Exit from mitosis in budding yeast requires a group of essential proteins--including the GTPase Tem1 and the protein phosphatase Cdc14--that downregulate cyclin-dependent kinase activity. We identified a mutation, net1-1, that bypasses the lethality of tem1 delta. NET1 encodes a novel protein, and mass spectrometric analysis reveals that it is a key component of a multifunctional complex, denoted RENT (for regulator of nucleolar silencing and telophase), that also contains Cdc14 and the silencing regulator Sir2. From G1 through anaphase, RENT localizes to the nucleolus, and Cdc14 activity is inhibited by Net1. In late anaphase, Cdc14 dissociates from RENT, disperses throughout the cell in a Tem1-dependent manner, and ultimately triggers mitotic exit. Nucleolar sequestration may be a general mechanism for the regulation of diverse biological processes.

The essential CDC14 gene of the budding yeast, Saccharomyces cerevisiae, encodes a 62-kDa protein containing a sequence that conforms to the active site motif found in all enzymes of the protein tyrosine phosphatase superfamily. Genetic studies suggest that Cdc14p may be involved in the initiation of DNA replication, but its precise cell cycle function is unknown. Recombinant Cdc14p was produced in bacteria, characterized, and shown to be a dual specificity protein phosphatase. Polyanions such as polyglutamate and double-stranded and single-stranded DNA bind to Cdc14p and affect its activity. Native molecular weights of 131,000 and 169,000 determined by two independent methods indicate that recombinant Cdc14p self-associates in vitro to form active oligomers. The catalytically inactive Cdc14p C283S/ R289A mutant is not able to suppress the temperature sensitivity of a cdc14 -1 ts mutant nor replace the wild type gene in vivo, demonstrating that phosphatase activity is required for the cell cycle function of Cdc14p. A distinctive COOH-terminal segment (residues 375-551) is rich in Asn and Ser residues, carries a net positive charge, and contains two tandem 21-residue repeats. This COOH-terminal segment is not required for activity, for oligomerization, or for the critical cell cycle function of Cdc14p.Genetic analyses of temperature-sensitive CDC mutants as well as biochemical studies using Xenopus oocytes have revealed that reversible protein phosphorylation is a major mechanism for regulating cell cycle progression (1). Transitions in the cell cycle are coordinated by changes in the activity of kinases and phosphatases and in the phosphorylation state of their target proteins. CDC genes as well as a number of other genes that have been linked to cell cycle progression in Saccharomyces cerevisiae encode protein kinases or protein phosphatases.The CDC14 gene (2) may encode a protein phosphatase, since the predicted sequence contains the highly conserved HCX-AGXXR(S/T) motif that is located in the active site of enzymes from the protein tyrosine phosphatase family (3-5). To date, the enzymatic properties of Cdc14p have not been studied, and the existence of intrinsic phosphatase activity has not been directly demonstrated. The CDC14 gene is essential (2), and at restrictive temperatures, cells carrying a thermosensitive mutation in the CDC14 gene arrest in late mitosis with elongated mitotic spindles and a single bud (6). Homozygous cdc14 mutants exhibit defects in meiosis and, depending on when they are switched to restrictive temperatures, arrest in either meiosis I or meiosis II (7).The precise cell cycle function(s) of Cdc14p is unknown. However, several genetic studies suggest that Cdc14p may be involved in initiating DNA replication (8 -11). For instance, the combination of cdc14-1 and orc2-1 mutations is lethal. The ORC2 gene encodes one of the six subunits of the origin of replication complex, which binds in an ATP-dependent manner to yeast origins and is required for initiation of DNA replication (12, 13)...

