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  • Although intact Chk is distributed throughout the nucleus an

    2019-12-02

    Although intact Chk is distributed throughout the nucleus and the cytoplasm (Fig. 1), the deletion of the SH2 domain greatly augments nuclear localization of Chk. The SH2 domain-deleted mutants might lose the ability to localize to the exterior of the nucleus due to a lack of the binding to a hypothetical tyrosine-phosphorylated protein that resides in the extranuclear region. Another possibility is that the deletion of the SH2 domain might induce a conformational change to facilitate nuclear import of Chk. Although these possibilities are mutually inclusive, further studies will be needed to understand the mechanism of nuclear localization of Chk. Induction of multi-lobulated nuclei is unexpectedly observed in ubiquitin e3 ligase expressing the kinase-inactive mutants as well as the kinase-active version (Fig. 2), indicating that the kinase domain but not the kinase activity is required for multi-lobulation of the nucleus. Construction of various deletion mutants showed that the minimum requirement in the Chk structure for multi-lobulation is both the N-terminal unique domain and the N-terminal portion of the kinase domain (Fig. 2). In addition, we have recently revealed that Lyn tyrosine kinase contains a subregion within the kinase domain, which is responsible for the trafficking of Lyn [39]. These results present a couple of examples of unconventional modules in the structure of tyrosine kinases for kinase activity-independent functions. Normal lamin organization appears to be required for DNA replication [40] through the interaction of lamins and lamin-associated proteins (LAP) with chromatin. Also, the normal lamin organization appears to be required for the chain elongation phase or initial organization of replication factors in DNA replication [40]. Lamins can interact with chromatin [41] via their connections to LAP2 [42], [43]. Interaction of LAP2 with lamins is involved in the control of initiation of DNA replication [44], suggesting that disorganization of lamins and LAP2 may affect DNA replication through induction of aberrant chromatin organization. Given that Chk is localized to the nucleus and chromosomes [17], we therefore speculate that the nuclear-localized Chk inhibits DNA replication presumably through lamin disorganization. In fact, we found that upon nuclear expression of Chk the peak of S-phase cells continues to shift from early S phase to late S or G2/M phase during the 4-day observation period (Fig. 4B). Intriguingly, time-lapse observations of living cells showed that proliferation is inhibited by expression of Chk in the nucleus (data not shown). After treatment with nocodazole, very few mitotic entries are found among Chk-expressing cells (data not shown). These results suggest that nuclear expression of Chk severely retards the cell cycle progression in S phase.
    Acknowledgments
    Main Text In enzymology, protein compartmentalization is often considered the “last refuge of a scoundrel.” Yet the spatial segregation of enzymes is a fundamental tenet of cell biology (Scott and Pawson, 2009). The union of these two seemingly contradictory ideologies comes into sharp focus during cell division. Progression through the G1, S, G2, and M phases of the cell cycle is a complex molecular dance that requires an extraordinarily high degree of fidelity in space and time. Such meticulous synchronization of numerous enzyme activities is one way to ensure accurate transfer of the genetic code. Conversely, failure to complete DNA replication, chromosome condensation, or cell division causes the cell to descend into biochemical limbo in the form of checkpoint arrest. This favors recovery from genotoxic stress and protects against mutations, chromosomal aberrations, or defects in genome maintenance that accumulate as cells venture down a perilous path toward malignancy. Although at least five independent molecular complexes sense and sort DNA damage, two protein kinase-signaling units modulate the key elements of checkpoint control (Harper and Elledge, 2007).