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54-664: In the G1 phase of the cell cycle, Sic1 binds tightly to the Cdc28-Clb complex and inhibits it. Low Cdc28-Clb activity leads to the disassembly of the mitotic spindle , the assembly of the prereplicative complex and initiation of bud formation in yeast. At the START point in the yeast cell cycle, the G1- cyclins Cln3 , Cln1 and Cln 2 activate Cdc28. The activated complex will phosphorylate Sic1 at multiple sites which leads to its degradation by

108-763: A catalytic Cdk subunit, i.e. Cks and a regulatory cyclin subunit, such as a G1 cyclin, controlling Cdk function by directing cyclin-cdk complex activity toward specific and significant substrates. Malfunctions of cdk-dependent associations lead to defects into the entry of mitosis for cells. Cks1 in the Cdk-independent pathway involves the recognition of substrates p27 and p21 by directly associating with E3 SCF when stimulated by certain mitogenic signals, such as TGF-β. Cks1-depleted breast cancer cells not only exhibit slowed G(1) progression, but also accumulate in G(2)-M due to blocked mitotic entry. Cdk1 expression, which

162-399: A positive feedback loop where Clb5-Cdk1 inhibition is continually decreased while Sic1 degradation is increased. The protein p27Kip1 is a human homologue of Sic1, both having a conserved inhibitory domain, but p27Kip1 inhibits G1 cyclins and not cyclin B. There are several human diseases that are linked to p27Kip1 and other cyclin kinase inhibitors: Thus, the human Cdk inhibitor p27Kip1

216-400: A process called condensation. Condensation begins in prophase and chromosomes are maximally compacted into rod-shaped structures by the time they are aligned in the middle of the spindle at metaphase. This gives mitotic chromosomes the classic "X" shape seen in karyotypes , with each condensed sister chromatid linked along their lengths by cohesin proteins and joined, often near the center, at

270-512: A process known as dynamic instability determines to a large extent the shape of the mitotic spindle and promotes the proper alignment of chromosomes at the spindle midzone. Microtubule-associated proteins (MAPs) associate with microtubules at the midzone and the spindle poles to regulate their dynamics. γ-tubulin is a specialized tubulin variant that assembles into a ring complex called γ-TuRC which nucleates polymerization of α/β tubulin heterodimers into microtubules. Recruitment of γ-TuRC to

324-439: A role in the response to osmostress. The stress-activated protein kinase (SAPK) Hog1 phosphorylates Sic1 at a single residue at the carboxyl terminus . This leads to downregulation of cyclin expression and Sic1 stabilization which arrests the cell cycle. Sic1 needs to be phosphorylated at multiple sites for ubiquitination -driven degradation (Fig. 2). The multiple phosphorylations are required for Sic1 to be recruited by Cdc4 to

378-399: A spindle-like structure with chromosomes aligned along the cell equator. In this model, microtubules are nucleated at microtubule organizing centers and undergo rapid growth and catastrophe to 'search' the cytoplasm for kinetochores. Once they bind a kinetochore, they are stabilized and their dynamics are reduced. The newly mono-oriented chromosome oscillates in space near the pole to which it

432-798: Is a protein that in humans is encoded by the CKS1B gene . The CKS1B protein binds to the catalytic subunit of the cyclin-dependent kinases and is essential for their biological function. The CKS1B mRNA is found to be expressed in different patterns through the cell cycle in HeLa cells , which reflects a specialized role for the encoded protein. CKS1B and CKS2 proteins have demonstrated principal roles in cell cycle regulation. Defined originally as suppressors of mutations in both fission and budding yeast Cdk1 genes, Cks molecules interact with Cdk1, Cdk2 and Cdk3. These Cdk-dependent enzyme complexes in cell cycle regulation frequently consist of Cdk molecules bound to

486-519: Is a crucial transition point in the cell cycle called the spindle assembly checkpoint . If chromosomes are not properly attached to the mitotic spindle by the time of this checkpoint, the onset of anaphase will be delayed. Failure of this spindle assembly checkpoint can result in aneuploidy and may be involved in aging and the formation of cancer. Cell division orientation is of major importance for tissue architecture, cell fates and morphogenesis. Cells tend to divide along their long axis according to

540-419: Is a molecule with disordered regions, which aids in the manipulation of phosphorylation site distances. For the following findings, Koivomagi et al. utilized a Sic1 construct with a T33 optimal consensus motif, acting as the primary phosphorylation site, and a suboptimal motif, acting as a secondary site. When limiting observations to only double-phosphorylated Sic1 constructs, a two-step phosphorylation process

