## 3 Results
### PTEN is a Novel SMN-Interacting Partner  
Since we showed that phosphorylation of hSMN S290 is detrimental for its stability and physiological function, we next sought to identify a phosphatase that dephosphorylates this residue and stabilizes SMN. We recently found that PKB/AKT is hyperphosphorylated in Smn-depleted NSC34 cells [29]. The main upstream regulators of PKB/AKT are the class I phosphatidylinositol-3-kinases (PI3 Ks) and phosphatase and tensin homolog (PTEN) [51]. Apart from its role as a tumor suppressor and antagonist of the PI3K/AKT pathway [52], PTEN is an important phosphatase during CNS development, regulating soma size, neurite branching and arborization, as well as axonal pathfinding, which involve its lipid and protein phosphatase activities [53]. _PTEN_ mutations or even subtle protein level changes are frequently linked to macrocephaly, autism spectrum disorders, epilepsy and Cowden syndrome [54; 55]. Interestingly, it has been described recently that PTEN binds to spliceosomal components including U2 AF in HEK293T cells to regulate alternative splicing and that PTEN depletion resulted in alternative splicing events linked to tumorigenesis [56]. Additionally, the interaction between PTEN and U2 AF as well as other spliceosomal components (e.g., SF3B2 or LSM5) was also confirmed in stressed neurons [57]. To analyze whether PTEN and SMN are endogenous interactors, we performed co-immunoprecipitation(IPs) with non-transfected and differentiated NSC34 cells (Figure 4A). After precipitation of phospho-PTEN (p-PTEN; S380/T382/T383), a strong signal of endogenous SMN could be detected by Western blot (IB; Figure 4A), indicating an endogenous interaction between PTEN and SMN. An immunoglobulin (IgG) control, beads alone as well as incubation of the membrane with an antibody for \(\alpha\)-tubulin served as negative controls. None of them showed any signals, thus suggesting a specific interaction between p-PTEN and SMN. Of note, since a pan-PTEN antibody, directed to the C-terminus of PTEN, did not work in the immunoprecipitation assays, we employed a phospho-PTEN (p-PTEN) antibody in these assays. PTEN exists in two conformations: (i) the open non-phosphorylated form that preferentially binds to the plasma membrane to dephosphorate PtdIns(3,4,5)P3 to PtdIns(4,5)P2, and (ii) a closed phosphorylated form preferentially localized in the cytosol and nucleus [58]. The IP results suggest that PTEN prefers to bind to SMN in the closed, phosphorylated conformation. To verify a physiological interaction between SMN and PTEN, we carried out immunocyt experiments in NSC34 cells with antibodies against SMN and pan-PTEN (Figure 4B). Endogenous SMN and PTEN co-localized within the cytosol and in growth cones but not in the nucleus (e.g., in gems or CBs). A quantitative evaluation of the co-localization by calculation of product of the differences of the mean (PDM)-values [59] revealed positive values confirming the qualitative co-localization patterns (Figure 4B). The results demonstrate an interaction between PTEN and SMN in a common complex, which localized to growth cones and the cytosol. We next addressed which domain of SMN is involved in PTEN binding. NSC34 cells were transfected with either of the following constructs fused to GFP: full-length hSMN 1-294, a C-terminal deletion mutant hSMN 1-239 (lacking residues 240-294) and an N-terminal deletion mutant hSMN 235-294 (Figure 4C) [30]. 18 h post-transfection, p-PTEN was pulled down and analyzed for co-precipitation of SMN using an antibody against GFP. hSMN 1-294 and N-terminal deleted hSMN 235-294 were co-immunoprecipitation whereas SMN, lacking its C-terminus (hSMN 1-239), was not detected (Figure 4D), indicating that PTEN binds to SMN at its C-terminus. We additionally tested whether SMN\(\Delta\)7, which lacks the last 16 amino acid residues compared to hSMN 1-294 and is the main SMN isoform in SMA due to alternative splicing, harbors PTEN binding properties. Indeed, SMN\(\Delta\)7 co-immunoprecipitation with PTEN (Figure 4F).