PKN activation via transforming growth factor-beta 1 (TGF-beta 1) receptor signaling delays G2/M phase transition in vascular smooth muscle cells.

The transition of vascular smooth muscle cells (VSMCs) from G2 phase into the M (mitosis) phase of the cell cycle is a tightly controlled process. As an arterial SMC prepares for a G2/M transition, the cell has primed the Cdc2/cyclinB1 complex for activation by the phosphorylation of threonine-161 residue on Cdc2. This phosphorylation is necessary but not sufficient for the VSMC to enter into the M phase. In order to enter into mitosis, a phosphatase, Cdc25C, must first dephosphorylate two other critical residues: tyrosine-15 and threonine-14. If Cdc25C phosphatase activity is blocked, VSMC entry into mitosis is delayed. However, how the activity of Cdc25C is regulated has not been fully illustrated. In an earlier published study we have demonstrated that exposure of the VSMC line, PAC-1, to Transforming growth factor-beta1 (TGF-beta1), activated PKN (a RhoA-dependent kinase). Here we show that exposure to TGF-beta1 delays the G2/M transition by 2 hrs in G1/S synchronized and released PAC-1 culture. This delay is abolished by the RhoA kinase inhibitors, HA1077 or Y-27632. More importantly, knockdown of PKN expression prevents the G2/M transition delay induced by TGF-beta1. Changes in PKN activity temporally correlates to the G2/M transition timing. Moreover, Cdc25C is phosphorylated by the TGF-beta1-activated PKN. PKN and Cdc25C coimmunoprecipitate with each other. Finally, PKN and Cdc25C colocalize to the nuclear region only during the critical period of time prior to entry into the M phase. Our data demonstrate that Cdc25C activity is negatively regulated by TGF-beta1-stimulated PKN. Once activated through TGF-beta1 signaling, PKN binds to and phosphorylates Cdc25C. The physical interaction and phosphorylation result in an inactivation of Cdc25C and delay the VSMC entry into the M stage of the cell cycle.

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