Engineered FKBP** permits rapid, reversible and tunable regulation of PTEN function in cellular models.

<p><b>(A)</b> FKBP**-PTEN is reversibly regulated in response to Shld1 treatment in cells. U87MG cells stably expressing FKBP**-PTEN were treated with vehicle or with Shld1 (1μM) for 24 h. One set of cells was then washed thoroughly with DMEM and cultured in Shld1-free culture media for further 24hs. The expression level of FKBP**-PTEN was examined by immunofluorescence staining. <b>(B)</b> upper panel, U87MG cells stably expressing FKBP**-PTEN were treated with Shld1 (1 μM) for different time periods; lower panel, Shld1 (1 μM) was removed after 24 h treatment, and cultured in Shld1 free culture media for different time periods as indicated. <b>(C)</b> PTEN protein levels and activity can be maintained at specific concentrations over a prolonged period of time in dependence of Shld1. U87MG cells stably expressing FKBP**-PTEN were cultured in media containing indicated concentrations of Shld1 for 24 h. <b>(D)</b> Stable FKBP**-PTEN expressing U87MG cells were analyzed in cell invasion (left) and colony formation (right) assays. In the cell invasion assay, data are shown as the mean ±SEM of the number of cells migrated in three independent experiments. Plating efficiency refers to the ratio of the number of colonies to the number of cells seeded, shown as mean ±SEM of three independent experiments.</p

Codon optimization of FKBP** establishes a conditional fine-tuning protein regulation system.

<p><b>(A)</b> The transcription of the destabilization cassette FKBP**-PTEN was subcloned downstream of LoxP-Stop-LoxP; transcription was driven by the ubiquitous CMV-enhanced chicken beta actin promoter (CAG). Presence of Cre induces FKBP**-PTEN gene transcription; however, the translated FKBP**-PTEN will be rapidly degraded. Addition of Shld1 confers FKBP**-PTEN stabilization in a tunable and reversible manner. <b>(B)</b> Cre-mediated cleavage of LoxP-Stop-LoxP (LSL) created a truncated FKBP**-PTEN fusion protein. Mouse forebrain neurons were nucleofected with LSL-FKBP**-PTEN and Cre-IresRFP (Cre (+)), or RFP (Cre (-)). Cells were treated with Shld1 (or control vehicle) overnight, before analyses of cell lysates using indicated antibodies. <b>(C)</b> Sequence alignment of FKBP** and LoxP. The third ATG codon region sequence (TATGCTA) of FKBP** (M3L^cta) is identical to the linker region of the LoxP stem-loop sequence (TATGCTA). Codon optimization of CTA with TTG (both code for amino acid Leu) will destroy the potential pseudo-cleavage site of Cre on FKBP**. <b>(D)</b> Codon optimization of M3L^cta to M3L^ttg in FKBP** abolished the Cre-dependent FKBP**-PTEN truncation and produced a Shld1 dependent PTEN fusion protein. The M3L^ttg modified LSL-FKBP**-PTEN construct was co-expressed with Cre-IresRFP (Cre (+)), or RFP (Cre (-)) in mouse forebrain neurons as before. <b>(E)</b> Combinatorial use of the Cre-LoxP system with the FKBP**-PTEN/Shld1 chemical-genetic protein control system in PTEN^flox/flox cells. Constructs were nucleofected into <i>PTEN</i>^{<i>loxp/loxp</i>} mouse primary neurons as before. Note that nucleofection of primary cells occurs with efficiencies at approx. 80%, and result in a residual endogenous PTEN signals detected by western blotting. <b>(F)</b> The generated system is able to couple PTEN-loss directly with the expression of tunable FKBP**-PTEN. Upon Cre-mediated recombination, <i>PTEN</i>^{<i>loxp/loxp</i>} cells will lose PTEN and, at the same time, activate expression of FKBP**-PTEN. In essence, endogenous PTEN is replaced by tuneable PTEN, which will enable to test whether PTEN-loss induced phenotypes can be rescued at different time-points (<i>t</i>_Shld1) and/or at different PTEN-concentrations (<i>d</i>_Shld1).</p

Engineered FKBP** permits regulation of PTEN function in the zebrafish.

<p><b>(A)</b> The migration and growth of stable FKBP**-PTEN expressing U87MG cell clones injected into zebrafish embryos can be inhibited by Shld1. Prior to microinjection into the perivitelline cavity of 2 days post fertilization (dpf) zebrafish embryos, cells were pre-stained with DiI. Embryos were then transferred into fish water with (n = 11) or without (n = 13) Shld1 (4 μM) and maintained for 24h. Images were taken immediately after injections (0 days post injections, dpi) and after 24 h (1dpi). <b>(B)</b> The migration indices and the increase in tumor mass size of transplanted cells were quantified. ** p<0.01; ***p<0.001.</p

Consequence of linker optimization on PTEN activity of the FKBP*-PTEN fusion protein.

