Table of contents

Miscellaneous cell cycle dependent proteins phosphorylated by cdc2

During early development, gene expression is controlled principally at the translational level. Oocytes of the surf clam Spisula solidissima contain large stockpiles of maternal mRNAs, which are translationally dormant or masked until meiotic maturation. Fertilization of the oocyte leads to rapid polysomal recruitment of the abundant cyclin and ribonucleotide reductase mRNAs at about the time they undergo cytoplasmic polyadenylation. Clam p82, a 3' UTR RNA-binding protein, and a member of the CPEB (cytoplasmic polyadenylation element binding protein: Drosophila homolog Orb) family, functions as a translational masking factor in oocytes and as a polyadenylation factor in fertilized eggs. In meiotically maturing clam oocytes, p82/CPEB is rapidly phosphorylated on multiple residues to a 92-kDa apparent size, prior to its degradation during the first cell cleavage. The protein kinase(s) that phosphorylates clam p82/CPEB were examined using a clam oocyte activation cell-free system that responds to elevated pH, mirroring the pH rise that accompanies fertilization. p82/CPEB phosphorylation requires Ca2+ (<100 muM) in addition to raised pH. Examination of the calcium dependency combined with the use of specific inhibitors implicates the combined and independent actions of cdc2 and MAP kinases in p82/CPEB phosphorylation. Calcium is necessary for both the activation and the maintenance of MAP kinase, whose activity is transient in vitro, as in vivo. While cdc2 kinase plays a role in the maintenance of MAP kinase activity, it is not required for the activation of MAP kinase. A model of clam p82/CPEB phosphorylation is proposed in which MAP kinase initially phosphorylates clam p82/CPEB, at a minor subset of sites that does not alter its migration, and cdc2 kinase is necessary for the second wave of phosphorylation that results in the large mobility size shift of clam p82/CPEB (Katsu, 1999).

Translational activation of several dormant mRNAs in vertebrate oocytes is mediated by cytoplasmic polyadenylation, a process controlled by the cytoplasmic polyadenylation element (CPE) and its binding protein CPEB. The translation of CPE-containing mRNAs does not occur en masse at any one time, but instead is temporally regulated. In Xenopus, partial destruction of CPEB controls the temporal translation of CPE-containing mRNAs. While some mRNAs, such as the one encoding Mos, are polyadenylated at prophase I, the polyadenylation of cyclin B1 mRNA requires the partial destruction of CPEB that occurs at metaphase I. CPEB destruction is mediated by a PEST box and Cdc2-catalyzed phosphorylation, and is essential for meiotic progression to metaphase II. CPEB destruction is also necessary for mitosis in the early embryo. These data indicate that a change in the CPEB:CPE ratio is necessary to activate mRNAs at metaphase I and drive the cells' entry into metaphase II (Mendez, 2002).

Consistent with cdc2/cyclin B being the protein kinase controlling entry of cells into mitosis, addition of cdc2/cyclin B kinase to extracts prepared from asynchronous cells or reconstituted transcription reactions causes a complete transcriptional shut-off. A reconstituted cell-free transcription system was used to investigate the molecular basis of mitotic repression of RNA polymerase I (pol I) transcription. SL1, the human pol-I-specific TBP-TAFI containing promoter-binding factor, is inactivated by cdc2/cyclin B-directed phosphorylation, and reactivated by dephosphorylation. Transcriptional inactivation in vitro is accompanied by phosphorylation of two subunits, e.g. TBP and hTAFI110. To distinguish whether transcriptional repression is due to phosphorylation of TBP, hTAFI110 or both, SL1 was purified from two HeLa cell lines that express either full-length TBP or only the core domain of TBP. Both TBP-TAFI complexes exhibit similar activity and both are repressed at mitosis, indicating that the variable N-terminal domain, which contains multiple target sites for cdc2/cyclin B phosphorylation, is dispensable for mitotic repression (Heix, 1999).

Transcription of 18S and 28S rRNA genes by RNA polymerase I requires the cooperative binding of the multimeric protein complex termed selectivity factor I (SL1) and upstream binding factor (UBF). SL1 confers RNA polymerase I selectivity and is composed of TATA-binding protein (TBP) and three TBP-associated factors: (TAF)I110, TAFI63, and TAFI48. However, the rRNA promoter lacks a TATA box, and SL1 does not bind efficiently to the rRNA promoter by itself. Stable binding of SL1 requires UBF, a 94/97-kDa polypeptide that binds to the upstream control element of the rRNA promoter. Protein-protein interaction studies reveal that mitotic phosphorylation impairs the interaction of SL1 with Upstream binding factor (UBF). Phosphorylation of hTAFI110 rather than TBP accounts for the loss of SL1 activity. The results suggest that phosphorylation of SL1 by cdc2/cyclin B is used as a molecular switch to prevent pre-initiation complex formation and to shut down ribosomal DNA transcription at mitosis (Heix, 1999).

poly(A) polymerase (PAP) adds a poly(A) tail to nearly every newly transcribed pre-mRNA, and is a part of a multisubunit complex that tightly couples precise pre-mRNA cleavage to subsequent poly(A) addition. p34cdc2/cyclin B (mitosis/meisois promoting factor, or MPF) hyperphosphorylates poly(A) polymerase during M-phase of the cell cycle, causing repression of its enzymatic activity. Mutation of three cyclin-dependent kinase (cdk) consensus sites in the PAP C-terminal regulatory domain prevent complete phosphorylation and MPF-mediated repression. PAP also contains four nearby non-consensus cdk sites that are phosphorylated by MPF. Remarkably, full phosphorylation of all these cdk sites is required for repression of PAP activity, and partial phosphorylation has no detectable effect. The consensus sites are phosphorylated in vitro at a 10-fold lower concentration of MPF than the non-consensus sites. Consistent with this, during meiotic maturation of Xenopus oocytes, consensus sites are phosphorylated prior to the non-consensus sites at metaphase of meiosis I, and remain so throughout maturation, while the non-consensus sites do not become fully phosphorylated until after 12 h of metaphase II arrest. It is proposed that PAP's multiple cdk sites, and their differential sensitivity to MPF, provide a mechanism to link repression specifically to late M-phase. This reflects a general means to control the timing of cdk-dependent regulatory events during the cell cycle. Progressive phosphorylation of PAP, first on consensus cdk sites and later on non-consensus sites, provides a mechanism for linking repression of PAP activity exclusively to late M-phase (Colgan, 1998)

Human single-stranded DNA-binding protein (HSSB, also called RPA), is a heterotrimeric complex that consists of three subunits: p70, p34, and p11. HSSB is essential for the in vitro replication of SV40 DNA and nucleotide excision repair. It also has important functions in other DNA transactions, including DNA recombination, transcription, and double-stranded DNA break repair. The p34 subunit of HSSB is phosphorylated in a cell cycle-dependent manner. Both Cdc2 kinase and the DNA-dependent protein kinase (DNA-PK) phosphorylate HSSB-p34 in vitro. Efficient phosphorylation of HSSB-p34 by DNA-PK requires Ku as well as DNA. The DNA-PK phosphorylation sites in HSSB-p34 have been mapped at Thr-21 and Ser-33. Kinetic studies demonstrate that a phosphate residue is first incorporated at Thr-21 followed by the incorporation of a second phosphate residue at Ser-33. Ser-29 was identified as the major Cdc2 kinase phosphorylation site in the p34 subunit (Niu, 1997).

