Centrosome Separation Driven by Actin- Microfilaments During Mitosis Is Mediated by Centrosome-Associated Tyrosine-Phosphorylated Cortactin
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1334 Research Article Centrosome separation driven by actin- microfilaments during mitosis is mediated by centrosome-associated tyrosine-phosphorylated cortactin Wenqi Wang*, Luyun Chen*, Yubo Ding, Jing Jin and Kan Liao‡ State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, Peopleʼs Republic of China *These authors contributed equally to this work ‡Author for correspondence (e-mail: [email protected]) Accepted 31 January 2008 Journal of Cell Science 121, 1334-1343 Published by The Company of Biologists 2008 doi:10.1242/jcs.018176 Summary The regulation of protein tyrosine phosphorylation is an phosphorylation sites in the truncated C-terminus of cortactin important aspect during the cell cycle. From G2-M transition and found that the C-terminus could no longer interfere with to mitotic anaphase, phosphorylation of Tyr421, Tyr466 and centrosome separation process. Our study shows that, cortactin Tyr482 of cortactin, an actin-filament associated protein, is phosphorylated at Tyr421, Tyr466 and Tyr482 mediates the dramatically induced. The phosphorylated cortactin is almost actin-filament-driven centrosome separation at G2-M transition exclusively associated with centrosomes or spindle poles by providing a bridge between the centrosome and actin- during mitosis. At G2-M transition prior to the breakdown of filaments. the nuclear envelope, two duplicated centrosomes migrate towards opposite ends of the nucleus to form the spindle poles. This centrosome-separation process and also the start of mitosis Supplementary material available online at are inhibited or delayed by the depolymerization of actin http://jcs.biologists.org/cgi/content/full/121/8/1334/DC1 filaments. Also inhibited is the separation of centrosomes when a truncated form of cortactin is expressed, whose C-terminus Key words: Cortactin, Cortactin tyrosine phosphorylation, contains the tyrosine phosphorylation region but lacks the actin- Centrosome separation, Actin-filament, Focal adhesion points, Src Journal of Cell Science binding domains. We introduced mutations at the tyrosine kinase Introduction regulation of cell motility and formation of lamellipodia and Cortactin is an actin-filament-binding protein (Olazabal and membrane ruffles (Lua and Low, 2005). Machesky, 2001; Weed and Parsons, 2001). It has initially been During mitosis the complex sequence of events is dominated by described as a tyrosine-phosphorylated protein in cells infected with the formation and activity of the microtubule spindle. The initiation v-Src and is a direct substrate of Src kinase (Kanner et al., 1990; of the microtubule spindle is governed by the centrosome, also Wu et al., 1991). Its association with F-actin stabilizes the known as the microtubule-spindle-organization center (Bornens, microfilament network (Huang et al., 1997; Olazabal and Machesky, 2002; Rieder et al., 2001; Kellogg et al., 1994; Joshi, 1993; Mishima 2001; Pant et al., 2006). Of the all the different domains in cortactin, et al., 2004). Consequently, the separation and positioning of the N-terminal acidic region, the F-actin-binding repeats, the helix centrosomes determines the formation and orientation of spindles. domain and the SH3 domain at the C-terminus are relatively The centrosome cycle progresses in a precise order: (1) the conserved from invertebrates to vertebrates (Weed and Parsons, centrosome is duplicated at G1-S transition, (2) cohesion between 2001). The proline-rich domain between the helix domain and C- two duplicated centrosomes is broken in G2 phase, (3) two terminal SH3 domain, however, is only conserved in vertebrates, centrosomes migrate apart in G2-M transition and the separated but not between vertebrates and invertebrates (Weed and Parsons, centrosomes initiate the spindle microtubules to form bipolar 2001). The proline-rich domain of Drosophila melanogaster has spindle in early mitotic phase (Meraldi and Nigg, 2002; Tsou and only 15% homology with its counterpart in mouse. The proline- Stearns, 2006). Therefore, the separation and positioning of rich domain of murine cortactin has six identified phosphorylation centrosomes are crucial for the correct formation of a bipolar spindle, sites (four tyrosine sites and two serine sites) (Huang et al., 1997; which governs the chromosome segregation and the fate of daughter Head et al., 2003; Lua and Low, 2005; Martin et al., 2006). All cells. four identified tyrosine phosphorylation sites are missing in In order to drive the centrosomes to opposite ends of nucleus, Drosophila cortactin (Genebank: AAF55840). The current motion force needs to be generated. It has been reported