Two LXXLL motifs in the N terminus of Mps1 are required for Mps1 nuclear import during G2/M transition and sustained spindle checkpoint responses

Spindle assembly checkpoint kinase Mps1 is spatially and temporally regulated during cell cycle progression. Mps1 is predominately localized to the cytosol in interphase cells, whereas it is concentrated on kinetochores in prophase and prometaphase cells. The timing and mechanism of Mps1 redistribution during cell cycle transition is currently poorly understood. Here, we show that Mps1 relocates from the cytosol to the nucleus at the G₂/M boundary prior to nuclear envelope breakdown (NEB). This timely translocation depends on two tandem LXXLL motifs in the N terminus of Mps1, and mutations in either motif abolish Mps1 nuclear accumulation. Furthermore, we found that phosphorylation of Mps1 Ser80 (which is located between the two LXXLL motifs) also plays a role in regulating timely nuclear entry of Mps1. Mps1 that is defective in LXXLL motifs has near wild-type kinase activity. Moreover, the kinase activity of Mps1 appears to be dispensable for nuclear translocation, as inhibition of Mps1 by a highly specific small-molecule inhibitor did not perturb its nuclear entry. Remarkably, translocation-deficient Mps1 can mediate activation of spindle assembly checkpoint response; however, it fails to support a sustained mitotic arrest upon prolonged treatment with nocodazole. The mitotic slippage can be attributed to precocious degradation of Mps1 in the arrested cells. Our studies reveal a novel cell cycle-dependent nuclear translocation signal in the N terminus of Mps1 and suggest that timely nuclear entry could be important for sustaining spindle assembly checkpoint responses.     

Two domains of the progesterone receptor interact with the estrogen receptor and are required for progesterone activation of the c-Src/Erk pathway in mammalian cells

In breast cancer cells, estrogens activate the Src/Erk pathway through an interaction of the estrogen receptor alpha (ERalpha) with the SH2 domain of c-Src. Progestins have been reported to activate also this pathway either via an interaction of the progesterone receptor isoform B (PRB) with ERalpha, which itself activates c-Src, or by direct interaction of PRB with the SH3 domain of c-Src. Here we identify two domains of PRB, ERID-I and -II, mediating a direct interaction with the ligand-binding domain of ERalpha. ERID-I and ERID-II flank a proline cluster responsible for binding of PRB to c-Src. In mammalian cells, the interaction of PRB with ERalpha and the progestin activation of the Src/Erk cascade are abolished by deletion of either ERID-I or ERID-II. These regions are not required for transactivation of a progesterone-responsive reporter gene. Mutations in the proline cluster of PRB that prevent a direct interaction with c-Src do not affect the strong activation of c-Src by progestins in the presence of ERalpha. Thus, in cells with ERalpha, ERID-I and ERID-II are necessary and sufficient for progestin activation of the endogenous Src/Erk pathway.     

Both TATA box and upstream regions are required for the nopaline synthase promoter activity in transformed tobacco cells

Summary Using a promoter expression vector system based on the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens, we have studied the molecular structure of the nopaline synthase (nos) promoter which is active constitutively in transformed plant tissues. The system uses the sensitive and reliable chloramphenicol acetyltransferase (CAT) assay for the analysis of promoter strength in plant cells. Two sets of mutants were generated by sequential deletion of the nos promoter region from both 5 and 3 ends. These promoter fragments were linked to the cat coding sequence within the expression vector. The strength of the mutant promoters was measured in transformed tobacco calli as CAT activity. 3 deletions up to-17 bp did not significantly affect the promoter strength. Further deletions into the TATA box region reduced the promoter strength by about ten-fold. Analysis of the 5 deletion mutants showed that an upstream region is required for the nos promoter activity in addition to the TATA box and CCAAT box regions.     

Conserved motifs II to VI of DNA helicase II from Escherichia coli are all required for biological activity.