Cells of the dipteran insect Chironomus contain a high mobility group protein that is homologous to the mammalian high mobility group proteins I/Y (HMGI/Y). These proteins facilitate the assembly of higher order nucleoprotein complexes. In proliferating cells, >30% of Chironomus HMGI is found to be phosphorylated. The phosphorylation sites map to Ser3, Ser22, and Ser72 and are found to be substrates for the kinases Cdc2 (and mitogen-activated protein [MAP]), MAP, and Ca2+/phospholipid-dependent protein kinase, respectively. In mitotically arrested cells, the extent of phosphorylation at Ser3 increases, whereas phosphorylation at Ser22 remains unchanged. In nondividing cells, phosphorylation at Ser3 and Ser22 is strongly reduced. The DNA binding affinity of Chironomus HMGI is not influenced by single phosphorylation at Ser3 or Ser22. In contrast, phosphorylation at both of these sites results in a 10-fold weakening of the binding activity and alters the mode of protein-DNA interaction. Since both human and murine HMGI/Y proteins (similar to insect HMGI protein) possess phosphorylation sites for Cdc2 and MAP kinases that intersperse the AT-hook DNA-binding motifs, these results may reflect a general mechanism that regulates the properties and function of this class of putative transcriptional regulators (Schwanbeck, 1997).

Mutations in the tumor suppressor gene APC invariably lead to the development of colorectal cancer. The vast majority of these mutations are nonsense or frameshifts resulting in nonfunctional, truncated APC protein products. Eleven cyclin-dependent kinase (CDK) consensus phosphorylation sites have been identified in the frequently deleted carboxyl-terminal region of APC; loss of these phosphorylation sites by mutation could therefore compromise the ability of APC to inhibit cell growth. This report demonstrates that immunoprecipitates of full-length, but not truncated, APC protein include a mitosis-specific kinase activity in vivo. Biochemical and Western analysis of these immunoprecipitates confirms the presence of the CDK p34(cdc2). APC is a substrate for recombinant human p34(cdc2)-cyclin B1. Modification of APC by p34(cdc2) implicates phosphorylation as a mechanism for regulating APC function via a link to the cell cycle (Trzepacz, 1997).

The lamin B receptor (LBR) is an integral protein of the inner nuclear membrane that is modified at interphase by a nuclear envelope-bound protein kinase (see Drosophila Lamin). This enzyme (RS kinase) specifically phosphorylates arginine-serine dipeptide motifs located at the NH2-terminal domain of LBR and regulates its interactions with other nuclear envelope proteins. To compare the phosphorylation state of LBR during interphase and mitosis, phosphopeptide mapping was performed using in vitro and in vivo 32P-labeled LBR; a series of recombinant proteins and synthetic peptides were analyzed. LBR undergoes two types of mitotic phosphorylation mediated by the RS and the p34(cdc2) protein kinases, respectively. The RS kinase modifies similar sites at interphase and mitosis (i.e. Ser76, Ser78, Ser80, Ser82, Ser84), whereas p34(cdc2) mainly phosphorylates Ser71. These findings clarify the phosphorylation state of LBR during the cell cycle and provide new information for understanding the mechanisms responsible for nuclear envelope assembly and disassembly (Nikolakaki, 1997).

Xenopus nuclear factor 7 (xnf7) is a maternally expressed putative transcription factor that exhibits phosphorylation-dependent changes in subcellular localization during early Xenopus development. Xnf7 is localized to the germinal vesicle (nucleus) of immature oocytes in a hypophosphorylated state. Xnf7 is phosphorylated during oocyte maturation and released to the cytoplasm. The protein is retained in the cytoplasm during early embryonic cleavage stages but returns to nuclei at the mid-blastula transition. Xnf7 is phosphorylated at two sites during oocyte maturation: the first, designated P1, consists of one threonine at position 103; the second, P2, consists of three clustered threonines at positions 209, 212, and 218. Phosphorylation of both sites is important in regulating xnf7 localization. The P1 site can be phosphorylated by cyclin B/Cdc2 in vitro. To further understand the mechanisms regulating subcellular localization of xnf7 during early development, kinases capable of catalyzing phosphorylation of the P2 site were purified from mature oocyte extracts. Mitogen-activated protein kinase phosphorylates Thr212; cyclin B/Cdc2 phosphorylates Thr 209 and Thr212. No other kinase in mature oocyte extracts phosphorylate the xnf7 P2 site to a significant extent. These results implicate mitogen-activated protein kinase and cyclin B/Cdc2 in regulating xnf7 localization during oocyte maturation. This also suggests that localization of xnf7 may be regulated by multiple kinase activation pathways (El-Hodiri, 1997).

Phosphorylation of the monomeric GTPase rab4 in mitotic cells leads to its relocalization, from the endosome membranes to the cytosol. To determine the mechanism underlying this change in distribution, an in vitro assay was established that reconstitutes specific binding of rab4 when endosome-containing membranes are incubated with rab4, complexed with its cytosolic chaperone, GDP dissociation inhibitor (GDI). rab4 is found to bind to a saturable receptor associated with highly purified endosomes. Membrane binding and nucleotide exchange are physically distinct, since an active soluble fragment of the rab4 receptor, but not rab4 nucleotide exchange activity, can be released from membranes by elastase cleavage. Interestingly, the soluble fragment can be used to fully reconstitute rab4 membrane binding. In vitro phosphorylation of rab4 by cdc2/cyclin B kinase does not affect formation of rab4-GDI complexes, but does completely inhibit rab4 binding to its receptor. In contrast, in vitro phosphorylation of membranes does not result in the dissociation of bound rab4, nor are mitotic membranes deficient with respect to binding non-phosphorylated rab4. Thus, mitotic cells appear to accumulate rab4 in the cytosol by phosphorylating rab4 during the soluble phase of its normal activity cycle, thereby preventing membrane attachment (Ayad, 1997).

Nucleolin is a major component of the nucleolus. In Xenopus laevis, a maternal store of nucleolin accumulates in the multiple nucleoli generated during oogenesis. This maternal nucleolin is distributed throughout the cytoplasm of the egg during oocyte maturation; after fertilization, it is gradually reaccumulated in the nuclei of the embryo. Cytoplasmic localization of nucleolin coincides with massive phosphorylation by p34cdc2 kinase, and nuclear translocation is accompanied by net dephosphorylation. Multiple phosphorylation consensus sites for the cell cycle-dependent p34cdc2 kinase and for protein kinase CK2 are clustered in the N-terminal domain of nucleolin. To assess the efficiency of the bipartite nuclear localization signal, fusion proteins were constructed consisting of maltose binding protein (MBP) and the nuclear localization signal of nucleolin. Either an acidic domain of nucleolin without phosphorylation sites, or an acidic domain containing 4 CK2 sites, or a cluster of 5 cdc2 sites was fused to the MBP-nuclear localization signal (MBP-NLS). Nuclear translocation of these constructs was tested in an in vitro system consisting of Xenopus egg extract and sperm nuclei. Nuclear targeting of MBP by the bipartite nuclear localization signal of nucleolin becomes significantly more efficient after addition of either CK2 sites or cdc2 sites to the MBP-NLS construct. Yet the cdc2 sites play a dual role. They enhance nuclear translocation exclusively in their dephosphorylated state and promote cytoplasmic localization when phosphorylated, thereby providing a powerful cell cycle-dependent regulatory element of the nuclear localization signal (Schwab, 1997).

