• 2018-07
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • In rodents but not humans two splice


    In rodents but not humans, two splice variants of Chk are identified as Ntk and Ctk, which possess and lack the N-terminal unique domain, respectively. While Ctk is abundantly expressed in the brain, Ntk is selectively restricted to myeloid and lymphoid cells, especially megakaryocytes [31]. This raises the possibility that Ntk, which possesses the N-terminal unique domain, may also play a role in the nucleus. We previously reported that overexpression of human Chk inhibits proliferation and induces polyploidization of an immature myeloid cell line [9]. Recent reports Immunology Inflammation Compound Library of showed with the Src inhibitors PP1 and PP2 that Src-family kinases are involved in megakaryocyte polyploidization [32], [33]. Microinjection of anti-Src antibody into NIH3T3 Immunology Inflammation Compound Library of at M phase inhibited cytokinesis and induced multinucleated cells [34]. Thus, inhibition of Src-family kinases, through tyrosine phosphorylation of the C-terminal negative tyrosine residue, may be involved in Chk-induced inhibition of proliferation leading to polyploidization. Although expression of Chk inhibits cell proliferation of a human breast carcinoma cell line in a kinase activity-dependent manner [17], [18], inactivation of Src-family kinases by Chk is also reported in a manner that is independent of the kinase activity of Chk, where the direct binding of Chk to Src-family kinases is involved [35]. Recently, Chk is shown to stimulate Erk1/2 activation via Ras-mediated signaling in a Src-independent manner [36]. Our Western blotting experiments showed that a majority of proteins tyrosine-phosphorylated by Chk have different molecular sizes compared to Src-family kinases (Fig. 4, Fig. 5). Expression of kinase-deficient Chk(K262R) did not increase tyrosine phosphorylation of proteins (Figs. 5A and B), whereas overexpression of a kinase-deficient Csk mutant elevates tyrosine phosphorylation of proteins possibly by activating Src-family kinases through inactivation of protein-tyrosine phosphatases [37]. These data suggest that the induction of tyrosine phosphorylation in Chk-expressing cells (Fig. 4, [5], Fig. 5, [6], Fig. 6) is directly mediated by the kinase activity of Chk but not Src-family kinases. It is therefore plausible that Chk can tyrosine-phosphorylate nuclear proteins other than Src-family kinases. Furthermore, Chk induces tyrosine phosphorylation of nuclear matrix proteins as well as cytoplasmic proteins (Fig. 4, Fig. 5, [6], Fig. 6). The nuclear matrix is non-chromatin structures of the nucleus upon which chromatin is organized, and this non-chromatin nuclear structures consist of the fibrogranular ribonucleoprotein network [38]. The nuclear matrix binds a variety of nuclear proteins and supports their assembly into functional macromolecular complexes involved in important nuclear processes, such as transcription, RNA splicing and DNA replication [39]. The tumor suppressor, retinoblastoma protein Rb is an example of the protein present in the nucleus through the binding to the nuclear matrix during G0 and G1 phase, and the interaction with the nuclear matrix is important for the ability of Rb to regulate cell cycle progression [40]. Our results show the association of Chk with the nuclear matrix via the region of N-terminal unique domain by using in situ binding assays (Fig. 1, [2], Fig. 2, [3], Fig. 3). Our results also show Chk-induced tyrosine phosphorylation of nuclear matrix proteins (Fig. 6). Recently, we showed that nuclear localization of Chk mutants induces multi-lobulation of the nucleus and retardation of DNA replication of COS-1 cells in a kinase activity-independent manner [19]. Taken together, these findings raise the intriguing possibility that inhibition of cell proliferation by Chk may involve both kinase activity-dependent and-independent mechanisms.
    Introduction Meiosis is a specialized cell division that produces haploid gametes to transmit genetic information from parent to progeny through sexual reproduction. During meiotic prophase, chromosomes are reorganized around a central axis and then pair, synapse, and recombine with their homologs. This process generates physical linkages known as chiasmata, which enable homologs to biorient on the meiotic spindle and to disjoin during meiosis I. A fundamental mystery is how these chromosomal events are coordinated during the meiotic cell cycle. In diverse organisms, defects in synapsis (assembly of the synaptonemal complex [SC]) or meiotic recombination trigger a delay or arrest in mid-prophase (Bishop et al., 1992, Edelmann et al., 1996, Ghabrial and Schüpbach, 1999, MacQueen et al., 2002, Pittman et al., 1998), indicative of surveillance mechanisms that monitor meiotic events. This meiotic checkpoint (also referred to as the “pachytene checkpoint” or the “meiotic recombination checkpoint”) typically prevents meiosis I division in cells that fail to form crossovers (MacQueen and Hochwagen, 2011, Roeder and Bailis, 2000, Subramanian and Hochwagen, 2014). Because induction of programmed DNA double-strand breaks (DSBs) is an integral aspect of the meiotic program, proteins involved in DNA damage response also play essential roles in meiotic checkpoints (Lydall et al., 1996).