Identification of a Myc-dependent step during the formation of active G1 cyclin-cdk complexes.

Abstract

Activation of conditional alleles of Myc can induce proliferation in quiescent cells. We now report that induction of Myc in density‐arrested fibroblasts triggers rapid hyperphosphorylation of the retinoblastoma protein and activation of both cyclin D1‐ and cyclin E‐associated kinase activities in the absence of significant changes in the amounts of cyclin‐cdk complexes. Kinase activation by Myc is blocked by inhibitors of transcription and requires intact DNA binding and heterodimerization domains of Myc. Activation of cyclin E‐cdk2 kinase in serum‐starved cells occurs in two steps. The first is induced by Myc and involves the release of a 120 kDa cyclin E‐cdk2 complex from a 250 kDa inactive complex that is present in starved cells. This is necessary, but not sufficient, to generate full kinase activity, as cdc25 phosphatase activity is limiting in the absence of external growth factors. In vivo cdc25 activity can be supplied by the addition of growth factors. In vitro recombinant cdc25a strongly activates the 120 kDa, but only poorly activates the 250 kDa cyclin E‐cdk2 complex. Our data show that two distinct signals, one of which is supplied by Myc, are necessary for consecutive steps during growth factor‐induced formation of active cyclin E‐cdk2 complexes in G(o)‐arrested rodent fibroblasts.

Malignant transformation by cyclin E and Ha-Ras correlates with lower sensitivity towards induction of cell death but requires functional Myc and CDK4

“…One possibility is that overexpression of D-or E-type cyclins leads to a higher associated kinase activity simply because more cyclin/CDK complexes are formed. However, higher cyclin/CDK kinase activity is not necessarily linked to higher amounts of the individual partner proteins because it depends on the activity of CAKs (CDK activating kinases) and the degree of phosphorylation (for a review see Draetta, 1997) and, in addition, cyclin/ CDK kinase complexes can be activated by releasing them from inactive high molecular weight complexes with inhibitors by still poorly de®ned biochemical steps (Steiner et al, 1995;Vlach et al, 1996). Thus, higher levels of a particular cyclin would not necessarily lead to the formation of more cyclin/CDK dimers with kinase activity.…”

“…It has been previously proposed that one signalling pathway of cyclin D/CDK4 functions via the phosphorylation of the Rb related pocket protein p107 and the release of the Myc/Max transcription factor (Beijersbergen et al, 1994(Beijersbergen et al, , 1995Gu et al, 1994). Taking into account recent ®ndings about the activation of the cyclin E/ CDK2 kinase by the induction of Myc through conditional alleles (Steiner et al, 1995) we had postulated that Myc acts downstream of cyclin D/ CDK4 and is regulated via the Rb related pocket protein p107 that is in turn uncontrolled by cyclin D/CDK4 activity and p16 (Haas et al, 1997). In this model, Myc was placed upstream of cyclin E/CDK2 (Steiner et al, 1995) and downstream of cyclin D/CDK4 (Haas et al, 1997).…”

“…Taking into account recent ®ndings about the activation of the cyclin E/ CDK2 kinase by the induction of Myc through conditional alleles (Steiner et al, 1995) we had postulated that Myc acts downstream of cyclin D/ CDK4 and is regulated via the Rb related pocket protein p107 that is in turn uncontrolled by cyclin D/CDK4 activity and p16 (Haas et al, 1997). In this model, Myc was placed upstream of cyclin E/CDK2 (Steiner et al, 1995) and downstream of cyclin D/CDK4 (Haas et al, 1997). This model would also provide an explanation for the requirement of CDK4 kinase activity for the oncogenic activity of cyclin E. While it is possible that high level expression of cyclin E titrates enough p27 away to activate endogenous cyclin E/CDK2 kinase activity, a block of CDK4 by p16 for instance would lead to sequestration and inhibition of Myc/Max through underphosphorylated p107 (Beijersbergen et al, 1994(Beijersbergen et al, , 1995.…”

“…In addition to the regulation of cyclin E or cyclin D expression through the modulation of the promotor, the enzymatic activity of D-and E-type cyclin/CDK complexes is regulated independently on a posttranscriptional level. The transcription factor Myc, for example, which is a paradigmatic immediate early factor in response to mitogen stimulation with a clear involvement in G1 progression (for a review see Meichle et al, 1992) may be responsible for cyclin E/ CDK2 activation without changing the steady state level of the cyclin E or CDK2 protein itself (Steiner et al, 1995). The cyclin dependent kinase inhibitors (CKIs) p27 and p21 both inhibit D-and E-type cyclin/CDK activity and at least D-type cyclin/CDK complexes seem to depend on the activity of Myc to exert their function in G1 progression or for their activity as oncoproteins (Roussel et al, 1995;Haas et al, 1997).…”

Pim-1 kinase stimulates c-Myc-mediated death signaling upstream of caspase-3 (CPP32)-like protease activation

“…It has been reported that the expression of p27 cyclin-dependent kinase (cdk) inhibitor is under negative posttranslational regulation by c-Myc (Steiner et al, 1995). Since p27 has been shown to confer resistance against anticancer agents to tumor cells (Croix et al, 1996), it appeared likely that p27 has antiapoptotic activity similar to other cdk inhibitors such as p16 and p21 (Wang and Walsh, 1996;Poluha et al, 1996).…”