There are two vertebrate nonmuscle myosin heavy chain (MHC) genes that encode two separate isoforms of the heavy chain: MHC-A and MHC-B. Recent work has identified additional, alternatively spliced isoforms of MHC-B cDNA with inserted sequences of 30 nucleotides (chicken and human) or 48 nucleotides (Xenopus) at a site corresponding to the ATP binding region in the MHC protein. The deduced amino acid sequence of these inserts contains a consensus sequence for phosphorylation by cyclin-p34cdc2 (cdc2) kinase. In cultured Xenopus XTC cells, two inserted MHC-B isoforms and a non-inserted MHC-A isoform have been identified. When myosin is immunoprecipitated from XTC cells and phosphorylated in vitro with cdc2 kinase, the kinase catalyzes the phosphorylation of both inserted MHC-B isoforms but not MHC-A. Isoelectric focusing of tryptic peptides generated from MHC-B that has been phosphorylated with cdc2 kinase reveals one major phosphopeptide, purified by reverse-phase high performance liquid chromatography and sequenced. The phosphorylated residue is Ser-214, the cdc2 kinase consensus site within the insert near the ATP binding region. The same site is phosphorylated in intact XTC cells during log phase of growth and in cell-free lysates of Xenopus eggs stabilized in second meiotic metaphase, but not interphase. Ser-214 phosphorylation is detected during maturation of Xenopus oocytes when the cdc2 kinase-containing maturation-promoting factor is activated, but not in G2 interphase-arrested oocytes. These results demonstrate that MHC-B phosphorylation is tightly regulated by cdc2 kinase during meiotic cell cycles. MHC-A and MHC-B isoforms are differentially phosphorylated at these stages, suggesting that they may serve different functions in these cells (Kelley, 1995).

Cyclin-dependent kinases (CDKs) are thought to initiate and coordinate cell division processes by sequentially phosphorylating key targets; in most cases these substrates remain unidentified. Using a screen that scores for phosphorylation of proteins, 20 mitotically phosphorylated proteins have been identified in Xenopus embryos; of these, 15 have sequence similarity to other proteins. Five have previously been shown to be phosphorylated during mitosis (epithelial-microtubule associated protein-115, Oct91, Elongation factor 1gamma, BRG1 and Ribosomal protein L18A); five are related to proteins postulated to have roles in mitosis (epithelial-microtubule associated protein-115, Schizosaccharomyces pombe Cdc5, innercentrosome protein, BRG1 and the RNA helicase WM6), and nine are related to transcription factors (BRG1, negative co-factor 2alpha, Oct91, S. pombe Cdc5, HoxD1, Sox3, Vent2, and two isoforms of Xbr1b). Of 16 substrates tested, 14 can be directly phosphorylated in vitro by the mitotic CDK, cyclin B-Cdc2, although three of these may be physiological substrates of other kinases activated during mitosis. Based on the examination of this broad set of mitotic phosphoproteins, three conclusions have been drawn about how the activation of CDKs regulates cell-cycle events: (1) Cdc2 itself appears to directly phosphorylate most of the mitotic phosphoproteins; (2) during mitosis most of the substrates are phosphorylated more than once and a number may be targets of multiple kinases, suggesting combinatorial regulation, and (3) the large fraction of mitotic phosphoproteins that are presumptive transcription factors, two of which have been previously shown to dissociate from DNA during mitosis, suggests that an important function of mitotic phosphorylation is to strip the chromatin of proteins associated with gene expression (Stukenberg, 1997).

Mitotic fragmentation of the Golgi apparatus can be largely explained by disruption of the interaction between GM130 and the vesicle-docking protein p115. A single serine (Ser-25) in GM130 has been identified as the key phosphorylated target and Cdc2 as the responsible kinase. MEK1, a component of the MAP kinase signaling pathway recently implicated in mitotic Golgi fragmentation, is not required for GM130 phosphorylation or mitotic fragmentation either in vitro or in vivo. It is proposed that Cdc2 is directly involved in mitotic Golgi fragmentation and that signaling via MEK1 is not required for this process (Lowe, 1998).

Mammalian p21-activated kinase 1 (Pak1) is a highly conserved effector for the small GTPases Cdc42 and Rac1. In lower eukaryotes, Pak1 homologs are regulated during the cell cycle by phosphorylation. Pak1 is phosphorylated during mitosis in mammalian fibroblasts. This phosphorylation occurs at a single site, Thr 212, within a domain that is unique to Pak1. Cdc2 phosphorylates Pak1 at the identical site in vitro, and inhibition of Cdc2 abolishes Pak1 mitotic phosphorylation in vivo, indicating that Cdc2 is the kinase responsible for phosphorylating Pak1 in mitotic cells. Expression of a Pak1 mutant in which Thr 212 is replaced with a phosphomimic (aspartic acid) has marked effects on the rate and extent of postmitotic spreading of fibroblasts. The mitotic phosphorylation of Pak1 does not alter the basal or Rac-stimulated activity of this kinase, but it does affect the coimmunoprecipitation of at least three proteins with Pak1. These findings are the first to implicate a mammalian Pak in cell cycle regulation and suggest that Pakl, as a result of phosphorylation by Cdc2, alters its association with binding partners and/or substrates that are relevant to the morphologic changes associated with cell division (Thiel, 2002).

The Pak kinases are targets of the Rho GTPases Rac and Cdc42, which regulate cell shape and motility. It is increasingly apparent that part of this function is due to the effect Pak kinases have on microtubule organization and dynamics. Overexpression of Xenopus Pak5 enhances microtubule stabilization, and Pak1 may inhibit a microtubule-destabilizing protein, Op18/Stathmin. A specific phosphorylation site has been identified on mammalian Pak1, T212, which is targeted by the neuronal p35/Cdk5 kinase. Pak1 phosphorylated on T212, Pak1T212(PO4), is enriched in axonal growth cones and colocalizes with small peripheral bundles of microtubules. Cortical neurons overexpressing a Pak1A212 mutant display a tangled neurite morphology, which suggests that the microtubule cytoskeleton is affected. Cyclin B1/Cdc2 phosphorylates Pak1 in cells undergoing mitosis. In the developing cortex and in cultured fibroblasts, Pak1T212(PO4) is enriched in microtubule-organizing centers and along parts of the spindles. In living cells, a peptide mimicking phosphorylated T212 accumulates at the centrosomes and spindles and causes an increased length of astral microtubules during metaphase or following nocodazole washout. It is proposed that the region surrounding phosphorylated T212 contains a protein binding site, since the phosphorylated peptide is enriched in spindles and MTOCs and competes with endogenous Pak1 for this location.Together these results suggest that similar signaling pathways regulate microtubule dynamics in a remodeling axonal growth cone and during cell division (Banerjee, 2002).