There are seven conserved motifs (IA, IB, and II to VI) in DNA helicase II of Escherichia coli that have high homology among a large family of proteins involved in DNA metabolism. To address the functional importance of motifs II to VI, we employed site-directed mutagenesis to replace the charged amino acid residues in each motif with alanines. Cells carrying these mutant alleles exhibited higher UV and methyl methanesulfonate sensitivity, increased rates of spontaneous mutagenesis, and elevated levels of homologous recombination, indicating defects in both the excision repair and mismatch repair pathways. In addition, we also changed the highly conserved tyrosine(600) in motif VI to phenylalanine (uvrD309, Y600F). This mutant displayed a moderate increase in UV sensitivity but a decrease in spontaneous mutation rate, suggesting that DNA helicase II may have different functions in the two DNA repair pathways. Furthermore, a mutation in domain IV (uvrD307, R284A) significantly reduced the viability of some E. coli K-12 strains at 30 degrees C but not at 37 degrees C. The implications of these observations are discussed.     

Cyclin-binding motifs are essential for the function of p21CIP1.

The cyclin-dependent kinase (Cdk) inhibitor p21 is induced by the tumor suppressor p53 and is required for the G1-S block in cells with DNA damage. We report that there are two copies of a cyclin-binding motif in p21, Cy1 and Cy2, which interact with the cyclins independently of Cdk2. The cyclin-binding motifs of p21 are required for optimum inhibition of cyclin-Cdk kinases in vitro and for growth suppression in vivo. Peptides containing only the Cy1 or Cy2 motif partially inhibit cyclin-Cdk kinase activity in vitro and DNA replication in Xenopus egg extracts. A monoclonal antibody which recognizes the Cy1 site of p21 specifically disrupts the association of p21 with cyclin E-Cdk2 and with cyclin D1-Cdk4 in cell extracts. Taken together, these observations suggest that the cyclin-binding motif of p21 is important for kinase inhibition and for formation of p21-cyclin-Cdk complexes in the cell. Finally, we show that the cyclin-Cdk complex is partially active if associated with only the cyclin-binding motif of p21, providing an explanation for how p21 is found associated with active cyclin-Cdk complexes in vivo. The Cy sequences may be general motifs used by Cdk inhibitors or substrates to interact with the cyclin in a cyclin-Cdk complex.     

Phosphatidylserine synthesis required for the maximal tryptophan transport activity in Saccharomyces cerevisiae

Saccharomyces cerevisiae cho1/pss mutants, which are severely impaired in phosphatidylserine (PS) synthesis, do not have detectable amounts of PS in their lipid fractions. Their derivatives with mutations that cause defects in tryptophan synthesis grew poorly in a medium containing 5 micrograms/ml of L-tryptophan, a concentration that met the requirements of tryptophanauxotrophic CHO1/PSS strains. The rates of tryptophan uptake of trp1 cho1/pss mutants were low at low tryptophan concentrations. This defect in the use of tryptophan was restored either by expression of CHO1/PSS or by introduction of a gene encoding tryptophan transporter, TAT1 or TAT2. These results indicate that PS synthesis is required for the maximal tryptophan-transporting activity of S. cerevisiae at low tryptophan concentrations.     

Histone H2A phosphorylation and H3 methylation are required for a novel Rad9 DSB repair function following checkpoint activation

In budding yeast, the Rad9 protein is an important player in the maintenance of genomic integrity and has a well-characterised role in DNA damage checkpoint activation. Recently, roles for different post-translational histone modifications in the DNA damage response, including H2A serine 129 phosphorylation and H3 lysine 79 methylation, have also been demonstrated. Here, we show that Rad9 recruitment to foci and bulk chromatin occurs specifically after ionising radiation treatment in G2 cells. This stable recruitment correlates with late stages of double strand break (DSB) repair and, surprisingly, it is the hypophosphorylated form of Rad9 that is retained on chromatin rather than the hyperphosphorylated, checkpoint-associated, form. Stable Rad9 accumulation in foci requires the Mec1 kinase and two independently regulated histone modifications, H2A phosphorylation and Dot1-dependent H3 methylation. In addition, Rad9 is selectively recruited to a subset of Rad52 repair foci. These results, together with the observation that rad9Delta cells are defective in repair of IR breaks in G2, strongly indicate a novel post checkpoint activation role for Rad9 in promoting efficient repair of DNA DSBs by homologous recombination.     

