Fig. 5. Cdc2 phosphorylation of caldesmon in injury-preconditioned Schwann cells. (A) Western blot analysis of phospho-caldesmon in cultured Schwann cells. Phospho-caldesmon protein was greatly increased in cultured Schwann cells with injury preconditioning for 7 days. As a control, Schwann cell lysate was prepared from the sciatic nerve in rats at postnatal day 3 (pnd3). (B) Identification of phospho-caldesmon protein in Schwann cells by western blot analysis. Caldesmon pulled down by immunoprecipitation of actin was used as substrate for the kinase reaction by exogenous Cdc2 enzyme (lane 1). Kinase reaction in the presence of roscovitine is shown in lane 2 and the reaction without Cdc2 in lane 3. (C) In vitro kinase assay for caldesmon phosphorylation by exogenous Cdc2. Caldesmon pulled down by immunoprecipitation of actin was used as a substrate for the kinase reaction. Inclusion or exclusion of roscovitine (rosco) or histone H1 in the incubation mixture are indicated respectively by + or –, respectively. Caldesmon phosphorylation by Cdc2 was decreased by the cdk inhibitor, roscovitine or by competition with the alternative substrate, histone H1. (D) Inhibition of caldesmon phosphorylation by dn-Cdc2 expression. Schwann cells were cotransfected with pGFP and pCMV5 vector or pGFP and pCMVdn-Cdc2. A cell group transfected with dn-Cdc2 was less positive for phospho-caldesmon immunostaining than the vector control. The graph shows a significant reduction of phospho-caldesmon-positive (+) cells among the GFP-positive (+) transfected cell population by dn-Cdc2-transfection. (n=3, mean ± s.e.m.). Western blot analysis shows significant suppression of phospho-caldesmon immunopositivity in Schwann cells that had been injury preconditioned and transfected with dn-Cdc2. (E) Subcellular localization of caldesmon in injury-preconditioned Schwann cells. The perinuclear distribution of caldesmon in vehicle-treated cultures contrasts with the peripheral distribution in cultures treated with 10 µM roscovitine (arrow). Bars, 30 µm (D); 20 µm (E). Actin was detected as an internal loading control.