Role of Substance P in The Pathogenesis of Spider Angiomas in Patients With Nonalcoholic Liver Cirrhosis

Entry into mitosis is regulated by the Cdc2 kinase complexed to B-type cyclins. We and others recently reported that cyclin B1/Cdc2 complexes, which appear to be constitutively cytoplasmic during interphase, actually shuttle continually into and out of the nucleus, with the rate of nuclear export exceeding the import rate (1-3). At the time of entry into mitosis, the import rate is increased, whereas the export rate is decreased, leading to rapid nuclear accumulation of Cdc2/cyclin B1. Although it has recently been reported that phosphorylation of 4 serines within cyclin B1 promotes the rapid nuclear translocation of Cdc2/cyclin B1 at G 2 /M, the role that individual phosphorylation sites play in this process has not been examined (3, 4). We report here that phosphorylation of a single serine residue (Ser 113 of Xenopus cyclin B1) abrogates nuclear export of cyclin B1. This serine lies directly within the cyclin B1 nuclear export sequence and, when phosphorylated, prevents binding of the nuclear export factor, CRM1. In contrast, analysis of phosphorylation site mutants suggests that coordinate phosphorylation of all 4 serines (94, 96, 101, and 113) is required for the accelerated nuclear import of cyclin B1/Cdc2 characteristic of G 2 /M. Additionally, binding of cyclin B1 to importin-␤, the factor known to be responsible for the slow interphase nuclear entry of cyclin B1, appears to be unaffected by the phosphorylation state of cyclin B. These data suggest that a distinct import factor must be recruited to enhance nuclear entry of Cdc2/cyclin B1 at the G 2 /M transition.In all eukaryotes studied to date, the onset of both mitosis and meiosis is regulated by the activation of MPF (maturation promoting factor), a heterodimeric complex that contains the serine/threonine kinase Cdc2 and a B-type cyclin. Coordination of the G 2 /M transition relies upon tight regulation of the kinase activity of Cdc2/cyclin B. During interphase, cyclin B is synthesized and forms complexes with the inactive Cdc2 monomer. Phosphorylation of complexed Cdc2 at two sites, Thr 14 and Tyr 15 , by the inhibitory kinases Wee1 and Myt1 keeps Cdc2 inactive until the time of entry into mitosis (5-13). At the G 2 /M transition, dephosphorylation of Cdc2 by the dual specificity phosphatase, Cdc25, promotes Cdc2/cyclin B activation, thus driving cells into mitosis (14 -17).The biological function of Cdc2/cyclin B is regulated not only at the level of enzymatic activity but also by changes in subcellular localization. During interphase, Cdc2/cyclin B1 kinase complexes are predominantly cytoplasmic; at the onset of mitosis, they transit precipitously to the nucleus, presumably to catalyze the nuclear events of mitosis (18). Cyclin B2, in contrast, remains cytoplasmic throughout the cell cycle (19). By deletion analysis, the region of cyclin B1 responsible for its interphase cytoplasmic localization maps to a domain near the N terminus of the protein termed the cytoplasmic retention sequence (CRS) 1 (19). Recently, we and others reported that the CRS ...

supporting

confidence: 55%

Combinatorial Control of Cyclin B1 Nuclear Trafficking through Phosphorylation at Multiple Sites

Yang

Song

Walsh

et al. 2001

“…As shown recently in the case of MADR2, its nuclear accumulation and signaling depend on it being phosphorylated (35). This does not appear to be an isolated case as more and more instances are being reported on the influence of protein phosphorylation on the cellular location of various proteins (34,36,37), particularly those associated with cell division. It is interesting to note that only the nuclear form of dUTPase is also phosphorylated (33).…”

Section: Discussionmentioning

confidence: 82%

Intracellular Location of Thymidylate Synthase and Its State of Phosphorylation

Samsonoff

Reston

McKee³

et al. 1997

Thymidylate synthase (TS), an enzyme that is essential for DNA synthesis, was found to be associated mainly with the nucleolar region of H35 rat hepatoma cells, as determined both by immunogold electron microscopy and by autoradiography. In the latter case, the location of TS was established through the use of [6-3 H]5-fluorodeoxyuridine, which forms a tight ternary complex of TS with 5-fluorodeoxyuridylate (FdUMP) and 5,10-methylenetetrahydrofolylpolyglutamate within the cell. However, with H35 cells containing 50 -100-fold greater amounts of TS than unmodified H35 cells, the enzyme, although still in the nucleus, was located primarily in the cytoplasm as shown by autoradiography and immunohistochemistry. In addition, TS was also present in mitochondrial extracts of both cell lines, as determined by enzyme activity measurements and by ternary complex formation with [ 32 P]FdUMP and 5,10-methylenetetrahydrofolate. Another unique observation is that the enzyme appears to be a phosphoprotein, similar to that found for other proteins associated with cell division and signal transduction. The significance of these findings relative to the role of TS in cell division remains to be determined, but suggest that this enzyme's contribution to the cell cycle may be more complex than believed previously.Thymidylate synthase (TS, EC 2.1.1.45) 1 is a unique enzyme in nature by virtue of the fact that one of the substrates in the reaction, CH 2 H 4 PteGlu serves to reductively methylate the second substrate, dUMP, to yield dTMP and H 2 PteGlu. Because dTMP plays an essential role in the synthesis of DNA, the enzyme has been a chemotherapeutic target since its discovery about 40 years ago (1). The DNA sequences of some 30 different species of TS have been clarified (2) establishing it as one of the most phylogenetically conserved proteins known. X-ray crystal structures for the Lactobacillus casei (3), Escherichia coli (4, 5), and human TSs (6) have been utilized to define the mechanism by which the substrates interact with the enzyme to form product and the nature of the inhibition affected by substrate analogues, as well as to aid in the rational design of potential chemotherapeutic agents (7).While much is known about the physical and enzymic properties of TS, less is known about how the enzyme is regulated within the cell, its structural location, and its potential interaction with other proteins. Recent studies indicate that this enzyme may be regulated at both the transcriptional (8) and translational levels of synthesis (9), while earlier studies suggested that TS forms multienzyme complexes involved in DNA synthesis both in T-even phage-infected E. coli (10, 11) and eukaryotic cells (12, 13). These findings are compounded further now by the enzyme's apparent association with the nucleolus of the cell and its state of phosphorylation, as will be described in this paper. In addition we will provide evidence that TS may be located also in the mitochondria of cells as has been described recently for a bifunctional dihydrof...

