The Stil protein regulates centrosome integrity and mitosis through suppression of Chfr

Abstract

SummaryStil (Sil, SCL/TAL1 interrupting locus) is a cytosolic and centrosomal protein expressed in proliferating cells that is required for mouse and zebrafish neural development and is mutated in familial microcephaly. Recently the Drosophila melanogaster ortholog of Stil was found to be important for centriole duplication. Consistent with this finding, we report here that mouse embryonic fibroblasts lacking Stil are characterized by slow growth, low mitotic index and absence of clear centrosomes. We hypothesized that Stil regulates mitosis through the tumor suppressor Chfr, an E3 ligase that blocks mitotic entry in response to mitotic stress. Mouse fibroblasts lacking Stil by genomic or RNA interference approaches, as well as E9.5 Stil -/-embryos, express high levels of the Chfr protein and reduced levels of the Chfr substrate Plk1. Exogenous expression of Stil, knockdown of Chfr or overexpression of Plk1 reverse the abnormal mitotic phenotypes of fibroblasts lacking Stil. We further demonstrate that Stil increases Chfr auto-ubiquitination and reduces its protein stability. Thus, Stil is required for centrosome organization, entry into mitosis and cell proliferation, and these functions are at least partially mediated by Chfr and its targets. This is the first identification of a negative regulator of the Chfr mitotic checkpoint. Journal of Cell Scienceleads to degradation of mitotic kinases including Plk1, delaying the activation of Cdc25c phosphatase, and consequently delayed activation of Cdk1 and entry into mitosis (Kang et al., 2002;Shtivelman, 2003;Yu et al., 2005). Ubiquitination of target proteins is required for Chfr activity also in a proteosome-independent manner (Matsusaka and Pines, 2004). Its subcellular localization might be cell-cycle-dependent. It has been shown to localize in nuclear promyelocytic leukemia (PML) bodies and to regulate nuclear dynamics and genomic stability (Daniels et al., 2004;Kwon et al., 2009;Oh et al., 2009). Other studies have demonstrated localization at the spindle poles during metaphase and interaction with microtubule-associated proteins (Burgess et al., 2008;Maddika and Chen, 2009). Chfr might auto-regulate itself by autoubiquitination (Chaturvedi et al., 2002). However, the upstream negative regulators of Chfr are unknown. Thus, the mechanisms of Chfr activity and regulation are incompletely understood. Because Stil is required for entry into mitosis and Chfr is known to regulate the G 2 -M transition, we hypothesized that Stil might be involved in the regulation of Chfr. The present study demonstrates that Stil has a role in centrosome organization, entry into mitosis and cell proliferation, and that these functions are at least partially mediated by Chfr. In this regard, we show that overexpression of Stil results in increased auto-ubiquitination and reduced protein stability of Chfr. Results Mitotic defects in mouse embryonic fibroblasts lacking StilMouse embryonic fibroblasts (MEFs) derived from Stil knockout embryos (1022M) are characterized by a low prolifer...

Cell-cycle-regulated expression of STIL controls centriole number in human cells

“…The same authors also reported mitotic spindle defects in HeLa cells, as confirmed in our present study. Similarly, STIL-deficient mouse embryonic fibroblasts were shown to display defects in cell cycle progression (Castiel et al, 2011). STIL-knockout (Stil -/-) mice die at embryonic day 10.5, with prominent axial midline defects and randomized cardiac looping, consistent with a block in Sonic hedgehog (Shh) signaling (Izraeli et al, 1999).…”

“…Moreover, STIL expression was reported to be high in lung cancer (Erez et al, 2004) and most interestingly, mutations in STIL were recently shown to cause primary microcephaly (Kumar et al, 2009). Additional studies point to a possible role for STIL in cell proliferation, mitotic regulation and centrosome integrity (Izraeli et al, 1997;Campaner et al, 2005;Erez et al, 2007;Castiel et al, 2011), but the precise function of this protein remains unknown.…”

Malformations of Cortical Development and Epilepsy

“…For example, patients with microcephaly associated with osteodysplastic primordial dwarfism (MOPD), such as Meier-Gorlin syndrome, have mutations affecting subunits of the origin recognition complex, which is loaded onto chromatin at DNA origins before the S phase of mitosis to license replication; in conjunction with additional components CDC6, CDT1, and MCM2 -7, it forms the prereplication complex, which initiates DNA replication . Other genes that, when mutated, result in microcephaly include those involved in microtubule formation (TUBA1A, TUBB2B, TUBB3, TUBG1) (Poirier et al , 2012Cushion et al 2013), those coding for microtubule-associated proteins (DYNC1H, KIF5C, NDE1) (McKenney et al 2010;Alkuraya et al 2011;Bakircioglu et al 2011;Poirier et al 2013), those involved in spindle organization and positioning (ASPM) (Desir et al 2008;Passemard et al 2009), those regulating centriole length (CENPJ) (Tang et al 2009;Al-Dosari et al 2010) or centrosome integrity (STIL) (Castiel et al 2011), those that repair genomic defects and regulate genomic integrity (CEP152) (Kalay et al 2011), and many others involved in the very complex process of cell replication. By a combination of clinical characteristics (Mahmood et al 2011), imaging characteristics ( Fig.…”

Identifying Protein-protein Interaction Sites Using Peptide Arrays

“…Note that each peptide appears twice since it exists in duplicate, which verifies the reliability of the results. The interaction between STIL and CHFR was previously demonstrated at the protein level 13,14 . The peptide array screening revealed 7 CHFR-derived peptides that bound STIL IDR, residues 500-650 (Figure 3 …”

“…It controls normal cell division and cell proliferation 13,[15][16][17][18][19] . STIL interacts with several proteins 18,20,21 , and most of the interactions occur through its central part, which is an intrinsically disordered region (IDR) 14 .…”