A mechanism that triggers neuronal apoptosis has been characterized. The cell cycle-regulated protein kinase Cdc2 is expressed in postmitotic granule neurons of the developing rat cerebellum and Cdc2 mediates apoptosis of cerebellar granule neurons upon the suppression of neuronal activity. Cdc2 catalyzes the phosphorylation of the BH3-only protein BAD at a distinct site, serine 128, and thereby induces BAD-mediated apoptosis in primary neurons by opposing growth factor inhibition of the apoptotic effect of BAD. The phosphorylation of BAD serine 128 inhibits the interaction of growth factor-induced serine 136-phosphorylated BAD with 14-3-3 proteins. These results suggest that a critical component of the cell cycle couples an apoptotic signal to the cell death machinery via a phosphorylation-dependent mechanism that may generally modulate protein-protein interactions (Konishi, 2002).

The Ral signaling pathway is critically involved in Ras-dependent oncogenesis. One of its key actors, RLIP/RalBP1, which participates in receptor endocytosis during interphase, is also involved in mitotic processes when endocytosis is switched off. During mitosis, RLIP76 is located on the duplicated centrosomes and is required for their proper separation and movement to the poles. Factors have been sought that associate with RLIP during mitosis. RLIP/RalBP1 interacts with an active p34cdc2.cyclinB1 (cdk1) enzyme and this interaction is crucial for the mitotic phosphorylation of Epsin (see Drosophila Liquid facets) that, once phosphorylated, is no longer competent for endocytosis. This latter phosphorylation is dependent on Ral signaling. It is proposed that RLIP/RalBP1 is used as a platform by the mitotic cdk1 to facilitate the phosphorylation of Epsin, which makes Epsin incompetent for endocytosis during mitosis, when endocytosis is switched off (Rosse, 2003).

Xenopus oocytes are arrested in meiotic prophase I and resume meiotic divisions in response to progesterone. Progesterone triggers activation of M-phase promoting factor (MPF) or Cdc2-cyclin B complex and neosynthesis of Mos kinase, responsible for MAPK activation. Both Cdc2 and MAPK activities are required for the success of meiotic maturation. However, the signaling pathway induced by progesterone and leading to MPF activation is poorly understood, and most of the targets of both Cdc2 and MAPK in the oocyte remain to be determined. Aurora-A is a Ser/Thr kinase involved in separation of centrosomes and in spindle assembly during mitosis. It has been proposed that in Xenopus oocytes Aurora-A could be an early component of the progesterone-transduction pathway, acting through the regulation of Mos synthesis upstream Cdc2 activation. This study addressed the question of Aurora-A regulation during meiotic maturation by using new in vitro and in vivo experimental approaches. Cdc2 kinase activity was found to be necessary and sufficient to trigger both Aurora-A phosphorylation and Aurora-A Histone H3 kinase activation in Xenopus oocyte. In contrast, these events are independent of the Mos/MAPK pathway. Aurora-A is phosphorylated in vivo at least on three residues that regulate differentially its kinase activity. Therefore, Aurora-A is under the control of Cdc2 in the Xenopus oocyte and could be involved in meiotic spindle establishment (Maton, 2003).

The success of cell division relies on the activation of its master regulator Cdc2-cyclin B, and many other kinases controlling cellular organization, such as Aurora-A. Most of these kinase activities are regulated by phosphorylation. Despite numerous studies showing that okadaic acid-sensitive phosphatases regulate both Cdc2 and Aurora-A activation, their identity has not yet been established in Xenopus oocytes and the importance of their regulation has not been evaluated. Using an oocyte cell-free system, it has been demonstrated that PP2A depletion is sufficient to lead to Cdc2 activation, whereas Aurora-A activation depends on Cdc2 activity. The activity level of PP1 does not affect Cdc2 kinase activation promoted by PP2A removal. PP1 inhibition is also not sufficient to lead to Aurora-A activation in the absence of active Cdc2. It is therefore conclude that in Xenopus oocytes, PP2A is the key phosphatase that negatively regulates Cdc2 activation. Once this negative regulator is removed, endogenous kinases are able to turn on the activator Cdc2 system without any additional stimulation. In contrast, Aurora-A activation is indirectly controlled by Cdc2 activity independently of either PP2A or PP1. This strongly suggests that in Xenopus oocytes, Aurora-A activation is mainly controlled by the specific stimulation of kinases under the control of Cdc2 and not by downregulation of phosphatase (Maton, 2005).

Cleavage furrow formation marks the onset of cell division during early anaphase. The small GTPase RhoA and its regulators ECT2 and MgcRacGAP (Drosophila homolog: RacGAP50C) have been implicated in furrow ingression in mammalian cells, but the signaling upstream of these molecules remains unclear. The inhibition of cyclin-dependent kinase (Cdk)1 is sufficient to initiate cytokinesis. When mitotically synchronized cells are treated with the Cdk-specific inhibitor BMI-1026, the initiation of cytokinesis is induced precociously before chromosomal separation. Cytokinesis is also induced by the Cdk1-specific inhibitor purvalanol A but not by Cdk2/Cdk5- or Cdk4-specific inhibitors. Consistent with initiation of precocious cytokinesis by Cdk1 inhibition, introduction of anti-Cdk1 monoclonal antibody results in cells with aberrant nuclei. Depolymerization of mitotic spindles by nocodazole inhibits BMI-1026-induced precocious cytokinesis. However, in the presence of a low concentration of nocodazole, BMI-1026 induces excessive membrane blebbing, which appears to be caused by formation of ectopic cleavage furrows. Depletion of ECT2 or MgcRacGAP by RNA interference abolishes both of the phenotypes (precocious furrowing after nocodazole release and excessive blebbing in the presence of nocodazole). RNA interference of RhoA or expression of dominant-negative RhoA efficiently reduces both phenotypes. RhoA was localized at the cleavage furrow or at the necks of blebs. It is proposed that Cdk1 inactivation is sufficient to activate a signaling pathway leading to cytokinesis, which emanates from mitotic spindles and is regulated by ECT2, MgcRacGAP, and RhoA. Chemical induction of cytokinesis will be a valuable tool to study the initiation mechanism of cytokinesis (Niiya, 2005).

Centrosomes in mammalian cells have been implicated in cytokinesis; however, their role in this process is poorly defined. A human coiled-coil protein, Cep55 (centrosome protein 55 kDa), is described that localizes to the mother centriole during interphase. Despite its association with TuRC anchoring proteins CG-NAP and Kendrin, Cep55 is not required for microtubule nucleation. Upon mitotic entry, centrosome dissociation of Cep55 is triggered by Erk2/Cdk1-dependent phosphorylation at S425 and S428. Furthermore, Cep55 locates to the midbody and plays a role in cytokinesis, as evidenced by the observation that its depletion by siRNA results in failure of this process. S425/428 phosphorylation is required for interaction with Plk1, enabling phosphorylation of Cep55 at S436. Cells expressing phosphorylation-deficient mutant forms of Cep55 undergo cytokinesis failure. These results highlight the centrosome as a site to organize phosphorylation of Cep55, enabling it to relocate to the midbody to function in mitotic exit and cytokinesis (Fabbro, 2005).