594-425: Is a potential tumor suppressor protein. If its expression is reduced, the result might be unregulated progression from G1 to S-phase which deregulates cell division and simplifies the formation of tumors. Mitotic spindle In cell biology , the spindle apparatus is the cytoskeletal structure of eukaryotic cells that forms during cell division to separate sister chromatids between daughter cells . It

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648-508: Is another transcription factor of Sic1, but remains inactive until G1. At the end of mitosis, Sic1 is involved in the inactivation of Cdc28-Clb. In order to be recognized by Cdc4 of the SCF complex , Sic1 has to be phosphorylated , often by Cyclin-Cdk complexes, at least at 6 of the 9 cdk sites (Fig. 2). Sic1 can also be phosphorylated by other kinases, such as Pho85-Pc11 , a kinase which becomes essential when Cln1 and Cln2 are absent. Sic1 has also

702-549: Is attached to nucleosomes via core histones H2A and H2B. Thus, a gradient of GTP-bound Ran is generated around the vicinity of mitotic chromatin. Glass beads coated with RCC1 induce microtubule nucleation and bipolar spindle formation in Xenopus egg extracts, revealing that the Ran GTP gradient alone is sufficient for spindle assembly. The gradient triggers release of spindle assembly factors (SAFs) from inhibitory interactions via

756-434: Is attached until a microtubule from the opposite pole binds the sister kinetochore. This second attachment further stabilizes kinetochore attachment to the mitotic spindle. Gradually, the bi-oriented chromosome is pulled towards the center of the cell until microtubule tension is balanced on both sides of the centromere ; the congressed chromosome then oscillates at the metaphase plate until anaphase onset releases cohesion of

810-508: Is considered the main mitotic kinase in mammalian cells and is activated by Cyclin B1. Aurora kinases are required for proper spindle assembly and separation. Aurora A associates with centrosomes and is believed to regulate mitotic entry. Aurora B is a member of the chromosomal passenger complex and mediates chromosome-microtubule attachment and sister chromatid cohesion. Polo-like kinase, also known as PLK, especially PLK1 has important roles in

864-479: Is critical for Sic1 multi-phosphorylation and degradation. The phospho-binding pocket of Cks1 is capable of binding independently to phosphorylated CDK sites on Sic1. Additionally, the binding affinity of Cks1 for phosphoserines is extremely weak, essentially making Cks1 binding dependent on the presence of phosphothreonines only. Thus, in Sic1 mutants with one Cdk1 phosphorylation site or only phosphoserines present, Cks1

918-418: Is mediated by kinetochores , which actively monitor spindle formation and prevent premature anaphase onset. Microtubule polymerization and depolymerization dynamic drive chromosome congression. Depolymerization of microtubules generates tension at kinetochores; bipolar attachment of sister kinetochores to microtubules emanating from opposite cell poles couples opposing tension forces, aligning chromosomes at

972-455: Is referred to as the mitotic spindle during mitosis , a process that produces genetically identical daughter cells, or the meiotic spindle during meiosis , a process that produces gametes with half the number of chromosomes of the parent cell. Besides chromosomes, the spindle apparatus is composed of hundreds of proteins . Microtubules comprise the most abundant components of the machinery. Attachment of microtubules to chromosomes

1026-415: Is unable to properly bind to the substrate and promote Sic1 multi-phosphorylation. This provides a strong argument for a processive phosphorylation mechanism instead of the previous theory of a random distributive phosphorylation model. In addition to requiring threonine, Cks1 binding to Sic1 can be enhanced with the introduction of a proline residue at the -2 position relative to the threonine residue. Sic1

1080-470: The SCF complex . When Sic1 is degraded, the Cdc28-Clb complex is no longer inhibited and the cell can enter the S/M-phase. Thus Sic1 inactivation is essential for transition into S phase (Fig.1). Cdc28 in complex with B-type cyclin (Cdc28-Clb) phosphorylates Swi5 , the transcription factor of Sic1. This promotes the export of Swi5 from the nucleus to the cytoplasm and avoids further transcription of

1134-688: The centromere . While these dynamic rearrangements are vitally important to ensure accurate and high-fidelity segregation of the genome, our understanding of mitotic chromosome structure remains largely incomplete. A few specific molecular players have been identified, however: Topoisomerase II uses ATP hydrolysis to catalyze decatenation of DNA entanglements, promoting sister chromatid resolution. Condensins are 5-subunit complexes that also use ATP-hydrolysis to promote chromosome condensation. Experiments in Xenopus egg extracts have also implicated linker Histone H1 as an important regulator of mitotic chromosome compaction. The completion of spindle formation

