D-type cyclin-dependent kinase activity in mammalian cells.

Abstract

D-type cyclin-dependent kinase activities have not so far been detected in mammalian cells. Lysis of rodent fibroblasts, mouse macrophages, or myeloid cells with Tween 20 followed by precipitation with antibodies to cyclins Dl, D2, and D3 or to their major catalytic partner, cyclin-dependent kinase 4 (cdk4), yielded kinase activities in immune complexes which readily phosphorylated the retinoblastoma protein (pRb) but not histone Hi or casein. Virtually all cyclin Dl-dependent kinase activity in proliferating macrophages and fibroblasts could be attributed to cdk4. When quiescent cells were stimulated by growth factors to enter the cell cycle, cyclin Dl-dependent kinase activity was first detected in mid G1, reached a maximum near the G1/S transition, and remained elevated in proliferating cells. The rate of appearance of kinase activity during G1 phase lagged significantly behind cyclin induction and correlated with the more delayed accumulation of cdk4 and formation of cyclin Dl-cdk4 complexes. Thus, cyclin Dl-associated kinase activity was not detected during the Go-to-G1 transition, which occurs within the first few hours following growth factor stimulation. Rodent fibroblasts engineered to constitutively overexpress either cyclin Dl alone or cyclin D3 together with cdk4 exhibited greatly elevated cyclin D-dependent kinase activity, which remained absent in quiescent cells but rose to supraphysiologic levels as cells progressed through G1. Therefore, despite continued enforced overproduction of cyclins and cdk4, the assembly of cyclin D-cdk4 complexes and the appearance of their kinase activities remained dependent upon serum stimulation, indicating that upstream regulators must govern formation of the active enzymes.D-and E-type cyclins govern the rate of progression of mammalian cells through the first gap phase (Gl) of the cell cycle and enforce the commitment of cells to replicate their chromosomal DNA (reviewed in reference 44). When quiescent cells enter the cell cycle, both classes of cyclins are synthesized during G, phase and associate with cyclin-dependent kinases (cdks) to form holoenzymes whose activities are presumed to facilitate entry into S phase.Cyclin E, first identified by complementation of G, cyclin deficiency in yeasts (22,25), is synthesized in late G, phase and associates with cdk2 to generate a histone Hi kinase that is maximally active during the Gl-to-S phase transition in mammalian cells (10, 23). The enforced overexpression of cyclin E in fibroblasts shortens their G, interval, decreases (but does not eliminate) their dependency on serum growth factors, and leads to a reduction in their size, indicating that cyclin E can be rate limiting for G1 progression (33). Its catalytic partner, cdk2, is also necessary for S-phase entry (34, 48), but it remains unclear whether this is due to its association with cyclin E and/or with cyclin A, which is expressed somewhat later during the cell cycle and steadily accumulates during the S and G2 phases (30,38,39). The activity of the cyclin E-c...

Identification of a p130 domain mediating interactions with cyclin A/cdk 2 and cyclin E/cdk 2 complexes

“…P130 has been shown to interact in vitro with D cyclins, presumably through a similar mechanism . Cyclin D/cdk4 complexes have been shown to phosphorylate pRB and p107 in vitro (Dowdy et al, 1993;Ewen et al, 1993;Kato et al, 1993;Li et al, 1993;Matsushime et al, 1994) and overexpression of D cyclins supports the phosphorylation of pRB, p107 and p130 in vivo Kato et al, 1993;Beijersbergen et al, 1995;Xiao et al, 1996;our unpublished results).…”

The antidiabetic drug metformin exerts an antitumoral effect in vitro and in vivo through a decrease of cyclin D1 level

“…Cyclin D1 functions are primarily to bind and activate the cyclin dependent kinase (CDK) 4/6, which then phosphorylates the retinoblastoma protein (Rb). Upon phosphorylation Rb releases the transcription factor E2F, which is then able to activate the transcription of genes required for G 1 /S phase transition (Matsushime et al, 1994;Weinberg, 1995). The cyclin D1/CDK4/6 complex is also able to sequester p27 kip1 and other CDK inhibitory proteins, thereby neutralizing their inhibitory capacity for cyclin E/CDK2 (Sherr and Roberts, 1995) whose activity is required for G 1 /S transition (Hunter, 1997).…”

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

“…In this regard, cyclin E is thought to function downstream of cyclin D1 and is rate limiting for G1/S transition (Ko et al, 1992;Dulic et al, 1992;Pagano et al, 1993;Ohtsubo et al, 1995). A considerable number of experimental data suggests that the tumour suppressor protein Rb is one of the substrates of the cyclin D1/CDK4 and the cyclin E/ CDK2 kinase (Dowdy et al, 1993;Ewen et al, 1993;Hinds et al, 1994;Kato and Sherr, 1993;Matsushime et al, 1994;Meyerson and Harlow, 1994;Hu et al, 1992;Sherr, 1995). According to this hypothesis, Rb is hyperphosphorylated by either D-type or E-type cyclin/ CDK kinases and allows progression to the late G1 phase by releasing transcription factors of the E2F family that prepare the entry into S-phase.…”