Cdk1 and Okadaic Acid-sensitive Phosphatases Control Assembly of Nuclear Pore Complexes in<i>Drosophila</i>Embryos

Onischenko, Evgeny; Губанова, Н. В.; Киселева, Елена; Hallberg, Einar

doi:10.1091/mbc.e05-07-0642

Cited by 67 publications

(65 citation statements)

References 54 publications

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“…The WGA-Nup persisted at the NE into late prometaphase, longer than Nup107. As reported previously (Onischenko et al, 2005), and in marked contrast to Nup107, the WGA-Nup became evenly distributed in the cytoplasm and showed no enrichment in the spindle area during metaphase and anaphase. In telophase, the first reappearance of both markers at the NE was nearly coincident, although mRFP-Nup107 reached its half-maximal intensity at the NE slightly earlier than the WGA-Nup ( Figure 5B).…”

supporting

confidence: 84%

“…These various aspects of NE and NPC remodeling during mitosis need to be spatially and temporally coordinated with other cell cycle events. Key regulators that contribute to this spatio-temporal coordination are mitotic kinases and phosphatases as well as the small GTPase Ran and the transport receptors of the karyopherin/importin ␤ family (for reviews, see Dasso, 2002;Harel and Forbes, 2004;Hetzer et al, 2005;Margalit et al, 2005; see also Onischenko et al, 2005). In addition, there is growing evidence that NPCs functionally participate in mitosis.…”

Section: Introductionmentioning

confidence: 99%

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In Vivo Dynamics ofDrosophilaNuclear Envelope Components

Katsani

Karess

Dostatni

et al. 2008

MBoC

120

128

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Nuclear pore complexes (NPCs) are multisubunit protein entities embedded into the nuclear envelope (NE). Here, we examine the in vivo dynamics of the essential Drosophila nucleoporin Nup107 and several other NE-associated proteins during NE and NPCs disassembly and reassembly that take place within each mitosis. During both the rapid mitosis of syncytial embryos and the more conventional mitosis of larval neuroblasts, Nup107 is gradually released from the NE, but it remains partially confined to the nuclear (spindle) region up to late prometaphase, in contrast to nucleoporins detected by wheat germ agglutinin and lamins. We provide evidence that in all Drosophila cells, a structure derived from the NE persists throughout metaphase and early anaphase. Finally, we examined the dynamics of the spindle checkpoint proteins Mad2 and Mad1. During mitotic exit, Mad2 and Mad1 are actively imported back from the cytoplasm into the nucleus after the NE and NPCs have reformed, but they reassociate with the NE only later in G1, concomitantly with the recruitment of the basket nucleoporin Mtor (the Drosophila orthologue of vertebrate Tpr). Surprisingly, Drosophila Nup107 shows no evidence of localization to kinetochores, despite the demonstrated importance of this association in mammalian cells. INTRODUCTIONIn eukaryotes, the nuclear envelope (NE) defines the limits between the nucleus and the cytoplasm. The outer NE membrane is considered to be structurally and functionally part of the endoplasmic reticulum network, whereas the inner membrane, with its distinct protein composition, provides anchoring points for the chromatin and nuclear lamina. Nuclear pore complexes (NPCs) are embedded at the points of fusion between the inner and outer NE membrane and represent the sole channels of transport across the NE. NPCs are composed of multiple copies of ϳ30 different proteins termed nucleoporins (Nups), most of which are organized into subcomplexes that associate with each other to build up the mature NPCs (for reviews, see Hetzer et al., 2005;Schwartz, 2005;Lim and Fahrenkrog, 2006;Tran and Wente, 2006). During cell division, the NE and NPCs are subjected to major rearrangements. However, the extent to which the NE and NPCs disassemble at mitotic entry varies among organisms (for reviews, see Margalit et al., 2005;Prunuske and Ullman, 2006). Unlike in most yeast and fungi, characterized by a "closed mitosis," NE disassembly is required in animal cells to allow spindle microtubule access to chromosomes. In vertebrates, cell division leads to complete NE breakdown at the prophase-prometaphase transition. During this "open mitosis," integral membrane proteins of the NE and the soluble subcomplexes of the NPCs redistribute throughout the endoplasmic reticulum and the mitotic cytoplasm (for reviews, see Hetzer et al., 2005;Margalit et al., 2005;Prunuske and Ullman, 2006). In Drosophila and Caenorhabditis elegans embryos, however, the NE only partially disassembles near spindle poles in early mitosis. NPCs disassemble during prometapha...