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1188-499: The +20 to +30 range. The introduction of the -2 proline residue enhances phosphorylation both in vitro by expanding the peak phosphorylation range, but does not increase phosphorylation activity at distances less than +10. This expansion of the peak phosphorylation range could possibly be attributed to enhanced binding of the priming site to Cks1. A simple Sic1 construct containing 5 phosphorylation residues (1 priming site and two phosphodegron pairs) revealed that any slight movement of

1242-502: The RXL motif of Clb5 is +16 to +20 positions relative to the optimal CDK motif. RXL positioning located N-terminal to the motif led to negligible amounts of phosphorylation. In contrast, moving the LLPP motif away from the priming site increases Cln2 phosphorylation, regardless of directionality. Cks1-dependent multi-phosphorylation occurs in a processive or semi-processive manner, evidenced by

1296-622: The S69/S76/S80 cluster, processive phosphorylation of these phosphodegron pairs are reliant on Cdk1 sites. Clb5 processivity is dependent on the T5 and T33 sites, while Cln2 processivity is dependent on T5. Reintroduction of various residues led to the discovery of the T33 residue serving as a docking site for the T45/T48 phosphodegron pair, which is able to promote Sic1 degradation to a certain extent in

1350-507: The SCF complex. The Cdc4 substrate recognition mechanism includes the interaction with consensus binding motifs on the surface of the folded and phosphorylated Sic1, the so-called Cdc4 phospho-degrons (CPD). It has been shown that the optimal consensus sequence for Cdc4 is a phosphorylated serine or threonine followed by a proline and a basic amino acid. However, none of the CPDs on the surface of

1404-437: The Sic1 show such a composition. Therefore, multiple phosphorylation of Sic1 is necessary to get high-affinity binding to Cdc4. Although this mechanism looks inefficient, it provides advantages for a cell because it is possible to measure the environmental Cln/cdc28 concentration. The number of phosphorylated sites corresponds to the concentration of Cln/cdc28 and Sic1 could be considered as a sensor for this protein. In contrast to

1458-457: The absence of centrosomes and kinetochores. Indeed, it has also been shown that laser ablation of centrosomes in vertebrate cells inhibits neither spindle assembly nor chromosome segregation. Under this scheme, the shape and size of the mitotic spindle are a function of the biophysical properties of the cross-linking motor proteins. The guanine nucleotide exchange factor for the small GTPase Ran (Regulator of chromosome condensation 1 or RCC1 )

1512-675: The absence of other phosphodegron pairs. The following is a proposed mechanism by Koivomagi et al. of the in vivo cascade to promote Sic1 phosphorylation and degradation. In late G1, Sic1 is inhibiting the Clb5-Cdk1 complex, simultaneously inhibiting its own degradation. The phosphorylation cascade proceeds by Cln2-Cdk1 phosphorylation of the T5 priming site. Following this, the T33, T45, and S76 residues are phosphorylated by Cln2-Cdk1, but no degron pairs are phosphorylated. However, these phosphorylated sites enhance Clb5-Cdk1 docking, leading to increased Sic1 phosphorylation at suboptimal sites and

1566-419: The cdk inhibitor. Cdc28-Clb also phosphorylates any Sic1 molecules still available and triggers their ubiquitin -dependent degradation, exactly like Cdc28-Cln. High Cdc28-Clb levels also initiate DNA replication and duplication of the spindle pole bodies (SPBs). Then the metaphase spindle assembles and chromosome segregation can occur. The transcription of Sic1 starts during telophase , mediated by Swi5. Aca2

1620-482: The cell equator and poising them for segregation to daughter cells. Once every chromosome is bi-oriented, anaphase commences and cohesin , which couples sister chromatids , is severed, permitting the transit of the sister chromatids to opposite poles. The cellular spindle apparatus includes the spindle microtubules , associated proteins, which include kinesin and dynein molecular motors, condensed chromosomes, and any centrosomes or asters that may be present at

1674-422: The chromosomes, their plus-ends embedded in kinetochores and their minus-ends anchored at the cell poles. The precise orientation of this complex is required to ensure accurate chromosome segregation and to specify the cell division plane. However, it remains unclear how the spindle becomes organized. Two models predominate the field, which are synergistic and not mutually exclusive. In the search-and-capture model ,

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1728-613: The distribution of cortical clues is set up by the adhesive pattern. In vivo polarity cues are determined by localization of Tricellular junctions localized at cell vertices. The spatial distribution of cortical clues leads to the force field that determine final spindle apparatus orientation and the subsequent orientation of cell division. CKS1B 1BUH , 1DKS , 1DKT , 2ASS , 2AST , 4YC6 1163 54124 ENSG00000173207 ENSMUSG00000028044 P61024 P61025 NM_001826 NM_016904 NP_001817 NP_058600 Cyclin-dependent kinases regulatory subunit 1

