Skp2 induction and phosphorylation is associated with the late G1 phase ofproliferating fat hepatocytes

The changes in phosphoproteins purified with the affinity peptide p9CKShs1 were analyzed from extracts of regenerating rat livers in order to define s ome G1 and G1/S regulations characteristic of mature hepatocytes stimulated to proliferate. We observed a 47 kDa phosphoprotein that occurred first at the end of G1 before peaking in the S phase. P47 was also found to be phos phorylated in late G1 in primary hepatocyte cultures stimulated with mitoge ns, P47 was still phosphorylated in extracts depleted of Cdc2, but to a les ser extent after Cdk2 depletion. This phosphoprotein was identified as Skp2 . (i) P47 shared the same electrophoretic mobility than Skp2, a cell cycle protein essential for S phase entry in human fibroblasts; (ii) Skp2, like P 47, started to be expressed and was highly phosphorylated during the G1/S t ransition of hepatocytes stimulated to proliferate in vivo and in vitro; (i ii) P47 was specifically immunoprecipitated by an antibody directed against Skp2, In addition, cyclin A/Cdk2 complexes from regenerating liver clearly interacted with Skp2. This is the first demonstration that Skp2 is induced and phosphorylated in the late G1 and S phase of hepatocytes in vivo in re generating liver as well as in vitro in mitogen-stimulated hepatocytes. (C) 1999 Federation of European Biochemical Societies.