Stress-mediated exit to quiescence restricted by increasing persistence in CDK4/6 activation

Abstract

Mammalian cells typically start the cell-cycle entry program by activating cyclin-dependent protein kinase 4/6 (CDK4/6). CDK4/6 activity is clinically relevant as mutations, deletions, and amplifications that increase CDK4/6 activity contribute to the progression of many cancers. However, when CDK4/6 is activated relative to CDK2 remained incompletely understood. Here, we developed a reporter system to simultaneously monitor CDK4/6 and CDK2 activities in single cells and found that CDK4/6 activity increases rapidly before CDK2 activity gradually increases, and that CDK4/6 activity can be active after mitosis or inactive for variable time periods. Markedly, stress signals in G1 can rapidly inactivate CDK4/6 to return cells to quiescence but with reduced probability as cells approach S phase. Together, our study reveals a regulation of G1 length by temporary inactivation of CDK4/6 activity after mitosis, and a progressively increasing persistence in CDK4/6 activity that restricts cells from returning to quiescence as cells approach S phase.

CDK2 activity determines the timing of cell-cycle commitment and sequential DNA replication

“…To explore how CDK4/6 and CDK2 coordinate the Rb/E2F pathway in relation to APC Cdh1 inactivation, we used live-cell reporters for CDK4/6, CDK2, and APC/C Cdh1 activities [8][9][10]30,31 in human epithelial MCF-10A cells (Fig. 1a).…”

“…1b). To account for CDK2 activity in S and G2 phase that can be captured by the CDK4/6 reporter, we previously derived a corrected CDK4/6 activity by subtracting a calculated fraction (35%) of the CDK2 reporter signal from the CDK4/6 reporter signal in MCF-10A cells 9 .…”

“…In asynchronously cycling cells after mitosis, cells continuously trigger CDK4/6 activation, Rb phosphorylation, and high E2F activity and immediately start CDK2 activation to continue cell-cycle entry 9,33 . Therefore, to explore the initiation of the Rb/E2F pathway by CDK4/6 and CDK2, we synchronized cells in quiescence (G0) by mitogen starvation for 48 hours.…”

“…In mitogen-starved cells, Pardee first defined a specific point in late G1, the restriction point, where cells commit to the cell cycle regardless of mitogen availability 5 . In addition to mitogenic signaling, stress signals including DNA damage and osmotic stress can also regulate cell-cycle entry [6][7][8][9] . In asynchronously cycling cells, recent studies demonstrated that while cells can commit to the next cell cycle in G2 phase of mother cells with respect to mitogen availability 10 , stress can reverse cell-cycle entry until inactivation of the anaphase-promoting complex/cyclosome-Cdh1 (APC/C Cdh1 ) at the G1/S transition 8,11 .…”

“…Here, we investigated regulation of the Rb/E2F pathway by CDK4/6 and CDK2 in relation to the commitment point by using a live-cell reporter for CDK4/6 activity 9 . We propose that CDK2-Rb feedback is the primary signaling network regulating cell-cycle commitment with respect to mitogenic and DNA damage signaling.…”