The centrosome is the principle microtubule organizing center of the mammalian cell, consisting of a pair of barrel-shaped microtubule assemblies which are nonidentical and are described as the mother and daughter centrioles. They (mainly the mother) are surrounded by pericentriolar material (PCM), which consists of a matrix of predominantly coiled-coil proteins. PCM is the main site for nucleation of cytoplasmic microtubules and microtubules that form the meiotic and mitotic spindles. The centrosome is structurally and functionally regulated in a cell cycle-dependent manner to form a bipolar spindle to ensure the proper segregation of replicated chromosomes into two daughter cells. Defects in the number, structure, and function of centrosomes can generate mono- or multipolar mitotic spindles and cytokinesis defects resulting in aneuploidy and chromosome instability: these are common characteristics of tumor cells. Therefore, it is not surprising that these centrosome abnormalities are frequently found in tumors and are usually associated with high cytological grade. Thus, it is critical to understand regulators of the centrosome cycle because it must be carefully coordinated with the cell cycle to complete cell division precisely (Fabbro, 2005).

Microtubule nucleation by the PCM requires the conserved complex, gamma-tubulin ring complex (gamma-TuRC), in metazoan organisms. In yeast, the large coiled-coil spindle pole body (SPB) protein Spc110p anchors gamma-tubulin ring complex, providing sites for microtubule nucleation. In mammalian cells, Kendrin, like its yeast homolog Spc110p, complexes with CG-NAP to provide a structural scaffold for gamma-TuRC (Takahashi, 2002). Nlp has also been implicated in gamma-TuRC anchorage (Casenghi, 2003). CG-NAP and Kendrin associate with several protein kinases and phosphatases, suggesting that microtubule nucleation may be regulated through phosphorylation of CG-NAP/Kendrin complexes or associated proteins thatremain to be defined (Fabbro, 2005).

In recent years, several studies have provided a link between centrosomes and cytokinesis. Acentrosomal cells have been shown to form mitotic spindles and progress through mitosis but fail to complete cytokinesis. The molecular understanding of centrosome function in cytokinesis is only beginning to emerge. During cytokinesis, the mother centriole has been shown to transiently reposition to the midbody correlating with bridge narrowing and microtubule depolymerization, while movement away from the midbody correlates with cell cleavage. It is proposed that the mother centriole regulates an as yet unidentified pathway anchored at the centrosome that is analogous to the mitotic exit pathways in budding yeast called the 'mitotic exit network,' which is anchored at the SPB and controls mitotic exit and cytokinesis. The siRNA silencing of a recently identified mother centriole component, centriolin, produces cytokinesis failure (Gromley, 2003), suggesting that it is a component of this pathway in mammalian cells. However, additional components and pathways that control cytokinesis will need to be identified to understand the precise role of centrosomes in this process (Fabbro, 2005).

This study reports the molecular characterization of a coiled-coil protein called Cep55. Cep55 localizes to the centrosome of interphase cells and to the midbody during cytokinesis. Characterization of Cep55-depleted cells reveals that Cep55 participates in membrane abscission to form two daughter cells. Furthermore, Cdk1, Erk2, and Plk1 cooperate in the mitotic phosphorylation of Cep55, and this modification is required for its correct mitotic localization and cytokinesis function to maintain genomic stability (Fabbro, 2005).

Interestingly, Cep55 does not remain associated with the centrosome during mitosis, and its displacement from the centrosome is triggered by its phosphorylation. Therefore, a model is proposed whereby the centrosome acts as a regulatory site to organize phosphorylation of Cep55 upon mitotic entry. This phosphorylated Cep55 is then able to locate to the midbody during the final stages of cell division to function in the signal transduction pathway(s) that results in mitotic exit and cytokinesis. Therefore, it is proposed that dephosphorylation of Cep55 may allow it to relocate to the centrosome upon entry into G1 (Fabbro, 2005).

Fluctuations in the activity of Cdks drive cells to progress through mitosis. Specifically, mitotic entry is promoted by elevated activity of Cdk1 when complexed with cyclin B1, while the exit from mitosis requires the inactivation of Cdk1 and the dephosphorylation of at least a subset of Cdk1 substrates. This study demonstrates that Cep55 is phosphorylated at S425 and S428 by Cdk1/cyclin B1 upon mitotic entry, which is when this kinase is found at the centrosome. Another kinase, Erk2, has been shown to localize to the mitotic centrosomes. S425 and S428 can also be phosphorylated by Erk2 upon mitotic entry. In addition to Cdk1 and Erk2, Cep55 is also phosphorylated by Plk1 and this phosphorylation event is dependent on prior phosphorylation by Cdk1 and/or Erk2 at S425 and S428. Plk1 locates to the midbody and its role during cytokinesis has been demonstrated in yeast, Drosophila, and mammals. These data strongly indicate that Cep55 and Plk1 colocalize at the midbody and that Plk1-dependent phosphorylation of Cep55 is required for completion of cytokinesis. In addition, the Plk1-dependent phosphorylation mutant S436A causes cytokinesis failure to the same extent as the Cdk1/Erk2-dependent mutant S425/428A, indicating that phosphorylation at S436 is absolutely required for the function of Cep55 during cytokinesis, whereas phosphorylation at S425 and S428 is not required for Cep55 cytokinesis function directly but is essential for Plk1-dependent phosphorylation at S436. These findings indicate that Cep55 and Plk1 may cooperate at the midbody to coordinate mitotic exit and cytokinesis. Thus, it is proposed that unphosphorylatable Cep55 may fail to bind or generate the required signal to downstream components of the mitotic exit pathway (Fabbro, 2005).

Cep55 localizes to the centrosome via its C-terminus and this same region associates in vivo with the centrosome proteins CG-NAP and Kendrin. In contrast to CG-NAP and Kendrin, which gain affinity for the centrosome to participate in gamma-TuRC anchorage (Takahashi, 2002), Cep55 loses affinity for the centrosome upon mitotic entry coinciding with its dissociation from CG-NAP and Kendrin. Thus, it is not surprising that no requirement is found for Cep55 for microtubule nucleation. Loss of Cep55 from the centrosome coincides with its phosphorylation at the C-terminal residues S425 and S428. It is plausible to suggest that phosphorylation of Cep55 by either Erk2 or Cdk1 causes a conformational change in the protein causing it to lose affinity for CG-NAP and Kendrin, consequently becoming displaced from the centrosome at the G2/M boundary. This in turn may enable S436 of Cep55 to be accessible to Plk1 for phosphorylation. It is hypothesized that the displacement of Cep55 from the centrosome enables CG-NAP and Kendrin to strongly anchor themselves to the centrosome by binding calmodulin (Takahashi, 2002). Consistent with this idea, Cep55 and calmodulin bind the same region of CG-NAP, and the CG-NAP/Kendrin/calmodulin interaction is thought to occur only during mitosis, which is when calmodulin is observed at the centrosome (Fabbro, 2005).