<p>(<b>A)</b> Rationale of the DD-technology: Fusion of PTEN with a modified human FKBP variant harboring the F36V and L106P point mutations (FKBP*) leads to degradation of the fusion protein. Presence of the FKBP* ligand Shld1 confers stabilisation of FKBP*-PTEN and results in inhibition of PI3K/Akt signaling. (<b>B)</b> The fusion protein FKBP*-PTEN shows no PTEN activity. PTEN-deficient U87MG cells were transiently co-transfected with FKBP*-PTEN and AKT-GFP. After one day, cells were treated with Shld1 for 12 hours at 1μM before cell lysis and samples analyses by western blotting using indicated antibodies. <b>(C)</b> Consequence of linker optimization on PTEN activity of the FKBP*-PTEN fusion protein. Schematic representation of linker optimization. FKBP*-linker-PTEN variants with different linker regions (A-E) were generated. Linker A—short flexible linker; Linker B—a helix forming linker. Linker C—long flexible linker. Linker D—long flexible linker containing the PWR motif. Linker E—the original linker present in the enzymatic active GFP-PTEN. <b>(D)</b> Analyses of the effect of different linkers on PTEN activity in FKBP*-PTEN fusion variants in U87MG cells. Constructs expressing FKBP*-PTEN variants or GFP-PTEN were co-transfected with AKT-GFP into a PTEN null U87MG cells. Stabilization of FKBP*-PTEN or GFP-PTEN in presence or absence of Shld1 was monitored by PTEN antibody. The phosphorylation level of AKT-GFP at Ser473 served as a fast and effective experimental protocol to control for PTEN activity towards PIP3-dependent signaling in transfected cells, only. Only constructs 4 and 5, which contain proline in the linker sequence, showed moderate PTEN activity towards PI3K signaling.</p

FKBP surface engineering restores PTEN activity of FKBP**-PTEN fusion protein.

<p><b>(A)</b> Electrostatic potential map of PTEN (PDB ID: 1d5r) and FKBP (PDB ID: 1BL4). Positively charged binding pocket of the PTEN PIP3 substrate is circled with a white dashed line; the negatively charged lump formed by E31/D32 residues on the FKBP surface is circled by a black dashed line. <b>(B)</b> Schematic representation of amino acid residue substitutions of FKBP* protein surface at E31/D32. <b>(C)</b> Different FKBP*-PTEN mutants were transiently co-tansfected with AKT-GFP into U87MG cells, the expression of fusion protein and pAKT were examined by Western blotting. <b>(D)</b> E31S/D32S amino acid substitutions on FKBP* (FKBP**) restores PTEN activity of the FKBP**-PTEN fusion protein. FKBP**-PTEN (or GFP/GFP-PTEN) was transiently co-expressed with AKT-GFP in U87MG cells. Cells were treated with Shld1 and analyzed as before.</p

pS647-Drebrin is negatively regulated by PTEN in neurons independently of PI3K.

<p>(A) Cortical neurons isolated from <i>PTEN</i>^{<i>flox/flox</i>} mice were transduced with increasing concentrations of Cre-expressing viruses at 13 DIV. Neuronal cell lysates were analysed at 19 DIV. Bar graph represents the relative band density of pS647-DBN and PTEN in three independent experiments with maximal cre-virus titer used, <u>+</u> sem. *p<0.05, *** < 0.001. (B) Rat cortical neurons were treated with the PI3K inhibitor LY294002 at indicated concentrations for 60 minutes before analyses of neuronal cell lysates. Bar graph represents the average band density of pS647-DBN in three independent experiments. (C) Hippocampal neurons were cultured for 1.5 DIV and labelled with anti-DBN, anti-pS647-DBN and anti-PTEN antibodies. (D) 18 DIV hippocampal neurons were stained with anti-DBN, anti-pS647-DBN and anti-PTEN. Top images show a specimen images at higher magnification. DBN is highly enriched in dendritic spines, but also found - albeit at lower levels - in the dendrite and the soma, whereas phosphorylation at S657 is more confined to the dendritic spine compartment. (E) Hippocampal neurons were cultured for 18 DIV and stained with anti-pS647-DBN and anti-PTEN antibodies. In dendritic spines (<b>arrows</b>), DBN is highly phosphorylated on pS647, whereas PTEN is mainly present in the dendrite process. Sporadically, PTEN is found in spines (<b>star</b>), which coincides with depleted pS647-Drebrin labeling. (F) Cortical neurons isolated from PTEN floxed/floxed mice were nucleofected with RFP or Cre-RFP and cultured for DIV3 before staining for pS647-DBN. Scale bars C, D, F: 10 µm; E: 2 µm.</p

pS647-Drebrin is de-phosphorylated in dependence of the PTEN phosphatase activity, but independent of PI3K signalling.