“…This shows that cyclin B phosphorylation is not essential for the catalytic activity of the complex and confirms similar data obtained indirectly (mutation of phosphorylation sites) in goldfish (29) and Xenopus (33) oocytes. Studies from Li et al (33,34) have shown the requirement of cyclin B 1 phosphorylation for nuclear localization and Xenopus oocyte maturation. Nuclear localization of cyclin B 1 is regulated by its phosphorylation at sites within the cytoplasmic retention signal domain (34,48).…”

Section: Discussionmentioning

confidence: 99%

“…Studies from Li et al (33,34) have shown the requirement of cyclin B 1 phosphorylation for nuclear localization and Xenopus oocyte maturation. Nuclear localization of cyclin B 1 is regulated by its phosphorylation at sites within the cytoplasmic retention signal domain (34,48). Phosphorylation of cyclin B 1 masking the cytoplasmic retention signal would therefore permit migration into the nucleus, allowing phosphorylation of nuclear substrates by MPF.…”

Section: Discussionmentioning

confidence: 99%

“…Mutational studies (29,32,33) have suggested that cyclin B phosphorylation is required neither for activity of the Cdc2 kinase, for Cdc2 binding, nor for cyclin B destruction in anaphase. More recently, Li et al (34) showed that if the residues were mutated to nonphosphorylatable residues, the Cdc2-cyclin B complex does not migrate from the cytoplasm to the nucleus and therefore loses its MPF activity. The kinase(s) responsible for cyclin B phosphorylations has not been identified so far.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Intra-M Phase-promoting Factor Phosphorylation of Cyclin B at the Prophase/Metaphase Transition

Borgne¹,

Østvold²,

Flament³

et al. 1999

in vivo phosphorylation as shown by phosphopeptide maps of in vivo and in vitro phosphorylated cyclin B. We demonstrate that Cdc2 itself is the cyclin B kinase; cyclin B phosphorylation requires Cdc2 activity both in vivo (sensitivity to vitamin K 3 , a Cdc25 inhibitor) and in vitro (copurification with Cdc2-cyclin B, requirement of Cdc2 dephosphorylation, and sensitivity to chemical inhibitors of cyclin-dependent kinases). Furthermore, cyclin B phosphorylation occurs as an intra-M phase-promoting factor reaction as shown by the following: 1) active Cdc2 is unable to phosphorylate cyclin B associated to phosphorylated Cdc2, and 2) cyclin B phosphorylation is insensitive to enzyme/substrate dilution. We conclude that, at the prophase/metaphase transition, cyclin B is mostly phosphorylated by its own associated Cdc2 subunit.Cell cycle events are regulated by the cyclin-dependent kinases (CDKs) 1 (for reviews, see Refs. 1-5). Cyclins are responsible for kinase activation, substrate specificity, and intracellular localization (6). The key regulator of the G 2 /M transition is the M phase-promoting factor (MPF), a complex constituted of the catalytic subunit p34 cdc2 (7-9) and the regulatory subunit cyclin B cdc13 (6, 10 -11). Activation of MPF at the onset of mitosis is associated with modifications of phosphorylation of its two subunits (11-13). In late prophase, Cdc2 is phosphorylated on three residues: Thr-14, Tyr-15, and Thr-161. Thr-161 phosphorylation is catalyzed by the Cdc2-activating kinase identified as a complex between Cdk7 (MO15), cyclin H, and MAT1 (14). This phosphorylation is necessary for Cdc2 activity (15, 16). The Thr-14 and Tyr-15 residues of Cdc2 are located in the ATP-binding pocket of the kinase (17). Following cyclin B binding to Cdc2 (6,12,18), Cdc2 becomes phosphorylated on Thr-14 by the Myt1 kinase (19,20) and on Tyr-15 by the Wee1/Mik1 or Myt1 kinases (20 -22). In yeast, only Tyr-15 is phosphorylated in G 2 (23). Cdc2 activation at prophase/metaphase transition requires dephosphorylation of both Thr-14 and Tyr-15. These dephosphorylations occur in two successive steps: first Thr-14 and then . The Cdc25 dual-specificity phosphatase dephosphorylates both residues (reviewed in Ref. 25). The Pyp3 tyrosine phosphatase acts on Tyr-15 in fission yeast (26). A positive feedback loop originating from Cdc2 leads to the autocatalytic amplification of the complex (27,28). This is possibly partially generated by the singly phosphorylated form of Cdc2, dephosphorylated on Thr-14/ phosphorylated on Tyr-15 (24).Simultaneously with Cdc2 dephosphorylation, cyclin B becomes phosphorylated as described in a variety of models such as yeast (6), starfish oocytes (13), sea urchin eggs (11), goldfish oocytes (29), Xenopus oocytes (30), and human cells (31). The residues phosphorylated in cyclin B 1 have been identified in Xenopus: Ser-2, Ser-94, 33). Mutational studies (29,32,33) have suggested that cyclin B phosphorylation is required neither for activity of the Cdc2 kinase, for Cdc2 binding, nor for cyclin B d...