1782-483: The enzyme complex, the primary site is phosphorylated and immediately shifted from the active site to the Cks1 binding pocket to allow for the additional phosphorylation of other CDK sites. The second mechanism proposes that the phosphorylated primary site binds to another location and is continuously bound while other CDK sites bind to the active site in a sequential manner for multi-phosphorylation. Simulations predict that

1836-472: The growing ends of microtubules and coordinates the binding of other +TIPs. Opposing the action of these microtubule-stabilizing proteins are a number of microtubule-depolymerizing factors which permit the dynamic remodeling of the mitotic spindle to promote chromosome congression and attainment of bipolarity . The kinesin -13 superfamily of MAPs contains a class of plus-end-directed motor proteins with associated microtubule depolymerization activity including

1890-442: The lack of intermediate Sic1 phosphorylation states in normal cells. This processivity is also dependent on the presence of the cyclin docking site since increasing the numbers of mutations in this site decreases the net phosphorylation rate. Processive phosphorylation has two plausible mechanisms where a single binding event leads to the phosphorylation of two or more sites. The first mechanism proposes that, without dissociating from

1944-439: The many sharp transitions of ultrasensitive kinase cascade feedback loops, this mechanism allows fine tuned regulation. Moreover, because multiple phosphorylations are required, the probability that Sic1 is degraded by random is small. Using multiple phosphorylation of Sic1, the cell evolved a strategy to highly regulate the onset of DNA replication that is absolutely vital to provide genetic stability. A simplified understanding of

1998-561: The midzone. CLIP170 was shown to localize near microtubule plus-ends in HeLa cells and to accumulate in kinetochores during prometaphase . Although how CLIP170 recognizes plus-ends remains unclear, it has been shown that its homologues protect against catastrophe and promote rescue, suggesting a role for CLIP170 in stabilizing plus-ends and possibly mediating their direct attachment to kinetochores. CLIP-associated proteins like CLASP1 in humans have also been shown to localize to plus-ends and

2052-452: The mitotic spindle as well as promoting chromosome segregation during anaphase. The activities of these MAPs are carefully regulated to maintain proper microtubule dynamics during spindle assembly, with many of these proteins serving as Aurora and Polo-like kinase substrates. In a properly formed mitotic spindle, bi-oriented chromosomes are aligned along the equator of the cell with spindle microtubules oriented roughly perpendicular to

2106-511: The outer kinetochore as well as to modulate the dynamics of kinetochore microtubules (Maiato 2003). CLASP homologues in Drosophila , Xenopus , and yeast are required for proper spindle assembly; in mammals, CLASP1 and CLASP2 both contribute to proper spindle assembly and microtubule dynamics in anaphase. Plus-end polymerization may be further moderated by the EB1 protein, which directly binds

2160-489: The pericentrosomal region stabilizes microtubule minus-ends and anchors them near the microtubule-organizing center . The microtubule-associated protein Augmin acts in conjunction with γ-TURC to nucleate new microtubules off of existing microtubules. The growing ends of microtubules are protected against catastrophe by the action of plus-end microtubule tracking proteins (+TIPs) to promote their association with kinetochores at

2214-504: The priming site can have significant effects on cell cycle progression. The priming site should be within the +12 to +16 range of both residues in the phosphodegron pair to maximize phosphorylation. Sic1 phosphorylation is initiated by the G1 cyclins, Cln1,2, and then completed by S-phase cyclins, Clb5,6 (Fig. 1). The docking motifs of the cyclin participate in Sic1 phosphorylation dynamics. S-phase cyclins use RXL docking while G1 cyclins use LLPP docking. Sic1 phosphorylation increases when

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2268-401: The probability of a second phosphorylation event after the first, without dissociation, is 40% and 20-40% for the first and second mechanism, respectively. Sic1 is targeted for degradation by SCF (Cdc4), which recognizes Sic1 phosphodegron pairs. These phosphodegron pairs are closely positioned paired phosphorylation residues that each have strong affinities for Cdc4. In a Sic1 construct with

2322-614: The regulation of Sic1 degradation involves the phosphorylation of multiple CDK sites, which consist of optimal and suboptimal consensus phosphorylation motifs. Recent studies conducted by Koivomagi et al. have revealed the many intricacies of the multi-phosphorylation reaction between the cyclin-CDK complex and the Sic1 protein. These studies unveil the important characteristics of the Sic1 CDK phosphorylation sites, which include priming sites, binding sites, degron pairs, distancing of phosphorylation sites, and relative site location. In addition,

