Abstract
According to current concepts, the cell cycle commitment after restriction (R) point passage requires the sustained stimulation by mitogens of the synthesis of labile d-type cyclins, which associate with cyclin-dependent kinase (CDK) 4/6 to phosphorylate pRb family proteins and sequester the CDK inhibitor p27kip1. In primary cultures of dog thyroid epithelial cells, the cAMP-dependent cell cycle induced by a sustained stimulation by thyrotropin or forskolin differs from growth factor mitogenic pathways, as cAMP does not upregulate d-type cyclins but increases p27 levels. Instead, cAMP induces the assembly of required cyclin D3-CDK4 complexes, which associate with nuclear p27. In this study, the arrest of forskolin stimulation rapidly slowed down the entry of dog thyrocytes into S phase and the phosphorylation of pRb family proteins. The pRb kinase activity, but not the formation, of the cyclin D3-CDK4-p27 complex was strongly reduced. Using two-dimensional gel electrophoresis, a phosphorylated form of CDK4 was separated. It appeared in response to forskolin and was bound to both cyclin D3 and p27, presumably reflecting the activating Thr-172 phosphorylation of CDK4. Upon forskolin withdrawal or after cycloheximide addition, this CDK4 phosphoform unexpectedly persisted in p27 complexes devoid of cyclin D3 but it disappeared from the more labile cyclin D3 complexes. These data demonstrate that the assembly of the cyclin D3-CDK4-p27 holoenzyme and the subsequent phosphorylation and activation of CDK4 depend on distinct cAMP actions. This provides a first example of a crucial regulation of CDK4 phosphorylation by a mitogenic cascade and a novel mechanism of cell cycle control at the R point.
Highlights
One division cycle [1]
The cell cycle commitment after restriction (R) point passage requires the sustained stimulation by mitogens of the synthesis of labile D-type cyclins, which associate with cyclindependent kinase (CDK) 4/6 to phosphorylate pRb family proteins and sequester the CDK inhibitor p27kip1
The accumulation of labile D-type cyclins (rather than cyclin E [16]) over an inhibitory threshold imposed by INK4 CDK inhibitors constitutes the sensor element of the sustained action of mitogens that leads to the phosphorylation of pRb-related proteins and to restriction point (R point) passage [7, 17,18,19]
Summary
One division cycle [1]. Whereas the progression through the pre-R portion of G1 strictly depends on the sustained cell stimulation by external growth factors and mitogens, the passage through the R point results in the triggering of an intrinsic machinery autonomously ordering cell cycle completion. The cell cycle commitment after restriction (R) point passage requires the sustained stimulation by mitogens of the synthesis of labile D-type cyclins, which associate with cyclindependent kinase (CDK) 4/6 to phosphorylate pRb family proteins and sequester the CDK inhibitor p27kip1.
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