Previous experiments with peptide fusion proteins suggested that cyclin A/Cdk1 and Cdk2 might exhibit similar yet distinct phosphorylation specificities. Using a physiological substrate, CDP/Cux, this notion has been confirmed. Proteolytic processing of CDP/Cux by cathepsin L generates the CDP/Cux p110 isoform at the beginning of S phase. CDP/Cux p110 makes stable interactions with DNA during S phase but is inhibited in G2 following the phosphorylation of serine 1237 by cyclin A/Cdk1. It is proposed that differential phosphorylation by cyclin A/Cdk1 and cyclin A/Cdk2 enables CDP/Cux p110 to exert its function as a transcriptional regulator specifically during S phase. Like cyclin A/Cdk1, cyclin A/Cdk2 interacts efficiently with recombinant CDP/Cux proteins that contain the Cut homeodomain and an adjacent cyclin-binding motif (Cy). In contrast to cyclin A/Cdk1, however, cyclin A/Cdk2 does not efficiently phosphorylate CDP/Cux p110 on serine 1237 and does not inhibit its DNA binding activity in vitro. Accordingly, co-expression with cyclin A/Cdk2 in cells does not inhibit the DNA binding and transcriptional activities of CDP/Cux p110. To confirm that the sequence surrounding serine 1237 is responsible for the differential regulation by Cdk1 and Cdk2, four amino acids flanking the phosphorylation site were replaced to mimic a known Cdk2 phosphorylation site present in the Cdc6 protein. Both cyclin A/Cdk2 and Cdk1 efficiently phosphorylates the CDP/Cux(Cdc6) mutant and inhibits its DNA binding activity. Altogether these results help explain why the DNA binding activity of CDP/Cux p110 is maximal during S phase and decreases in G2 phase (Santaguida, 2005).

AML1 (RUNX1) regulates hematopoiesis, angiogenesis, muscle function, and neurogenesis. Previous studies have shown that phosphorylation of AML1, particularly at serines 276 and 303, affects its transcriptional activation. Phosphorylation of AML1 serines 276 and 303 can be blocked in vivo by inhibitors of the cyclin-dependent kinases (CDKs) Cdk1 and Cdk2. Furthermore, these residues can be phosphorylated in vitro by purified Cdk1/cyclin B and Cdk2/cyclin A. Mutant AML1 protein that cannot be phosphorylated at these sites (AML1-4A) is more stable than wild-type AML1. AML-4A is resistant to degradation mediated by Cdc20, one of the substrate-targeting subunits of the anaphase-promoting complex (APC). However, Cdh1, another targeting subunit used by the APC, can mediate the degradation of AML1-4A. A phospho-mimic protein, AML1-4D, can be targeted by Cdc20 or Cdh1. These observations suggest that both Cdc20 and Cdh1 can target AML1 for degradation by the APC but that AML1 phosphorylation may affect degradation mediated by Cdc20-APC to a greater degree (Biggs, 2006).

A predominantly nuclear RNA-binding protein, HuR translocates to the cytoplasm in response to stress and proliferative signals, where it stabilizes or modulates the translation of target mRNAs. Evidence is presented that HuR phosphorylation at S202 by the G2-phase kinase Cdk1 influences its subcellular distribution. HuR is specifically phosphorylated in synchronous G2-phase cultures; its cytoplasmic levels increased by Cdk1-inhibitory interventions and declined in response to Cdk1-activating interventions. In keeping with the prominently cytoplasmic location of the nonphosphorylatable point mutant HuR(S202A), phospho-HuR(S202) was shown to be predominantly nuclear using a novel anti-phospho-HuR(S202) antibody. The enhanced cytoplasmic presence of unphosphorylated HuR is linked to its decreased association with 14-3-3 and to its heightened binding to target mRNAs. These findings suggest that Cdk1 phosphorylates HuR during G2, thereby helping to retain it in the nucleus in association with 14-3-3 and hindering its post-transcriptional function and anti-apoptotic influence (Kim, 2008).

The cell cycle transition from interphase into mitosis is best characterized by the appearance of condensed chromosomes that become microscopically visible as thread-like structures in nuclei. Biochemically, launching the mitotic program requires the activation of the mitotic cyclin-dependent kinase Cdk1 (cyclin-dependent kinase 1), but whether and how Cdk1 triggers chromosome assembly at mitotic entry are not well understood. This study reports that mitotic chromosome assembly in prophase depends on Cdk1-mediated phosphorylation of the condensin II complex. Thr 1415 of the CAP-D3 subunit was identified as a Cdk1 phosphorylation site, which proved crucial as it was required for the Polo kinase Plk1 (Polo-like kinase 1) to localize to chromosome axes through binding to CAP-D3 and thereby hyperphosphorylate the condensin II complex. Live-cell imaging analysis of cells carrying nonphosphorylatable CAP-D3 mutants in place of endogenous protein suggested that phosphorylation of Thr 1415 is required for timely chromosome condensation during prophase, and the Plk1-mediated phosphorylation of condensin II facilitates its ability to assemble chromosomes properly. These observations provide an explanation for how Cdk1 induces chromosome assembly in cells entering mitosis, and underscore the significance of the cooperative action of Plk1 with Cdk1 (Abe, 2011).

Cdc2 targets N-myc

Myc family transcription factors are destabilized by phosphorylation of a conserved amino-terminal GSK-3ß motif. In proliferating cerebellar granule neuron precursors (CGNPs), Sonic hedgehog signaling induces N-myc expression, and N-myc protein is stabilized by insulin-like growth factor-mediated suppression of GSK-3ß. N-myc phosphorylation-mediated degradation is a prerequisite for CGNP growth arrest and differentiation. This study investigated whether N-myc phosphorylation and turnover are thus linked to cell cycle exit in primary mouse CGNP cultures and the developing cerebellum. Phosphorylation-induced turnover of endogenous N-myc protein in CGNPs increases during mitosis, due to increased priming phosphorylation of N-myc for GSK-3ß. The priming phosphorylation requires the Cdk1 complex, whose cyclin subunits are indirect Sonic hedgehog targets. These findings provide a mechanism for promoting growth arrest in the final cycle of neural precursor proliferation competency, or for resetting the cell cycle in the G1 phase, by destabilizing N-myc in mitosis (Sjostrom, 2005).

Increased S54 phosphorylation is strongly associated with N-myc destabilization. Further, the mitotic kinase Cdk1, in complex with cyclins A and B1, mediates N-myc S54 phosphorylation in primary CGNPs. N-myc is thus primed for GSK-3ß-mediated phosphorylation, which promotes degradation of c-myc and other cell cycle regulatory proteins. This model is consistent with permitting primary neural precursor exit from the cell cycle before G1 is reentered. This allows differentiation to begin, in accordance with intrinsic programs (Sjostrom, 2005).

Shh directly induces N-myc expression and indirectly affects N-myc posttranslational modification, mediated by its indirect targets, cyclins A and/or B. Mitotic degradation of N-myc permits neuronal precursor cell cycle exit in the absence of Shh signaling or in the case of an intrinsic program-directed shift toward differentiation (Sjostrom, 2005).