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supporting

confidence: 84%

Section: Introductionmentioning

confidence: 99%

In Vivo Dynamics ofDrosophilaNuclear Envelope Components

Katsani

Karess

Dostatni

et al. 2008

MBoC

120

128

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“…We chose to focus on the nuclear envelope. Mitotic disassembly of the nuclear lamina and of the nuclear pore complex (NPC) are known to be imposed in large part by Cdk activity (33,34).…”

Section: Many Phosphorylation Sites Identified Lack An Optimal Cdk Comentioning

confidence: 99%

Covalent capture of kinase-specific phosphopeptides reveals Cdk1-cyclin B substrates

Blethrow

Glavy²,

Morgan³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

291

290

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We describe a method for rapid identification of protein kinase substrates. Cdk1 was engineered to accept an ATP analog that allows it to uniquely label its substrates with a bio-orthogonal phosphate analog tag. A highly specific, covalent capture-andrelease methodology was developed for rapid purification of tagged peptides derived from labeled substrate proteins. Application of this approach to the discovery of Cdk1-cyclin B substrates yielded identification of >70 substrates and phosphorylation sites. Many of these sites are known to be phosphorylated in vivo, but most of the proteins have not been characterized as Cdk1-cyclin B substrates. This approach has the potential to expand our understanding of kinase-substrate connections in signaling networks.P rotein kinases regulate a vast array of biological processes through phosphorylation of protein substrates. A comprehensive map of all phosphorylation sites and kinase-substrate pairs would greatly facilitate the study of signaling networks. This goal faces two fundamental challenges. First, all protein kinases use ATP as a cofactor to phosphorylate their targets, and thus the direct substrates of a single kinase cannot be easily traced in protein mixtures containing multiple kinases. Second, phosphorylation often occurs at low stoichiometry and on low-abundance proteins. This makes substrate and phosphorylation site identification very challenging. Powerful methods have been developed to address these dual problems (1, 2). Kinase-substrate pairs can be tested in multiplexed phosphorylation assays by using immobilized arrays of purified proteins. These high-throughput chip-based assays have the added benefit of presenting low-abundance proteins at easily detectable levels and have provided a first-generation map of protein phosphorylation in Saccharomyces cerevisiae (3). However, these assays do not currently allow identification of phosphorylation sites and have not been adapted to organisms with more complex proteomes. Prediction of high-likelihood kinase substrates can sometimes be achieved by using knowledge of the substrate sequence motif preferences of individual kinases, but only when these preferences are strong (4, 5). Finally, thousands of in vivo phosphorylation sites from metazoan organisms have been identified in proteomic screens (6-9), but for most of these sites the responsible upstream kinases remain unknown.We have demonstrated a chemical and genetic solution to the common use of ATP by all kinases. Our approach relies on engineering a kinase to accept unnatural ATP analogs by modification of the ATP-binding pocket (10, 11). The analogs are very poor substrates for wild-type kinases; thus, an analog-sensitive kinase (or as-kinase) can be used to specifically radiolabel its substrates in cell extracts while preserving important aspects of biological context, such as the integrity of protein complexes. Coupling this approach with the use of libraries of genetically encoded affinity-tagged proteins has facilitated identification of low-abundanc...

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“…A recent study using temperature-sensitive Cdk1 Drosophila embryos showed that Cdk1 activity is required for keeping NPCs dissociated during mitosis, while the reassembly may be phosphatase-dependent (18). The goal of the present study is to map and characterize the cell-cycle-dependent phosphorylation of the main components of the Nup107-160 subcomplex to throw light on the regulation of disassembly and reassembly of the NPC.…”

mentioning

confidence: 95%

Cell-cycle-dependent phosphorylation of the nuclear pore Nup107–160 subcomplex

Glavy

Krutchinsky²,

Cristea³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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The nuclear pore complex (NPC) mediates macromolecular transport between the nucleus and the cytoplasm. Many NPC proteins (nucleoporins, Nups) are modified by phosphorylation. It is believed that phosphorylation regulates the breakdown of the nuclear envelope at mitosis and the disassembly of the NPC into different subcomplexes. In this study, we examined the cell-cycledependent phosphorylation of the Nup107-160 subcomplex, a core building block of the NPC. Using in vivo 32 P labeling in HeLa cells, we found that Nup107, Nup96, and Nup133 are phosphorylated during mitosis. To precisely map the phosphorylation sites within the complex, we used a comprehensive multiple-stage MS approach (MS, MS 2 , and MS 3 ), establishing that Nup160, Nup133, Nup96, and Nup107 are all targets of phosphorylation. We determined that the phosphorylation sites are clustered mainly at the N-terminal regions of these proteins, which are predicted to be natively disordered. In addition, we determined the cell-cycle dependence of the phosphorylation of these sites by using stable isotope labeling and MS 2 analysis. Measurement of the site-specific phosphorylation ratios between mitotic and G1 cells led us to conclude that several phosphorylation events of the subcomplex are mainly mitotic. Based on these results and our finding that the entire Nup107-160 subcomplex is stable throughout the cell cycle, we propose that phosphorylation does not affect interactions within the Nup107-160 subcomplex, but regulates the association of the subcomplex with the NPC and other proteins. mitosis ͉ nucleoporin ͉ mass spectrometry ͉ nuclear pore complex ͉ mammalian

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Cdk1 and Okadaic Acid-sensitive Phosphatases Control Assembly of Nuclear Pore Complexes inDrosophilaEmbryos

Cited by 67 publications

References 54 publications

In Vivo Dynamics ofDrosophilaNuclear Envelope Components

In Vivo Dynamics ofDrosophilaNuclear Envelope Components

Covalent capture of kinase-specific phosphopeptides reveals Cdk1-cyclin B substrates

Cell-cycle-dependent phosphorylation of the nuclear pore Nup107–160 subcomplex

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