that actin knowledge regarding the function of cortactin and its tyrosine microfilaments and myosin II are required for centrosome separation phosphorylation mostly concerns actin-network organization, and positioning during mitosis, thus making them ideal candidates Cortactin and centrosome separation 1335 for providing the migration force (Rosenblatt et al., 2004; Burakov constant throughout the cell cycle (Fig. 1B). Similar phosphorylation et al., 2003; Waters et al., 1993). When F-actin is depolymerized and dephosphorylation on Tyr466 and Tyr482 was also observed by addition of latrunculin, centrosome separation is blocked and (data not shown). cells enter mitosis with unseparated centrosomes (Uzbekov et al., In the mitotic phase, the spindle structure is predominantly 2002). In HeLa cells, one major pathway for centrosome separation formed by microtubules, whereas no distinct structure is formed by that occurs prior to nuclear-envelope breakdown is dependent on actin-filaments. As an F-actin-associated protein, cortactin was an intact actin cytoskeleton (Whitehead et al., 1996). These studies evenly distributed in cytoplasm of mitotic cells (Fig. 2A and indicate or imply that actin-filaments provide the essential driving supplementary material Fig. S1). However, P-Tyr-cortactin was force for centrosome separation, which means that a regulatory almost exclusively located at the mitotic spindle poles, identified attachment of actin-filaments to centrosome is required at G2-M by staining for γ-tubulin (Fig. 2B) (Joshi, 1993). Individual transition. Here we show that cortactin phosphorylation at tyrosine antibodies against cortactin phosphorylated at Tyr421 (P-Tyr421- residues is induced in mitotic cells and phospho-cortactin (P- cortactin), Tyr466 (P-Tyr466-cortactin) or Tyr482 (P-Tyr466- cortactin) is associated with centrosomes or spindle poles at G2-M cortactin) (anti-P-Tyr421-cortactin, anti-P-Tyr466-cortactin or anti- transition until mitotic anaphase. We found that the cortactin C- P-Tyr482-cortactin antibodies, respectively) were used to stain terminal region, which can bind to centrosomes but does not mediate spindle poles (Fig. 2B). When mitotic spindles were revealed using the F-actin association, inhibited the centrosome separation prior anti-α-tubulin antibody, P-Tyr-cortactin was found to be located to nuclear envelope breakdown. We also found P-cortactin to be exactly at the spindle poles (Fig. 2C). P-cortactin, which is anchored at the tips of actin fibers in interphase cell. These associated with the spindle poles, was phosphorylated at least on observations indicate that centrosome or spindle-pole-associated P- one of the three Src kinase sites, at Tyr421, Tyr466 or Tyr482 (Fig. cortactin provide the anchor on the centrosome or spindle pole for 2B) (Kanner et al., 1990; Wu et al., 1991; Huang et al., 1997). F-actin attachment. However, this association did not enrich the spindle poles with just phosphorylated tyrosine residues (Fig. 2D). The spindle poles were Results distinct for P-Tyr-cortactin (Fig. 2B,C). Even the overexposed Induction of tyrosine phosphorylation of cortactin, and immunofluorescence images show that P-Tyr-cortactin was only association of P-Tyr-cortactin with centrosomes or spindle detected at spindle poles (supplementary material Fig. S1). poles in G2-M cells Besides the staining for P-Tyr-cortactin at centrosomes and In our study of hormone-induced 3T3-L1 adipocyte differentiation spindle poles, we detected an interaction between P-Tyr-cortactin and mitotic clonal expansion (Jin et al., 2000), an accumulated and the centrosomal protein γ-tubulin (Fig. 1C). Anti-P-Tyr421- tyrosine-phosphorylated protein was identified as P-Tyr-cortactin cortactin antibody immunoprecipitated γ-tubulin in mitotic cells and (data not shown). In hydroxylurea synchronized 3T3-L1 cells, we anti-γ-tubulin antibody immunoprecipitated P-Tyr-cortactin (Fig. observed cell-cycle-dependent phosphorylation of cortactin at 1C). In addition, centrosomal fraction isolated by sucrose-density Tyr421 (Fig. 1A,B). Levels of P-Tyr-cortactin were low in gradient from nocodazole-arrested mitotic cells contained P-Tyr- interphase, G1-S transition or S phase cells. However, it was cortactin (Fig. 1D) (Moudjou and Bornens, 1994). Furthermore, in dramatically increased after cells reached G2 phase. The P-cortactin cells expressing eGFP-tagged cortactin, GFP-cortactin fusion Journal of Cell Science was then dephosphorylated when the cells finished mitosis and protein was visualized at the spindle pole (supplementary material returned to G1 phase (Fig. 1B). Cortactin protein levels were Fig. S1). Fig. 1. Cell-cycle dependent cortactin phosphorylation at Tyr421. (A) Hydroxylurea-synchronized 3T3-L1 cells. Int, control interphase cells; HU, cells arrested