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

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

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

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

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

Analysis of the PTEN:DBN interaction by FRET in PC12 cells using multiphoton FLIM.

<p>(A) Images show the lifetime maps of FRET across cells using a pseudocolour scale (blue, normal GFP lifetime; red, FRET). GFP-PTEN (<b>top left</b>) and GFP-PTENΔD (<b>bottom left</b>) demonstrate normal GFP lifetime in the absence of acceptor, other images show co-expression of GFP-PTEN (or GFP-PTENΔD) with mCherry-DBN. Bar graph representing the average FRET efficiency of 16 cells over 3 independent experiments <u>+</u> sem. *p<0.01. (B) Cells were incubated for 1 hour with scrambled control peptide (control) or a small peptide derived from the PTENC2 domain (D-loop), before FRET analyses. Both peptides are fused to the antennapedia (Ant) internalisation sequence. Bar graphs represent the average FRET efficiency of 13 cells over 3 independent experiments <u>+</u> sem. *p<0.01. (C) Hippocampal neurons were transfected at 7DIV and incubated at 10DIV with Ant-control or Ant-D-loop peptides. Bar graphs represent the average FRET efficiency of 14 cells over 3 independent experiments <u>+</u> sem. **p<0.005, ***p<0.001. Scale bars: 10 µm.</p

Membrane depolarisation induced dissocation of PTEN-DBN and increases the phosphorylation of pS647-DBN.

<p>(A) Neurons were transfected at 7 DIV with mCherry-DBN and GFP-PTEN and incubated until 10 DIV, before treatment with KCl (20 mM) and FLIM measurements. Bar graphs represent the average FRET efficiency of 22 cells over 3 independent experiments + sem. *p<0.001. Scale bar: 10 µm (B) Cortical neurons from floxed PTEN mice expressing RFP or RFP-Cre were cultured for 10 DIV and treated with KCl (20 mM). (C) 11 DIV rat hippocampal neurons were treated with KCl (20 mM) for 3 minutes in the presence or absence of Wortmannin (wm) at 100 nM. Bar graph shows the relative band density of pS647-DBN in three independent experiments +sem; *p<0.05. (D) Schematic diagram illustrating signaling relationships. DBN phosphorylation at S647 is suppressed through physical interactions with PTEN. Synaptic activity induces a transient dissociation of the PTEN:DBN complex and allows maximal DBN-S647 phosphorylation to occur.</p