## 4 Discussion  
In this study, we have shown for the first time that phosphorylation of serine 290 is detrimental to the stability of SMN. Moreover, PTEN is a protein phosphatase dephosphorylating SMN and thereby stabilizing the protein. Interestingly, AAV-mediated _Pten_ knockdown resulted in increased survival of motoneurons isolated from SMN27 SMA mice [66]. In the same mice, ameliorated neuromuscular junction pathology, motor performance, and modestly prolonged lifespan were observed when _Pten_  
Figure 5: PTEN inhibition and knockdown decrease SMN levels via proteasomal degradation. **(A,B)** NSC34 cells were differentiated and treated with DMSO or PTEN inhibitor bpV(HOpic) for 6 h prior to lysis. Western blot analysis determined SMN protein levels and SMN compared to housekeeping protein was quantified by densitometry (mean \(\pm\) SEM, \(n=5\), paired two-tailed \(t\)-test, \(p=0.0551\)). **(C)** Relative _Smm_ expression of DMSO- or bpV(HOpic)-treated cells (6 h) was analyzed by qRT-PCR (mean \(\pm\) SEM, \(n=3\), paired two-tailed \(t\)-test, ns = not significant). **(D)** Cells were differentiated and treated with DMSO or bpV(HOpic) and in the absence or presence of the proteasome inhibitor MG132 for 6 h. SMN ubiquitination was analyzed upon SMN immunoprecipitation. **(E–G)** NSC34 cells were transfected with siRNA for _Pten_ (si_Pten_) or control (siCtrl) and differentiated. Western blot analysis determined PTEN and SMN levels **(E)** together with densitometric quantification of PTEN **(F)** and SMN **(G)** compared to housekeeping protein (mean \(\pm\) SEM, \(n=5\), paired two-tailed \(t\)-test, \({}^{*}p<0.05\), \({}^{**}p<0.01\)). **(H)** Relative _Smm_ expression in control or PTEN-depleted cells analyzed by qRT-PCR (mean \(\pm\) SEM, \(n=3\), paired two-tailed \(t\)-test, ns). **(I,J)** Quantification of SMN-positive nuclear bodies (NBs) of control or PTEN-depleted cells (mean \(\pm\) SEM, \(n=3\), multiple \(t\)-test, \({}^{*}p<0.05\)). had been systemically knocked down [67]. Mechanistically, PTEN depletion increased the antiapoptotic activity of AKT as well as AKT's downstream effector mTOR, which may be the primary neuroprotective effects following _Pten_ knockdown [68]. Although we show that PTEN depletion is detrimental for SMN stability, such effects were not detected in these previous studies. However, (i) the effect of PTEN depletion on SMN levels had not been analyzed in WT, (ii) SMNA7 does not harbor residue S290, and (iii) we have analyzed SMN stability in a motoneuron-like cell line in contrast to muscle cells in other studies. The _C. elegans_ PTEN homologue DAF-18 and PTEN share conserved sequences [69] and DAF-18 depletion increases motoneuron regeneration in _C. elegans_[70], corroborating published data regarding PTEN depletion. However, effects of DAF-18 loss in _smn-1_ background of the _C. elegans_ SMA model could be limited due to differences in sequence similiarity between SMN of _C. elegans_ and human as our rescue experiment with hSMN WT has shown. Thus, further in vivo investigations are needed to elucidate the functional role of PTEN in SMA. A small number of phosphatases have already been discovered that regulate SMN localization and function [21; 23; 25]. In this study, we identified PTEN as a novel endogenous interactor and phosphatase of SMN and show that PTEN is necessary for formation of SMN-positive NBs. Our results additionally suggest that PTEN regulates splicing complexes on another level that is by promoting the cytosolic SMN complex and snRNP precursor formation, since SMN protein stability and NB numbers decreased upon PTEN knockdown due to phosphorylation changes. Here, we found that hSMN residues S290 and S292 are phosphorylation sites and show that S290 plays a physiological function in vivo, a function that has been conserved in evolution. Our results show that PTEN binding is modulated by S290. However, this site is not an exclusive regulator since SMNA7 could partially bind PTEN.