In the budding yeast Saccharomyces cerevisiae, the multifunctional protein Net1 is implicated in regulating the cell cycle function of the Cdc14 protein phosphatase. Genetic and cell biological data suggest that during interphase and early mitosis Net1 holds Cdc14 within the nucleolus where its activity is suppressed. Upon its transient release from Net1 at late anaphase, active Cdc14 promotes exit from mitosis by dephosphorylating targets in the nucleus and cytoplasm. In this paper we present evidence supporting the proposed role of Net1 in regulating Cdc14 and exit from mitosis. We show that the NH 2 -terminal fragment Net1(1-600) directly binds Cdc14 in vitro and is a highly specific competitive inhibitor of its activity (K i ‫؍‬ 3 nM) with five different substrates including the physiologic targets Swi5 and Sic1. An analysis of truncation mutants indicates that the Cdc14 binding site is located within a segment of Net1 containing residues 1-341. We propose that Net1 inhibits by occluding the active site of Cdc14 because it acts as a competitive inhibitor, binds to a site located within the catalytic domain (residues 1-374), binds with reduced affinity to a Cdc14 C283S mutant in which an active site Cys is replaced, and is displaced by tungstate, a transition state analog known to bind in the catalytic site of protein-tyrosine phosphatases.

We reported previously that endogenous p38 MAPK activity is elevated in invasive breast cancer cells and that constitutive p38 MAPK activity is important for overproduction of uPA in these cells (Huang, S., New, L., Pan, Z., Han, J., and Nemerow, G. R. (2000) J. Biol. Chem. 275, 12266 -12272). However, it is unclear how elevated endogenous p38 MAPK activity is maintained in invasive breast cancer cells. In the present study, we found that blocking ␣ v integrin functionality with a functionblocking monoclonal antibody or down-regulating ␣ v integrin expression with ␣ v -specific antisense oligonucleotides significantly decreased the levels of active p38 MAPK and inhibited cell-associated uPA expression in invasive breast cancer MDA-MB-231 cells. These results suggest a function link between ␣ v integrin, p38 MAPK activity, and uPA expression in invasive tumor cells. We also found that vitronectin/␣ v integrin ligation specifically induced p38 MAPK activation and uPA up-regulation in invasive MDA-MB-231 cells but not in non-invasive MCF7 cells. Finally, using a panel of melanoma cells, we demonstrated that the cytoplasmic tail of ␣ v integrin subunit is required for ␣ v integrin ligation-induced p38 MAPK activation.The degradation of extracellular matrix and basement by tumor-associated proteases is an essential process required for cancer cell invasion and metastasis (1, 2). Urokinase plasminogen activator (uPA) 1 is of particular importance because uPA, through interaction with the uPA receptor (uPAR), facilitates the conversion of plasminogen into plasmin and the activation of metalloproteinases (3, 4). These proteases then allow cancer cells to degrade the surrounding matrix proteins and migrate to the distant sites (5). The overexpression of uPA is detected in various malignancies including breast, prostate, and colon cancers (6 -8). Recent studies (9 -13) have shown further that a high level of uPA in tumors is associated with a rapid disease progression and poor prognosis in breast cancers. In addition, studies performed in experimental models both in vitro and in vivo demonstrate that the levels of uPA are closely associated with the degree of tumor cell invasion (14 -18). Blocking uPA expression or disruption of uPA binding to uPAR has been found to inhibit significantly tumor cell invasion and metastasis in various tumor models (19 -22). From these facts, it is apparent that uPA plays a key role in tumor progression and metastasis.Integrins are heterodimers composed of noncovalently associated ␣ and ␤ subunits. There are at least 14 ␣ and 8 ␤ subunits, forming at least 21 different integrins, which are the major receptors for extracellular matrix proteins (23). The ␣ v integrins are a major subfamily with restricted tissue/cell distribution (23). They have classical integrin functions such as mediating cell attachment and spreading (24, 25), facilitating cell migration (26), and ligand-receptor internalization (27, 28). They also play an important role in tumor progression and metastasis by mediating a...

Pizzo DP, Ientile R, Colombatto S, Toninello A, Satriano J. The arginine metabolite agmatine protects mitochondrial function and confers resistance to cellular apoptosis.