Cyclin A- and Cyclin E-Cdk Complexes Shuttle between the Nucleus and the Cytoplasm

Abstract

Cyclins A and E and their partner cyclin-dependent kinases (Cdks) are key regulators of DNA synthesis and of mitosis. Immunofluorescence studies have shown that both cyclins are nuclear and that a proportion of cyclin A is localized to sites of DNA replication. However, recently, both cyclin A and cyclin E have been implicated as regulators of centrosome replication, and it is unclear when and where these cyclin-Cdks can interact with cytoplasmic substrates. We have used live cell imaging to study the behavior of cyclin/Cdk complexes. We found that cyclin A and cyclin E are able to regulate both nuclear and cytoplasmic events because they both shuttle between the nucleus and the cytoplasm. However, we found that there are marked differences in their shuttling behavior, which raises the possibility that cyclin/Cdk function could be regulated at the level of nuclear import and export. In the course of these experiments, we have also found that, contrary to published results, mutations in the hydrophobic patch of cyclin A do affect Cdk binding and nuclear import. This has implications for the role of the hydrophobic patch as a substrate selection motif. INTRODUCTIONSuccessive waves of cyclin-dependent kinase (Cdk) activity control progress through the eukaryotic cell cycle. Cdks are activated by binding a member of the cyclin family and phosphorylation by Cdk-activating kinase. The cyclin-Cdks that have been most strongly implicated in controlling entry into, and progress through, DNA replication are cyclins A and E. Both cyclins bind to Cdk2 (Elledge and Spottswood, 1991;Tsai et al., 1991;Koff et al., 1992), and the levels of both cyclins are strictly regulated throughout the cell cycle, by transcriptional and proteolytic mechanisms. Cyclin E levels are primarily dictated by the rate of its transcription because it is an unstable protein that is rapidly degraded by two different pathways of ubiquitin-dependent proteolysis (Clurman et al., 1996;Won and Reed, 1996;Singer et al., 1999;Wang et al., 1999;Winston et al., 1999;Nakayama et al., 2000). In contrast, cyclin A is stable until cells enter mitosis. Cyclin A levels first start to increase at the beginning of S phase and continue to rise throughout S and G2 phases until prometaphase, when they are rapidly degraded by ubiquitin-dependent proteolysis (Pines and Hunter, 1990;Hunt et al., 1992).There is extensive evidence to indicate that the activation of cyclin E/Cdk2 leads to the initiation of DNA replication. Cyclin E/Cdk2 activity is maximal at G1/S (Koff et al., 1992), replication of DNA in vitro is dependent on cyclin E/Cdk2 activity (Jackson et al., 1995;Strausfeld et al., 1996;Krude et al., 1997) and, in vivo, cyclin E is essential for DNA replication in Drosophila (Sauer et al., 1995). Cyclin A/Cdk2 has also been demonstrated to promote DNA replication (Girard et al., 1991;Pagano et al., 1992;Zindy et al., 1992;Strausfeld et al., 1996). However, cyclin A may be more significant in regulating progression through S phase (Connell-Crowley et al., 1998), perhap...

Nuclear Localization of the Cell Cycle Regulator CDH1 and Its Regulation by Phosphorylation

“…In this regard, cyclin A is a known substrate of the CDH1-activated APC (59 -61), whereas cyclin E is degraded primarily through the SCF complex (62, 63), but not through APC. Consistent with previous studies (57,64), ambient cyclin A and cyclin E are predominantly nuclear (Fig. 2B, panels 2 and 5).…”

“…The nuclear localization of APC subunits has been documented (58,66). Meanwhile, the nuclear trafficking of CDKs and cyclins, including cyclin A and cyclin B1, has important roles in cell cycle regulation (57,64). In addition, the CDH1 phosphatase CDC14, which is sequestered in the nucleolus in budding yeast (32), is a nuclear protein in human cells (67).…”

Identification of the Nuclear Localization Signal inXenopusCyclin E and Analysis of Its Role in Replication and Mitosis

“…Because cyclin E and importin-␣ proteins purified from bacteria are able to associate without incubation in egg extracts, we can exclude the possibility that the phosphorylation of either protein by members of the MAP kinase family is required for their interaction. It is possible that Cdk2/cyclin E complexes shuttle in and out of somatic cell nuclei (Jackman et al, 2002), and perhaps MAP kinase activity is necessary to downregulate the export arm of this shuttling. It would be interesting to identify the pathway(s) of cyclin E localization in somatic cells and how they contribute to the biological function of the protein.…”

“…This was not because of reduced binding of cyclin A to the Cdk2, because cyclin A strongly activated the histone H1 kinase activity of the Texas Red-labeled GSTCdk2 preparation (data not shown). It is possible that the Texas Red labeling procedure had a greater inhibitory effect on the import of Cdk2/cyclin A than Cdk2/cyclin E, because the import pathways appear to be different for the two cyclins (Jackman et al, 2002). We therefore tested whether endogenous newly synthesized cyclin A was imported into nuclei.…”

The Biochemistry of Mitosis

“…This is a conserved feature of mitosis in animal cells in which the B-type cyclins have a nuclear export sequence in their amino terminus (Hagting et al 1998;Toyoshima et al 1998;Yang et al 1998) and an ill-defined nuclear import (Yang et al 1998;Takizawa et al 1999;Jackman et al 2002); as a result, the cyclin B-Cdk complex constantly shuttles between the nucleus and the cytoplasm in interphase, but the bulk of the protein is cytoplasmic. Coincident with its activation 20 min before NEBD (Lindqvist et al 2007;Gavet and Pines 2010b), a large fraction of cyclin B -Cdk1 moves quickly into the nucleus (Pines and Hunter 1991;Ookata et al 1992;Hagting et al 1998), and it was reported that Plk1 is required for this by inhibiting the nuclear export of human cyclin B1 -Cdk1 (Toyoshima-Morimoto et al 2002).…”