Mitogen-dependent progression through the first gap phase (G1) and initiation of DNA synthesis (S phase) during the mammalian cell division cycle are cooperatively regulated by several classes of cyclin-dependent kinases (CDKs) whose activities are in turn constrained by CDK inhibitors (CKIs). CKIs that govern these events have been assigned to one of two families based on their structures and CDK targets. The first class includes the INK4 proteins (inhibitors of CDK4), so named for their ability to specifically inhibit the catalytic subunits of CDK4 and CDK6. Four such proteins [p16 (Serrano et al. 1993), p15 (Hannon and Beach 1994), p18 (Guan et al. 1994; Hirai et al. 1995), and p19 (Chan et al. 1995; Hirai et al. 1995)] are composed of multiple ankyrin repeats and bind only to CDK4 and CDK6 but not to other CDKs or to D-type cyclins. The INK4 proteins can be contrasted with more broadly acting inhibitors of the Cip/Kip family whose actions affect the activities of cyclin D-, E-, and A-dependent kinases. The latter class includes p21 (Gu et al. 1993; Harper et al. 1993; El-Deiry et al. 1993; Xiong et al. 1993a; Dulic et al. 1994; Noda et al. 1994), p27 (Polyak et al. 1994a,b; Toyoshima and Hunter 1994), and p57 (Lee et al. 1995; Matsuoka et al. 1995), all of which contain characteristic motifs within their amino-terminal moieties that enable them to bind both to cyclin and CDK subunits (Chen et al. 1995, 1996; Nakanishi et al. 1995; Warbrick et al. 1995; Lin et al. 1996; Russo et al. 1996). Based largely on in vitro experiments and in vivo overexpression studies, CKIs of the Cip/Kip family were initially thought to interfere with the activities of cyclin D-, E-, and A-dependent kinases. More recent work has altered this view and revealed that although the Cip/Kip proteins are potent inhibitors of cyclin Eand A-dependent CDK2, they act as positive regulators of cyclin Ddependent kinases. This challenges previous assumptions about how the G1/S transition of the mammalian cell cycle is governed, helps explain some enigmatic features of cell cycle control that also involve the functions of the retinoblastoma protein (Rb) and the INK4 proteins, and changes our thinking about how either p16 loss or overexpression of cyclin D-dependent kinases contribute to cancer. Here we focus on the biochemical interactions that occur between CKIs and cyclin Dand E-dependent kinases in cultured mammalian cells, emphasizing the manner by which different positive and negative regulators of the cell division cycle cooperate to govern the G1-to-S transition. To gain a more comprehensive understanding of the biology of CDK inhibitors, readers are encouraged to refer to a rapidly emerging but already extensive literature (for review, see Elledge and Harper 1994; Sherr and Roberts 1995; Chellappan et al. 1998; Hengst and Reed 1998a; Kiyokawa and Koff 1998; Nakayama 1998; Ruas and Peters 1998).

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CDK inhibitors: positive and negative regulators of G1-phase progression

The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases

The retinoblastoma protein and cell cycle control

As key regulators of the cell cycle, the cyclin-dependent kinases must be tightly regulated by extra- and intracellular signals. The activity of cyclin-dependent kinases is controlled by four highly conserved biochemical mechanisms, forming a web of regulatory pathways unmatched in its elegance and intricacy.

Principles of CDK regulation

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Inhibitors of mammalian G1 cyclin-dependent kinases.

Human cyclin E, originally identified on the basis of its ability to function as a G1 cyclin in budding yeast, associated with a cell cycle-regulated protein kinase in human cells. The cyclin E-associated kinase activity peaked during G1, before the appearance of cyclin A, and was diminished during exit from the cell cycle after differentiation or serum withdrawal. The major cyclin E-associated kinase in human cells was Cdk2 (cyclin-dependent kinase 2). The abundance of the cyclin E protein and the cyclin E-Cdk2 complex was maximal in G1 cells. These results provide further evidence that in all eukaryotes assembly of a cyclin-Cdk complex is an important step in the biochemical pathway that controls cell proliferation during G1.

Formation and activation of a cyclin E-cdk2 complex during the G1 phase of the human cell cycle

We have analyzed the activation of human cyclin-dependent kinases in a cell-free system. Human CDC2, cyclin-dependent kinase 2 (CDK2), cyclin A, and cyclin B1 were produced in insect cells by infection with recombinant baculoviruses. CDC2 or CDK2 monomers in lysates of infected cells could be activated by the addition of lysates containing cyclin A or B1. CDC2 activation by cyclin B1, as well as CDK2 activation by cyclins A and B1, was accompanied by the formation of high molecular weight complexes. In contrast, CDC2 did not bind effectively to cyclin A. CDC2 activation by cyclin B1 was studied in detail and was found to be accompanied by phosphorylation of CDC2 on Threonine 161. The binding of CDC2 to cyclin B1 also occurred under conditions where CDC2 phosphorylation was prevented, resulting in an inactive complex that could then be phosphorylated and activated on addition of cell extract. Highly purified CDC2 and cyclin B1 also formed inactive complexes that could be activated in an ATP-dependent fashion by unidentified components in crude cell extracts. These data suggest that the CDC2 activation process begins with cyclin binding, after which CDC2 phosphorylation, catalyzed by a separate enzyme, leads to activation.

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Activation of human cyclin-dependent kinases in vitro.

The cyclin-dependent protein kinases (CDKs) are activated by association with cyclins and by phosphorylation at a conserved threonine residue by the CDK-activating kinase (CAK). We have studied the binding of various human CDK and cyclin subunits in vitro, using purified proteins derived from baculovirus-infected insect cells. We find that most CDK-cyclin complexes known to exist in human cells (CDC2-cyclin B, CDK2-cyclin A, and CDK2-cyclin E) form with high affinity in the absence of phosphorylation or other cellular components. One complex (CDC2-cyclin A) forms with high affinity only after CAK-mediated phosphorylation of CDC2 at the activating threonine residue. CDC2 does not bind with high affinity to cyclin E in vitro, even after phosphorylation of the CDC2 subunit. Thus, phosphorylation is of varying importance in the formation of high-affinity CDK-cyclin complexes.

D Desai

Papers

Formation and activation of a cyclin E-cdk2 complex during the G1 phase of the human cell cycle

Activation of human cyclin-dependent kinases in vitro.

Effects of phosphorylation by CAK on cyclin binding by CDC2 and CDK2.