<p>(A) Alignment of S647 DBN. DBN comprises of an N-terminal ADF/cofilin homology domain (ADF), a coiled-coil (C–C) domain, a proline-rich stretch (P) and an unstructured C-terminus. (B) Adult rat brain homogenate was analysed by western blot analysis and probed with anti-DBN antibody or with the affinity purified pS647-DBN antibody. (C) Brain homogenate was incubated on ice or at 37°C for 1 hour to activate endogenous phosphatases, before western blotting and analyses. (D) Cortical neurons were nucleofected with control or DBN specific shRNA. Neuronal lysate was analysed at 3 DIV. (E) Bacterially expressed and purified His-DBN was probed for anti-DBN and anti-pS647-DBN antibodies. (F) Cell lysates prepared from YFP-DBN or YFP-S647A-DBN expressing HEK293 cells were analysed by western blotting using indicated antibodies. (G) 19 DIV hippocampal neurons were stained with pan-drebin (mouse) and pS647-DBN antibodies (rabbit) in the presence of control serum or with the pS647-DBN peptide. Scale bar: 10 µm. (H) GFP-PTEN or the GFP-PTEN CS mutant was co-expressed with Flag-DBN in U87MG cells. Bar graph represents the average band density of pS647-DBN/DBN in 3 independent experiments <u>+</u> sem. *p<0.05. (I) Flag-DBN expressing U87MG cells were treated with wortmannin (wm) for 1 hour at 100 nM. (J) As in (H), but cells were co-transfected with Flag-DBN and GFP-PTEN, or with Flag-DBN and GFP-PTENΔD. Bar graph (right) represents the average band density of pS647-DBN/DBN in 3 independent experiments <u>+</u> sem. *p<0.05.</p

Drebrin induced dendritic protrusions do not require PI3K.

<p>Rat hippocampal neurons were transfected at 7 DIV with YFP or YFP-DBN. The PI3K inhibitor wortmannin was applied at 100 nM following transfection. At 17 DIV, neurons were fixed and the length of individual dendrite protrusions was determined from the base to the tip of the spine head using Neuron-J. To avoid spine variability, we restricted the analysis to the primary and secondary dendrites 100-150 µm away from the soma. Each data point represent the relative length of at least 220 protrusions measured over 3 independent experiments <u>+</u> sem. ***p<0.001. Scale bar: 3 µm.</p

Phosphorylation of DBN at S647-Drebrin is increased by synaptic activity.

<p>(A) Cortical neurons were treated with 10 µM Y27632 (Rock inhibitor), 20 µM LY294002 (PI3K inhibitor), 10 µM PD98059 (MAPK inhibitor), 10 µM DRB (CK2 inhibitor), 10 µM Lavendustin A (general kinase inhibitor) or 2 µM CT99021 (GSK-3 inhibitor) and analyzed by western blotting using indicated antibodies. The anti-pMAPK and anti-pCRMP2 blot verified the activity of MAPK and GSK-3 inhibitors, respectively. (B) Hippocampal neurons were treated with KCl (20 mM) for 10 minutes, or with high frequency electrical stimulation (HFS) at 100 Hz, 1 s, twice with a 20 s interval. (C) Neurons were treated with KCl (20 mM) for indicated periods of times. Bar graph represents the average band density of pS647-DBN/DBN at each time point in 3 independent experiments <u>+</u> sem *p<0.05. (D) Organotypic hippocampal slice cultures were exposed to 5 µM gabazine (n = 21) or 500 nM TTX (n = 29) and network activity was recorded for 1 hour in stratum pyramidale of the CA3 subfield. TTX-exposed cultures showed no spontaneous activity whereas gabazine exposed cultures developed seizure-like events. A single event indicated by the asterisk is magnified. Bars demonstrate the frequency (left) of events over a time period of 1000 s and their amplitude (right). (E) Organotypic hippocampal slice cultures were exposed to gabazine or TTX for 1 hour lysed and analysed with the indicated antibodies. Bar graph shows the relative band density of pS647-DBN/DBN in <u>></u>7 independent samples <u>+</u> sem; *p<0.05.</p

The PTEN-DBN interaction requires an intact PTEN D-loop.

<p>(A) Coomassie blue stained gel of immunoprecipitated PTEN from E18 rat brain and liver. The arrow indicates the band excised from the gel for mass spectrometry analysis, which was identified as drebrin (DBN). (B) E18 rat brain lysate was incubated with anti-PTEN antibody or control IgG; IPs were analyzed with indicated antibodies. (C) GFP-PTEN (or control GFP) and Flag-DBN (or control Flag) were co-expressed in HEK293 cells. Following anti-flag (left) or anti-GFP (right) IP, co-precipitates were analyzed with anti-PTEN and anti-DBN antibodies. (D) Domain structure of PTEN and PTEN-deletion constructs. PTEN consists of an N-terminal phosphatase domain that can act on both protein and lipid substrates. The C-terminal domain contains a C2 domain and a cluster of phosphorylation sites. In the extreme C-terminus of PTEN a binding motif for PDZ domains is present. (E, F) HEK293 cells were co-transfected with indicated constructs before immunoprecipitation as described in (C). Blots were analyzed with indicated antibodies. (E) In comparison to full-length PTEN, the PTENC2 domain shows increased binding to DBN. (F) The PTEN: DBN interaction requires an intact D-loop (aa 281-312). Bar graph represents the average band density of GFP-PTEN/ FLAG-DBN in the co-IP in three different experiments <u>+</u> sem. *p<0.05 relative to wt PTEN.</p