PKN Activation via Transforming Growth Factor-β1 (TGF-β1) Receptor Signaling Delays G2/M Phase Transition in Vascular Smooth Muscle Cells

Abstract

Previouisly published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=3985 Key woRDSPKN, TGF-β 1, smooth muscle, G 2 /M phase, cell cycle ACKnowleDGeMenTSWe thank Dr. Yoshitaka Ono for providing the PKN constructs. AbSTRACTThe transition of vascular smooth muscle cells (VSMCs) from G 2 phase into the M (mitosis) phase of the cell cycle is a tightly controlled process. As an arterial SMC prepares for a G 2 /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-β1 (TGF-β1), activated PKN (a RhoA-dependent kinase). Here we show that exposure to TGF-β1 delays the G 2 /M transition by 2 hrs in G 1 /S synchronized and released PAC-1 culture. This delay is abolished by the RhoA kinase inhibitors, HA1077 or Y-27632. More importantly, RNAi knockdown of PKN expression prevents the G 2 /M transition delay induced by TGF-β1. Changes in PKN activity temporally correlates to the G 2 /M transition timing. Moreover, Cdc25C is phosphorylated by the TGF-β1-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-β1-stimulated PKN. Once activated through TGF-β1 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.

RETRACTED: Theaflavins induce G2/M arrest by modulating expression of p21waf1/cip1, cdc25C and cyclin B in human prostate carcinoma PC-3 cells

“…De-phosphorylation of these negative regulatory sites is needed to activate the cdc2/cyclin B complex. Cdc25C is a major phosphatase that de-phosphorylates both sites on cdc2 to activate them (Su et al, 2007;Roshak et al, 2000). The decreased expression of cdc25C by TF treatment, as shown here, inhibited de-phosphorylation of cdc2 (Thr14 and Tyr15), resulting in inhibition of the cdc2/cyclin B complex formation.…”

Identification of an Interaction between the TPα and TPβ Isoforms of the Human Thromboxane A2 Receptor with Protein Kinase C-related Kinase (PRK) 1

“…Interestingly, in a follow-up study, PRK1 was found to specifically phosphorylate the related TRAF1, which lacks the JNK/IKK signaling effector domain but not its interactant TRAF2 or other TRAF members, leading to the recruitment of TRAF1 to the TNF receptor, silencing the receptor complex by PRK1 (55). Moreover, within the vasculature, PRK1 has been implicated in the mediation of VSM-specific gene expression promoting VSM differentiation, such as in response to transforming growth factor-␤1 (56,57). Keeping in mind the central role of TXA 2 within the vasculature, including the mediation of inflammatory disease, coupled with the finding herein of a direct interaction with and activation of PRK1, it will be of significant interest to investigate the possible interplay between those critical pathways, be it at the cellular or (patho)physiologic levels.…”

Bromelain inhibits nuclear factor kappa‐B translocation, driving human epidermoid carcinoma A431 and melanoma A375 cells through G2/M arrest to apoptosis

“…Since the major regulator of G 2 to M transition is the M phase-promoting factor (MPF) [ 41,42], we proceeded by studying the effect of bromelain on this complex in terms of formation (via cdc2 and cyclin B1 levels) and phosphorylation status in A431 cells. Cdc25C is known as major phosphatase which dephosphorylates both sites of cdc2 to activate them [43,44]. We saw that cdc25C-phosphorylation (Ser198), normally required for its activation, was inhibited by bromelain which thereby curtailed the phosphorylation of cdc2 at Tyr15, inhibiting cdc2/cyclin B complex formation.…”