The data indicate an increase in phosphorylation-associated N-myc degradation during mitosis, which occurs over an interval of less than 1 hr. N-myc is a short-lived protein with a half-life of approximately 40 min, while the cell cycle in mouse CGNPs at PN4 lasts 16-18 hr. How is N-myc disposed of during interphase in proliferating CGNPs? There is growing evidence that myc protein stability regulation may also involve regions outside of the amino-terminal myc box 1 domain containing T50 and S54. Interactions between the myc box 2 domain and the F box protein Skp2 promote c-myc proteolysis during the G1-to-S phase transition, indicating that myc metabolism may be regulated in a cell cycle-dependent manner. With regard to N-myc, these mechanisms have yet to be verified in primary neural precursors (Sjostrom, 2005).

GSK-3ß-mediated phosphorylation targets c-myc for degradation through a mechanism involving the F box protein Fbw7 in a variety of cell lines. A similar mechanism for N-myc degradation was suggested. Whether Fbw7, or another F box protein with an appropriate spatiotemporal expression pattern, plays a role in N-myc proteolysis in primary cells and during cerebellar development in vivo remains to be determined. To act upon N-myc at T50, GSK-3ß must first be primed by phosphorylation at S54. Basal levels of N-myc S54 phosphorylation could be mediated by nonmitotic kinases, such as Erk, outside mitosis. N-myc was also identified as a substrate for the neural-specific kinase Cdk5. However, neither Erk nor Cdk5 activity was specific for S54. It was found that the Cdk1 complex contains a potent, specific N-myc S54 kinase (Sjostrom, 2005).

Cdk1 heterodimerizes with cyclin A and cyclin B1. Cell-free in vitro assays have shown that cyclin A in complex with Cdk1 and p107 can phosphorylate GST-c-myc fusion proteins. Both cyclin A and cyclin B1 immunoprecipitate specific activity toward N-myc, and both cyclins are expressed in Shh-treated CGNPs during mitosis. Cyclin A has also been found in interphase CGNPs, consistent with its participation in Cdk2 complexes during S phase. The lack of S54 phosphorylation in G1/S-arrested cells indicates that cyclin A:Cdk2 does not phosphorylate N-myc. Although cyclin A:Cdk1 complexes may have some substrates distinct from those of cyclin B:Cdk1 complexes, it has been shown that many Cdk substrates are indifferent as to the cyclin subunit of the cyclin:Cdk1 complex. These analyses suggest that, in primary CGNPs, N-myc S54 can be targeted by either cyclin A:Cdk1 or cyclin B1:Cdk1 (Sjostrom, 2005).

Early studies reported that c-myc protein synthesis and modification is not altered during mitosis. Later studies demonstrated mitosis-specific c-myc phosphorylation. These studies were carried out in cell lines with flexible cell cycle exit and reentry capacity. This work with N-myc has been conducted in primary neuronal precursors with a defined intrinsic program for irreversible cell cycle exit and subsequent differentiation. Many previous myc turnover studies have relied on overexpression, while this study focused on regulation of endogenous N-myc stability, in primary cultures and in vivo. The finding that N-myc is largely degraded at the conclusion of the primary neural precursor cell cycle provides insight as to how enhanced stability of N-myc protein can contribute to brain tumorigenesis, by enhancing CGNPs' capacity for ongoing division. Increased activity of IGF2, which is predicted to stabilize N-myc, is associated with increased proliferation in primary neural precursors and in mouse and human medulloblastomas. Thus, future analysis of how N-myc turnover is regulated during CGNP expansion in vivo will yield greater understanding of normal brain development and brain tumor biology (Sjostrom, 2005).

Cdc2 and damage checkpoint control

DNA damage and replication checkpoints in eukaryotic cells ensure that progression through the cell cycle is restrained while chromosomes undergo repair or replication. Damage and replication defects are recognized by the putative protein complex containing protein kinases such as human ATM and ATR, fission yeast Rad3, and budding yeast Mec1p. These kinases contain a lipid kinase motif, are highly conserved through evolution, and are thought to convey the checkpoint signal by phosphorylation to other downstream kinases, Chk1 and Cds1, through which regulators of the major cell cycle machinery are regulated. Chk1 kinase becomes phosphorylated in response to DNA damage, and this phosphorylation depends on Rad3 kinase. In fission yeast, Chk1 is targeted to restrain the Cdc25 mitotic inducer. Cds1 is activated upon replication arrest and DNA damage occurring in S phase. It is unknown, however, whether the checkpoint signal is sent directly from the checkpoint Rad complex to Chk1 in vivo. A fission yeast protein, Crb2/Rhp9, resembling budding yeast Rad9p and a human tumor suppressor protein BRCA1, is required for the damage checkpoint. Chk1 kinase is not activated in the crb2 deletion mutant (designated Deltacrb2) after DNA damage. Crb2 was identified by genetic and two-hybrid interactions with Cut5/Rad4 essential for replication and replication checkpoint. They form the complex in vitro, but the stable complex has not been found in vivo. Crb2 also shows genetic and two-hybrid interactions with Chk1. Hyperphosphorylation of Crb2 after DNA damage depends on the presence of Rad3 kinase, but not Chk1. Overproduction of Chk1, however, rescues the phenotype of Deltacrb2 damage. These results suggested that Crb2 might be placed upstream of Chk1 and downstream of Rad3. Alternatively, Crb2 and Rad3 may be independently required for the activation of Chk1 (Esashi, 1999 and references).

The C-terminal half of Crb2 contains two BRCT motifs present in a wide range of proteins implicated in DNA damage and repair. The BRCT domain of Crb2 is highly similar to that of budding yeast Rad9p, and to a lesser degree, to human BRCA1 and 53BP1. Plasmid carrying the RAD9 gene, however, fails to rescue the UV sensitivity of Deltacrb2. Crb2 and Rad9p are hyperphosphorylated after DNA damage, revealing upper band shift in the immunoblot patterns. Crb2 is phosphorylated not only when damaged but also during the normal mitotic cell cycle. A Cdc2 site, T215, was identified in the N terminus of Crb2. This T215 site undergoes stage-specific phosphorylation in the cell cycle and also is phosphorylated when damaged. The nonphosphorylatable T215A mutant abolishes hyperphosphorylation of Crb2 upon damage, and it allows cells to enter the arrested state but fails to reenter cell cycle progression. Thus fission yeast Crb2, which is required for the damage checkpoint, is the target of Cdc2 during normal mitosis and also in response to damage. Cdc2 kinase phosphorylation is necessary for reentering the cell cycle after the checkpoint. It may be noteworthy that finding Cdk phosphorylation sites for which phenotypes can be adduced is fairly unusual. Since only dividing cells undergo checkpoint arrest, active participation of Cdc2 in checkpoint control may have universal significance (Esashi, 1999).

Why is the Cdc2 site implicated in checkpoint control? The requirement of Cdc2 kinase in checkpoint control was unexpected. Cdc2 was considered to be the target of repression during checkpoint arrest because checkpoint was not activated in the presence of hyperactive Cdc2. This study, however, demonstrates that Cdc2 phosphorylation is involved in reestablishing cell cycle progression after checkpoint. The fact that the T215A mutant has problems reentering the cell cycle is clear from the results obtained. Furthermore, the level of thymine dimer generated by UV irradiation is reduced in the T215A mutant at a rate similar to that in wild-type cells. Difficulty in reentering the cell cycle thus may not be due to remaining damages but, rather, it may be caused by the failure of Crb2 to be the target of Cdc2 kinase (Esashi, 1999).

