Axons were traced in the GFP or Tuj1 pictures and numbers and lengths of filopodia were traced in the F-actin images using the NeuronJ plug-in of FIJI (Meijering et?al., 2004, https://imagescience.org/meijering/software/neuronj/). that PRG2 is both sufficient and necessary to account for the ability of neurons to generate axon filopodia and branches in dependence on PI3K/PI(3,4,5)P3 and PTEN. Our data indicate that PRG2 is part of a neuronal growth program that induces collateral branch growth in axons by conferring local inhibition of PTEN. mutations are associated with sporadic cancers; overgrowth syndromes, such as PTEN hamartoma tumor syndrome; and autism spectrum disorders (Huang et?al., 2016, Kwon et?al., 2006). Precise spatiotemporal regulation of PI3K/PTEN-generated PI(3,4,5)P3 is essential for the proper reorganization of the plasma membrane actin cytoskeleton to support cell morphology and migration in different cell types (Haugh et?al., 2000, Iijima et?al., 2002, Martin-Belmonte et?al., 2007). In neurons, in particular, localized production of Triptolide (PG490) PI(3,4,5)P3 is associated with hallmarks of neuronal morphology such as induction and elongation of neurites, dendritic spine morphogenesis and function, and axon branch morphogenesis (Gallo, 2013, Horiguchi et?al., 2006, Kreis et?al., 2014, Mnager et?al., 2004, Shi et?al., 2003). In axons, localized production of PI(3,4,5)P3 supports the initiation of F-actin patches, which give rise to filopodia protrusions along the axon shaft (Gallo, 2013, Ketschek and Gallo, 2010, Spillane et?al., 2012). These filopodial protrusions are considered precursors for axon branches, which mature by subsequent invasion and stabilization of microtubules and ultimately form the basis of neuronal connectivity in the adult brain (Kalil and Dent, 2014). Although generation of Triptolide (PG490) PI(3,4,5)P3 during axon growth and branching has been well documented (Kalil and Dent, 2014, Ketschek and Gallo, 2010), the mechanisms involving PTEN regulation deserve further attention, given that they can either expand or Rabbit polyclonal to Hsp90 blunt the signaling output of the PI3K pathway by altering PI(3,4,5)P3 plasma membrane concentration and localization (Kreis et?al., 2014). This is particularly relevant because PTEN is highly abundant in neurons and extremely efficient in confining and limiting PI3K-dependent growth in the axon, especially during early development when axons elongate to reach their targets (Chadborn et?al., 2006, Christie et?al., 2010, Drinjakovic et?al., 2010, Zhang et?al., 2013). How neurons overcome that growth barrier to allow collateral branching is not fully understood. Here, we identify and characterize a PTEN membrane protein association, which controls PTEN activity. The protein complex incorporates plasticity-related gene 2 (PRG2), a transmembrane protein belonging to the family of lipid phosphate phosphatase-related Triptolide (PG490) (also known as LPPR) proteins. Our data indicate that PRG2 associates with PTEN and organizes PI(3,4,5)P3-mediated cellular responses in neurons during axon filopodia initiation and branch formation. Results PTEN Interacts with the Neuronal Membrane Protein PRG2 We set out to identify proteins involved in regulating the sub-cellular localization and/or function of PTEN in neurons using mass spectrometry (van Diepen et?al., 2009). Among the protein interactions, we identified PRG2 as a PTEN binding partner. We first confirmed the PRG2-PTEN association by performing endogenous co-immunoprecipitation in embryonic day 18 (E18) rat brain lysates as well as in days (DIV) 9 cortical neuron cultures (Figure?1A). PRG2 (or LPPR3) is a member of the LPPR protein family whose members show high homology with bioactive lipid-inactivating phosphatases but lack catalytic activity (McDermott et?al., 2004, Sigal et?al., 2007, Strauss and Br?uer, 2013). PRG2 is closely related to its better functionally characterized homolog PRG1 (Br?uer et?al., 2003, Liu et?al., 2016, Trimbuch et?al., 2009); both proteins share the common domain structure with six transmembrane regions forming an extracellular oriented pseudo-LPP catalytic motif and an additional large Triptolide (PG490) C-terminal cytosolic domain (Figure?1B). Unique within this family, PRG2 bears a highly acidic stretch consisting of 20 glutamic acid residues in its C-domain (poly-E-box). To characterize whether the PRG2-specific poly-E-box region mediates.