Figure 1.
Cdh1 phosphorylation at Cdk sites promotes Cdh1 stability. A, Schematic of nine conserved sites of potential Cdk phosphorylation in Cdh1 (S/T-P), numbered according to human Cdh1. B, Lysates of 293T cells transfected with GFP–Cdh1 WT, 9A, or 9D were immunoblotted using a polyclonal antibody to Cdh1 or Erk, the latter to serve as a loading control. Quantitation of Cdh1 levels normalized by Erk revealed that the levels of the 9D and 9A mutant proteins were, respectively, increased by 122% and reduced by 57% relative to wild-type Cdh1 in 293T cells (average of 4 experiments). C, Lysates of Neuro2A cells transfected with GFP–Cdh1 WT, 9A, or 9D and treated with MG132 (5 μm) or the vehicle control DMSO were immunoblotted for GFP or Erk. The levels of the 9D and 9A mutant proteins were, respectively, increased by 150% and reduced by 37% relative to wild-type Cdh1 in Neuro2A cells (average of 2 experiments). MG132 treatment, respectively, increased WT, 9A, and 9D levels by 80, 134, and 9%. D, Lysates of granule neurons transfected with pEGFP–C1, GFP–Cdh1 WT, GFP–Cdh1 9A, or GFP–Cdh1 9D were immunoblotted for GFP or Erk. The 9D Cdh1 mutant is expressed at higher levels than wild-type Cdh1, which is higher than the 9A Cdh1 mutant in primary granule neurons. E, Lysates of Neuro2A cells or granule neurons treated with roscovitine (20 μm) or DMSO were immunoblotted using a monoclonal antibody to Cdh1 and the Erk or 14-3-3β antibody. Roscovitine reduces Cdh1 levels by 60% in both Neuro2A and neurons (average of 2 experiments each). F, Lysates of granule neurons treated with MG132 (10 μm), roscovitine (20 μm), both MG132 and roscovitine, or DMSO were immunoblotted using the Erk antibody or the Cdh1 monoclonal antibody. Roscovitine decreased Cdh1 levels by 50%, whereas MG132 increased Cdh1 levels by almost twofold (average of 2 experiments). The combination of MG132 and roscovitine treatment restored Cdh1 levels to 1.3-fold above control Cdh1 levels.