## 3 Results
### PTEN Dephosphorylates SMN In Vitro  
We showed that PTEN binds the C-terminus of SMN and that specific patient mutations do not influence binding, whereas mimicking phosphorylation at hSMN S290 abolishes the interaction with PTEN. Next, we wanted to evaluate whether PTEN not only binds to but also dephosphorylates SMN. To test this, NSC34 cells were treated with the PTEN inhibitor bpV(HOpic), a bisperoxovanadium (bpV) compound [61], or solvent for two hours. Subsequently, SMN protein was analyzed by 2D Western blot (1 st dimension: isoelectric point determined by isoelectric focusing; 2 nd dimension: apparent molecular weight separation by SDS-PAGE) (Figure 4G). Additional lysates treated with shrimp alkaline phosphatase (SAP) were included as controls to show that the SMN distribution pattern in the gel is caused by phosphorylation. If PTEN inhibition leads to decreased SMN dephosphorylation, an increase of acidic spots is expected due to negatively charged phospho-groups.  
PTEN inhibition with bpV(HOpic) indeed led to a shift in SMN intensity from the neutral to the acidic side compared to control. The most prominent spot in both conditions (spot #7, arrows in Figure 4G,H) served as a reference spot for quantification (Figure 4H). In contrast, neutral SMN spots adjacent to the core spot became weaker in intensity. This shift upon PTEN inhibition therefore indicates that PTEN is a _bona fide_ phosphatase for SMN.  
Pten Knockdown Negatively Influences SMN Stability and Reduces the Number of SMN-Positive Nuclear Bodies  
We next wondered about functional effects mediated by altered SMN phosphorylation. Since we have shown that (i) hSMN S290 regulates PTEN binding, (ii) PTEN dephosphorylates SMN, and (iii) hSMN S290D is unstable, we wanted to test the hypothesis that PTEN inhibition negatively influences SMN stability. To address this point, we first analyzed the endogenous SMN protein level after 6 h of PTEN inhibition with bpV(HOpic). Interestingly, the level of SMN protein but not of mRNA was markedly decreased (\(\sim\)60%, \(p=0.0551\)) compared to control (Figure 5A-C). SMN has a typical half-life of about 6-24 h depending on the cell type [62; 63; 64; 26] and is mainly degraded by the proteasome [65; 26].  
As we consistently observed a SMN half-life of 6-7 h (Figure 2E) and SMN reduction after 6 h of PTEN inhibition, we wondered whether phosphorylation primes SMN for ubiquitination and proteasomal degradation. To investigate this, cells were treated with DMSO or bpV(HOpic) and with or without the proteasome inhibitor MG132 for 6 h followed by immunoprecipitation of SMN and analysis of its poly-ubiquitination state by Western blot. We observed concomitant lowered SMN levels and increased SMN poly-ubiquitination upon PTEN inhibition (Figure 5D). Since the SMN reduction upon PTEN inhibition was not significant, we genetically knocked down _Pten_ using an RNAi approach. Treatment with siRNA against murine _Pten_ (si_Pten_) led to 85% PTEN protein level reduction compared to control (siCtrl) (Figure 5E,F). Concomitantly with _Pten_ knockdown, SMN protein levels significantly decreased by about 50% (Figure 5G), whereas mRNA was unaffected (Figure 5H), similar to the PTEN pharmacological inhibition experiment. Taken together, we propose that PTEN is necessary to dephosphorylate and stabilize SMN, thus preventing its degradation.  
Since we have shown in our model that SMN levels are reduced and that SMN phosphorylation is altered upon PTEN depletion or inhibition, we wondered whether _Pten_ knockdown also influences SMN-positive NB numbers. As expected, we observed that _Pten_ knockdown in NSC34 cells had a negative impact on the number of NBs (Figure 5,I,J). The percentage of PTEN-depleted cells without NBs significantly increased by two-fold compared to control cells. Additionally, the percentage of cells with 1-4 NBs was consistently decreased compared to control cells. Our results indicate that PTEN is necessary for the presence of SMN-positive NBs.