Cyclin-dependent Kinase 5 Promotes Myc Stabilization Through Inhibitory Phosphorylation of GSK-3β in Ewing Sarcoma

Glycogen synthase kinase-3β (GSK-3β) is a serine/threonine kinase known to function as a tumor suppressor by promoting ubiquitination and degradation of the Myc protein. In this study, we investigated the role of cyclin-dependent kinase 5 (CDK5) in regulating GSK-3β kinase activity, as well as Myc phosphorylation and ubiquitination.

Four Ewing sarcoma cell lines were used in this study. Cells were transfected with a CDK5 expression vector, and cell proliferation, cell-cycle progression, and apoptosis were analyzed. We examined whether CDK5 alters the phosphorylation status of GSK-3β. In addition, siRNA-mediated knockdown and expression vectors were used to modulate CDK5 and GSK-3β expression, and the effects on Myc ubiquitination were evaluated.

Compared with control cells, CDK5-overexpressing Ewing sarcoma cells showed increased proliferative capacity and enhanced cell-cycle progression. Under these conditions, dephosphorylation of GSK-3β at Tyr216 and phosphorylation at Ser9 as well as Ser389 - patterns consistent with the inactive form of GSK-3β - were observed. Concomitantly, ubiquitination of the Myc protein was reduced. In contrast, cells transfected with a GSK-3β expression vector exhibited increased Myc phosphorylation and ubiquitination.

These findings suggest that functional impairment of GSK-3β and subsequent upregulation of Myc may promote the proliferation of Ewing sarcoma cells. Myc is normally phosphorylated by GSK-3β, which triggers its ubiquitination and proteasomal degradation. Although CDK5 is recognized as a cell-cycle-related kinase, our results demonstrate that CDK5 is associated with increased inhibitory phosphorylation of GSK-3β, resulting in functional suppression of its activity. As a result, GSK-3β-mediated phosphorylation of Myc is attenuated, leading to reduced ubiquitination and accumulation of Myc. Regulation of Myc expression through CDK5-mediated modulation of GSK-3β plays a critical role in controlling Ewing sarcoma cell proliferation and may represent a promising therapeutic target.