Macrophages are required for the dextran-sulfate induced activation of B lymphocytes.

The proliferative response induced by the B cell activating ligand dextran-sulfate (DxS) requires the presence of macrophage-produced factors or the macrophage-substituting compound 2-mercaptoethanol. Thus, the mechanism for activation of mouse B splenic lymphocytes is different for DxS than for other polyclonal B cell activators such as lipopolysaccharides, which can trigger B cells directely in the absence of the helper factors. It is suggested that the disappearance of the DxS-induced response could be studied in a simple functional tests for the efficiency of macrophage depletion obtained by different techniques.     

Catalytic activity and chaperone function of human protein-disulfide isomerase are required for the efficient refolding of proinsulin.

Protein-disulfide isomerase (PDI) catalyzes the formation, rearrangement, and breakage of disulfide bonds and is capable of binding peptides and unfolded proteins in a chaperone-like manner. In this study we examined which of these functions are required to facilitate efficient refolding of denatured and reduced proinsulin. In our model system, PDI and also a PDI mutant having only one active site increased the rate of oxidative folding when present in catalytic amounts. PDI variants that are completely devoid of isomerase activity are not able to accelerate proinsulin folding, but can increase the yield of refolding, indicating that they act as a chaperone. Maximum refolding yields, however, are only achieved with wild-type PDI. Using genistein, an inhibitor for the peptide-binding site, the ability of PDI to prevent aggregation of folding proinsulin was significantly suppressed. The present results suggest that PDI is acting both as an isomerase and as a chaperone during folding and disulfide bond formation of proinsulin.     

Two nonadjacent regions in enteroaggregative Escherichia coli flagellin are required for activation of toll-like receptor 5

Flagellin is the major structural protein of the flagella of Gram-negative bacteria. Recent work has demonstrated that flagellin is a potent trigger of innate immune responses in a number of eukaryotic cells and organisms, including both mammals and plants. In several different human epithelial cell lines, this innate immune response involves toll-like receptor 5 (TLR5). The mechanisms by which flagellin activates TLR5 and the importance of this interaction in other model systems of flagellin-induced inflammation remain unknown. In this work, random and site-directed mutagenesis of the inflammatory flagellin from enteroaggregative Escherichia coli identified two regions in the conserved D1 domain that are required for interleukin-8 release and TLR5 activation. In contrast, large regions of the variable domain could be excised without reducing the inflammatory activity. In addition, regions of the protein analogous to epitopes that trigger innate immune responses in plants are not involved in Caco-2 flagellin responses. These results highlight the complexity of the interaction between bacterial flagellin and its eukaryotic recognition partners and provide the basis for further studies to characterize the innate immune response to flagellin.     

Analysis of the DXD motifs in human xylosyltransferase I required for enzyme activity

Human xylosyltransferase I (XT-I) is the initial enzyme involved in the biosynthesis of the glycosaminoglycan linker region in proteoglycans. Here, we tested the importance of the DXD motifs at positions 314-316 and 745-747 for enzyme activity and the nucleotide binding capacity of human XT-I. Mutations of the 314DED316 motif did not have any effect on enzyme activity, whereas alterations of the 745DWD747 motif resulted in reduced XT-I activity. Loss of function was observed after exchange of the highly conserved aspartic acid at position 745 with glycine. However, mutation of Asp745 to glutamic acid retained full enzyme activity, indicating the importance of an acidic amino acid at this position. Reduced substrate affinity was observed for mutants D747G (Km=6.9 microm) and D747E (Km=4.4 microm) in comparison with the wild-type enzyme (Km=0.9 microm). Changing the central tryptophan to a neutral, basic, or acidic amino acid resulted in a 6-fold lower Vmax, with Km values comparable with those of the wild-type enzyme. Despite the major effect of the DWD motif on XT-I activity, nucleotide binding was not abolished in the D745G and D747G mutants, as revealed by UDP-bead binding assays. Ki values for inhibition by UDP were determined to be 1.9-24.6 microm for the XT-I mutants. The properties of binding of XT-I to heparin-beads, the Ki constants for noncompetitive inhibition by heparin, and the activation by protamine were not altered by the generated mutations.