Mphase

CDK1/cyclin A and CDK1/cyclin B

Mitosis is characterized by nuclear envelope breakdown and chromosome alignment on the mitotic spindle. Sister chromatid separation follows as they move towards opposite poles of the spindle permitting the formation of daughter nuclei. Central to mitotic entry and orchestration of mitotic events is the activation of CDK1. As mentioned above, CDK kinase activity is controlled by phosphorylation and de-phosphorylation events in addition to cyclin binding (reviewed in [79]). Critical de-phosphoryla-tion events occur at the G2/M transition when de-phosphorylation events outnumber phosphoryla-tion events (cdc25 phosphatase activity exceeds Wee1/Myt1 kinase activity). Regulation of CDK1 kinase activity must be strictly coordinated as constitutive CDK1 activation was found to prevent chromosomal condensation, nuclear envelope re-assembly, and cell division [80].

The formation of CDK1 /cyclin A and CDK1 /cyclin B complexes is important for M-phase initiation, progression, and exit. Associations between cyclins A and B and CDK1 are initiated during G2 and are required for activation of chromosome condensation, nuclear envelope breakdown, and spindle assembly (reviewed in [81]).

The periodic activation of maturation promoting factor (MPF), identified as CDK1 /cyclin B complexes, results in the phosphorylation of critical mitotic regulators such as cyclin B and securin [82]. The upregulation of cyclin B promotes CDK1/cyclin B complex formation responsible for activation of the APC E3 ubiquitin ligase, which in turn controls mitotic cyclin degradation [83,84]. APC functions as a multi-subunit ubiquitin ligase complex and in conjunction with APC activator proteins (e.g., cdc20, cdh1), contributes to cell cycle regulation via the timed, sequential ubiquitination, and proteolysis of cyclins A and B as well as securin (reviewed in [85]). The APC itself is regulated by the spindle or kine-tochore (point of chromosome attachment to mitotic spindle fibers) checkpoint which prevents sister chromatid separation until all chromosomes are correctly aligned on the mitotic spindle (reviewed in [86]). Early in S-phase, the Early Mitotic Inhibitor 1 (emi1) protein inhibits the APC by binding to cdc20 [87-89] (Fig. 9.6). Proteolysis of emi1 in prophase renders the APC active and capable of association with cdc20 and destruction of cyclin A occurs [90]. Upon exit from mitosis, the APC retains activity and binds to cdh1 which is held inactive by CDK1 [91,92]. It is not yet known to what extent the APC is phosphorylated in order to remain active.

Mitotic Checkpoints

APC, bound to the cdc20 subunit controls the activity of the Mad2 and BubR1, regulatory components of the spindle assembly checkpoint (reviewed in [91]). It has been demonstrated that the spindle checkpoint machinery is required to recruit APC to the kinetochores [93]. Recently, the RASSF1A tumor suppressor, the expression of which is frequently silenced in cancers, was found to act as an inhibitor of cdc20 preventing mitotic progression due to premature APC activation [94].

A critical target of mitotic control is separase, responsible for anaphase progression [95]. Separase is inactive when bound to securin which is in turn inhibited by the APC (Fig. 9.6). When kinetochores are properly attached to the spindle, APC binds to cdc20 and targets securin for degradation, liberating separase. Relieved from securin binding, separase is free to cleave cohesin molecules between sister

figure 9.6 The Anaphase-Promoting Complex (APC). Maturation-promoting factor (MPF) consisting of CDKl/cyclin B complexes initiates entry into mitosis late in S-phase. cdc25A phosphatase contributes to maintenance of CDKl/cyclin B activity. At the same time, the APC co-factor cdc20, rendered inactive by binding to emi-1, is liberated by targeted degradation of emi-1. The active APC/cdc20 ubiquitin ligase targets mitotic molecules such as cyclin B and securin for degradation by ligation of ubiquitin moieties (encircled Ub). Securin normally functions to maintain separase in an inactive state. Once activated, separase promotes sister chromatid separation. Late in M-phase, APC associates with cdhl targeting cdc20 as well as cdc25A for degradation. See Plate 9.6 in Color Plate Section.

figure 9.6 The Anaphase-Promoting Complex (APC). Maturation-promoting factor (MPF) consisting of CDKl/cyclin B complexes initiates entry into mitosis late in S-phase. cdc25A phosphatase contributes to maintenance of CDKl/cyclin B activity. At the same time, the APC co-factor cdc20, rendered inactive by binding to emi-1, is liberated by targeted degradation of emi-1. The active APC/cdc20 ubiquitin ligase targets mitotic molecules such as cyclin B and securin for degradation by ligation of ubiquitin moieties (encircled Ub). Securin normally functions to maintain separase in an inactive state. Once activated, separase promotes sister chromatid separation. Late in M-phase, APC associates with cdhl targeting cdc20 as well as cdc25A for degradation. See Plate 9.6 in Color Plate Section.

chromatids facilitating the metaphase to anaphase transition. Another APC complex activator, cdhl, is phosphorylated by CDKl until anaphase at which point cdhl is subjected to de-phosphorylation events facilitating its binding to APC [96]. APC-cdhl is responsible for the degradation of cdc20, inactivating the APC-cdc20 complex and orchestrating the cell's exit from M-phase [97]. Expression of APC-cdhl in quiescent cells suggests its importance in Gl regulation. In fact, APC-cdhl complexes are readily detectable and abundant in tissues composed of post-mitotic cells (e.g., brain). APC-cdhl directly affects cell cycle during G0/Gl by preventing accumulation of S- and M-phase cyclins [98]. Identification of the APC and its role in cell cycle regulation is a relatively new field of study. Mutations in APC components have not yet been identified in human brain tumors.

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