Phosphorylation of the Actin Binding Protein Drebrin at S647 Is Regulated by Neuronal Activity and PTEN
Figure 1
The PTEN-DBN interaction requires an intact PTEN D-loop.
(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 + sem. *p<0.05 relative to wt PTEN.