freunde--SMA Deutschland (to P.C.) and Italian Telethon Foundation (to E.D.S. GGP16203). We acknowledge support by the German Research Foundation (DFG) and the Open Access Publication Fund of Hannover Medical School (MHH). **Acknowledgments:** In memoriam to [PERSON] (1936-2020), University of Gottingen, Germany. We thank [PERSON] (Hannover Medical School) for providing the ubiquitin antibody. We thank the Shared Facility Mass Spectrometry of the Charite for support in acquisition and analysis of the data. We thank [PERSON] (IBBR, Naples, Italy) for technical support, [PERSON] (Columbia University, New York, USA) and [PERSON] (University of Calgary, Canada) for plasmids. We thank [PERSON] and other members of the Claus lab for discussions.