Why does T215A result in prolonged cell cycle delay? One possible explanation is that the T215A-unphosphorylatable Crb2 mutant protein retains the ability to inhibit Cdc2 kinase or loses the ability to activate Cdc2 kinase through downregulating Chk1, so that irradiated cells cannot reestablish cell cycle progression, bringing cells into permanent arrest. The T215A mutant is UV sensitive for the opposite reason than is the crb2 null: the null fails to arrest in response to UV and therefore dies (the usual checkpoint defect), but the T215A mutant arrests and fails to recover. It is proposed that T215 phosphorylation by Cdc2 is required to turn off the checkpoint system -- that is, repair of the lesions may not be sufficient. T215 phosphorylation by Cdc2 may be a part of a feedback loop to reestablish the cell cycle. A small population of Cdc2 that is active after the completion of DNA repair may phosphorylate Crb2, which subsequently reduces Chk1 kinase activity, thereby accelerating mitotic entry. Cdc2 may be an antagonist of Crb2, while Crb2 activates Chk1 for restraining Cdc2 (Esashi, 1999).

DNA replication in higher eukaryotes requires activation of a Cdk2 kinase by Cdc25A, a labile phosphatase subject to further destabilization upon genotoxic stress. A distinct, markedly stable form of Cdc25A, is described that plays a previously unrecognized role in mitosis. Mitotic stabilization of Cdc25A reflects its phosphorylation on Ser17 and Ser115 by cyclin B-Cdk1, modifications required to uncouple Cdc25A from its ubiquitin-proteasome-mediated turnover. Cdc25A binds and activates cyclin B-Cdk1, accelerates cell division when overexpressed, and its downregulation by RNA interference (RNAi) delays mitotic entry. DNA damage-induced G2 arrest, in contrast, is accompanied by proteasome-dependent destruction of Cdc25A, and ectopic Cdc25A abrogates the G2 checkpoint. Thus, phosphorylation-mediated switches among three differentially stable forms ensure distinct thresholds, and thereby distinct roles for Cdc25A in multiple cell cycle transitions and checkpoints (Mailand, 2002).

Arguably the most unexpected finding of this study was that entry into mitosis is accompanied by an abrupt switch, which converts Cdc25A from a labile to a stable protein. Hence, while acceleration of the basal Cdc25A protein turnover participates in response to genotoxic stress or stalled replication in interphase, uncoupling of Cdc25A from the ubiquitin-proteasome-dependent degradation plays an important role once the cells become committed to undergo cell division. Whereas destruction of the phosphatase guards against premature mitosis of cells with an incompletely replicated and/or damaged genome, stabilization of Cdc25A ensures that even in the absence of de novo mRNA synthesis, Cdc25A is abundant and active during initial stages of mitotis. Both reduction and extension of the Cdc25A protein half-life rely on phosphorylations within its N-terminal regulatory region. It has been shown that in S-phase cells, phosphorylation of Ser123 by the ATM-Chk2 kinase cascade (see loki) is required to induce degradation of Cdc25A in response to ionizing radiation. Such a mechanism may also operate in the DNA damage-induced G2 checkpoint. The mitotic stabilization of Cdc25A also reflects site-specific phosphorylation. Both Ser17 and Ser115 fulfill the minimum requirements for a CDK consensus site, and phosphorylation of Ser17 and Ser115 is mediated by cyclin B-Cdk1 in vitro, and is detectable in vivo only after entry into mitosis when the cyclin B-Cdk1 complex becomes physiologically active. Although the integrity of Ser17 and Ser115 appears crucial for Cdc25A stabilization, the data do not exclude the potential involvement of other residues. Thus, while concomitant mutation of Ser17 and Ser115 does confer marked instability in mitosis, the protein turnover of such a Cdc25A(2A) mutant could still be accelerated moderately by roscovitine, and the Ser17/Ser115-deficient protein still undergoes a partial mitosis-associated electrophoretic mobility shift. This indicates that cyclin B-Cdk1 might phosphorylate more residues than those identified by mass spectrometry analysis. Moreover, the fact that the endogenous Cdc25A protein remains partly shifted in roscovitine-treated mitotic cells suggests that other kinases (such as Plk1), specifically activated at the G2/M transition, assist in inducing and/or maintaining the stability of Cdc25A (Mailand, 2002).

Cdc2 and vacuole inheritance

In budding yeast, vacuole inheritance is tightly coordinated with the cell cycle. The movement of vacuoles and several other organelles is actin-based and is mediated by interaction between the yeast myosin V motor Myo2 and organelle-specific adaptors. Myo2 binds to vacuoles via the adaptor protein Vac17, which binds to the vacuole membrane protein Vac8. This study shows that the yeast cyclin-dependent kinase Cdk1 phosphorylates Vac17 and that phosphorylation of Vac17 parallels cell cycle-dependent movement of the vacuole. Substitution of the Cdk1 sites in Vac17 decreases its interaction with Myo2 and causes a partial defect in vacuole inheritance. This defect is enhanced in the presence of Myo2 with mutated phosphorylation sites. Thus, Cdk1 appears to control the timing of vacuole movement. The presence of multiple predicted Cdk1 sites in other organelle-specific myosin V adaptors suggests that the inheritance of other cytoplasmic organelles may be regulated by a similar mechanism (Peng, 2008).

Multiple Cdk1 cyclins might be required for Vac17 phosphorylation; deletion of a single cyclin does not affect Vac17 phosphorylation. Phosphorylation of Vac17 increases early in the cell cycle and continues to increase through S phase. In addition, the levels of the M phase cyclin Clb2 peak after the peak of Vac17 phosphorylation. It is likely that the G1 and/or S phase cyclins are required for Vac17 phosphorylation. In an earlier study, deletion of CLN3 resulted in a partial defect in vacuole inheritance. However, using the same strains, little difference was observed between wild-type and mutant cells. In a second strain background (LWY3250), cln3delta had no effect on vacuole inheritance. Thus, the cyclins required for coordination of vacuole movement with the cell cycle are unknown (Peng, 2008).

Notably, another Myo2-binding protein, Kar9, is a substrate of Cdk1. Phosphorylation of Kar9 prevents its binding to the spindle pole that is distal from the bud, and therefore ensures that only one pole is oriented toward the bud. These studies of Vac17 raise the possibility that Cdk1 phosphorylation of Kar9 may regulate its attachment to Myo2. Likewise, other yeast myosin V adaptors, such as Mmr1 and Inp2, contain multiple putative Cdk1 sites. It is possible that these myosin V adaptors are phosphorylated by Cdk1, and like Vac17, may regulate a cell cycle-dependent interaction with myosin V. The fact that each myosin V adaptor is dedicated to the movement of a single organelle suggests that they are ideal candidates to regulate movement of each cargo. Thus, Cdk1 phosphorylation may be a common regulatory mechanism for coordination of organelle inheritance with the cell cycle (Peng, 2008).

Table of contents

cdc2: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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