2376-495: The search-and-capture mechanism in which centrosomes largely dictate the organization of the mitotic spindle, this model proposes that microtubules are nucleated acentrosomally near chromosomes and spontaneously assemble into anti-parallel bundles and adopt a spindle-like structure. Classic experiments by Heald and Karsenti show that functional mitotic spindles and nuclei form around DNA-coated beads incubated in Xenopus egg extracts and that bipolar arrays of microtubules are formed in

2430-464: The sister chromatids. In this model, microtubule organizing centers are localized to the cell poles, their separation driven by microtubule polymerization and 'sliding' of antiparallel spindle microtubules with respect to one another at the spindle midzone mediated by bipolar, plus-end-directed kinesins. Such sliding forces may account not only for spindle pole separation early in mitosis, but also spindle elongation during late anaphase. In contrast to

2484-414: The so-called Hertwig rule . The axis of cell division is determined by the orientation of the spindle apparatus. Cells divide along the line connecting two centrosomes of the spindle apparatus. After formation, the spindle apparatus undergoes rotation inside the cell. The astral microtubules originating from centrosomes reach the cell membrane where they are pulled towards specific cortical clues. In vitro ,

2538-581: The spindle is predominantly organized by the poleward separation of centrosomal microtubule organizing centers (MTOCs). Spindle microtubules emanate from centrosomes and 'seek' out kinetochores; when they bind a kinetochore they become stabilized and exert tension on the chromosomes. In an alternative self assembly model, microtubules undergo acentrosomal nucleation among the condensed chromosomes. Constrained by cellular dimensions, lateral associations with antiparallel microtubules via motor proteins, and end-on attachments to kinetochores, microtubules naturally adopt

2592-418: The spindle maintenance by regulating microtubule dynamics. By the end of DNA replication , sister chromatids are bound together in an amorphous mass of tangled DNA and protein. Mitotic entry triggers a dramatic reorganization of the duplicated genome, resulting in sister chromatids that are disentangled and separated from one another. Chromosomes also shorten in length, up to 10,000-fold in animal cells, in

2646-428: The spindle poles depending on the cell type. The spindle apparatus is vaguely ellipsoid in cross section and tapers at the ends. In the wide middle portion, known as the spindle midzone, antiparallel microtubules are bundled by kinesins . At the pointed ends, known as spindle poles, microtubules are nucleated by the centrosomes in most animal cells. Acentrosomal or anastral spindles lack centrosomes or asters at

2700-411: The spindle poles, respectively, and occur for example during female meiosis in most animals. In this instance, a Ran GTP gradient is the main regulator of spindle microtubule organization and assembly. In fungi , spindles form between spindle pole bodies embedded in the nuclear envelope , which does not break down during mitosis. The dynamic lengthening and shortening of spindle microtubules, through

2754-505: The studies also emphasize the influence of other factors on Sic1 phosphorylation, including the Cks1 phospho-binding pocket, cyclin docking motifs, and Cdk1 active site specificity. All of these mechanisms contribute to the dynamics of the sequence of events leading to Sic1 degradation and initiation of S-phase . Apart from being an often-overlooked component of the Cdk1 cyclin complex, Cks1

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2808-440: The transport proteins importin β/α. The unbound SAFs then promote microtubule nucleation and stabilization around mitotic chromatin, and spindle bipolarity is organized by microtubule motor proteins. Spindle assembly is largely regulated by phosphorylation events catalyzed by mitotic kinases. Cyclin dependent kinase complexes (CDKs) are activated by mitotic cyclins, whose translation increases during mitosis. CDK1 (also called CDC2)

2862-573: The well-studied mammalian MCAK and Xenopus XKCM1. MCAK localizes to the growing tips of microtubules at kinetochores where it can trigger catastrophe in direct competition with stabilizing +TIP activity. These proteins harness the energy of ATP hydrolysis to induce destabilizing conformational changes in protofilament structure that cause kinesin release and microtubule depolymerization. Loss of their activity results in numerous mitotic defects. Additional microtubule destabilizing proteins include Op18/ stathmin and katanin which have roles in remodeling

2916-497: Was observed, where the first step was primary site phosphorylation. However, the secondary site must be located towards the C-terminus of the protein relative to the primary site for phosphorylation to occur. Secondary site phosphorylation is also sensitive to positioning. Peak phosphorylation rates were found between the +12 to +16 amino acid distances, with a distinct increase around the +10 to +12 range and gradual decrease across

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