From the cross of shp1-7 with ipl1-321 carrying a

From the cross of shp1-7 with ipl1-321 carrying a centromeric plasmid for the expression of the indicated wild-type and mutant SHP1 alleles was analyzed at the indicated temperatures. The ability of the shp1 mutant gene products to bind Cdc48 is indicated at the right. (d) Hyper-Tubastatin A web phosphorylation of histone H3 in shp1-7. The phosphorylation state of histone H3 in the indicated WT and mutant strains at 35uC was analyzed by Western blot using an antibody recognizing phosphorylated residue Ser10 (pH3) and total 25033180 H3, respectively. The ratio of the signal intensities (pH3/total H3) is given at the bottom. doi:10.1371/journal.pone.0056486.gsuppressed the G2/M accumulation of the mutant cells (Fig. 5b, middle and bottom rows). Upon GLC7 over-expression, the cell cycle distribution of shp1-7 (46 G1/S, 53 G2/M) and shp1-a1 cells (42 G1/S, 57 G2/M) approached that of wild-type cells without GLC7 over-expression (43 G1/S, 54 G2/M). Unbalanced Ipl1 and Glc7 activities give rise to chromosome segregation defects [50,53,59], suggesting that shp1 mutants may be impaired in chromosome segregation as well. Indeed, yeast cells depleted of Shp1 were recently shown to exhibit defective chromosome bi-orientation [31]. Using strains containing a lacO array integrated at the LEU2 locus of chromosome III and expressing GFPLacI and the spindle pole body marker Spc42Mars, we analyzed chromosome segregation in wild-type and shp1 mutants by live-cell fluorescence microscopy (Fig. 5cd). Compared to wild-type, cultures of shp1-7 and shp1-a1 contained significantly more large budded cells with a short spindle and unseparated chromosomes III, and significantly less cells with a long spindle and two separated chromosomes III (Fig. 5c). This finding is fully consistent with the metaphase to anaphase delay described above. Of note, shp1-7 and shp1-a1 also showed a significant increase in cells with chromosome segregation defects (15?0 of largebudded cells in LED-209 biological activity comparison to 3 in the wild-type), as well as some aberrant spindles, confirming that Shp1 is required for faithful chromosome segregation. Importantly, and in line with the FACS data shown in Fig. 5b, over-expression of GLC7 in the shp1 mutants suppressed both the metaphase to anaphase delay and the chromosome segregation defects. Taken together, these results demonstrate for the first time that nuclear Glc7 activity is reduced in shp1 and that the mitotic phenotype of shp1 results from limiting Glc7 activity.Dam1 hyper-phosphorylation causes growth defects of shp1 mutantsThe phosphorylation state of the kinetochore protein Dam1 is critical for proper microtubule attachments during mitosis [55,78?80]. Since Dam1 has been identified as a common substrate of Ipl1 kinase and Glc7 phosphatase activities [54?6,81,82], we next analyzed the phosphorylation state of Dam1 in shp1 mutants. To this end, shp1, glc7 and ipl1 mutants were shifted to 35uC, and phosphorylation of Dam1 was analyzed by Western blot (Fig. 6a). Compared to wild-type cells, Dam1 was indeed hyper-phosphorylated in shp1-7, as judged by the reduction of the faster migrating non-phosphorylated form and the relative increase of the slower migrating phosphorylated form of Dam1. Of note, the increase of Dam1 phosphorylation in shp1 was comparable to that observed in glc7-129 cells. As expected, ipl1-321 cells exhibited strongly reduced Dam1 phosphorylation under these conditions. It has previously been shown that the hypo-phosphorylation of Dam1 in ipl1.From the cross of shp1-7 with ipl1-321 carrying a centromeric plasmid for the expression of the indicated wild-type and mutant SHP1 alleles was analyzed at the indicated temperatures. The ability of the shp1 mutant gene products to bind Cdc48 is indicated at the right. (d) Hyper-phosphorylation of histone H3 in shp1-7. The phosphorylation state of histone H3 in the indicated WT and mutant strains at 35uC was analyzed by Western blot using an antibody recognizing phosphorylated residue Ser10 (pH3) and total 25033180 H3, respectively. The ratio of the signal intensities (pH3/total H3) is given at the bottom. doi:10.1371/journal.pone.0056486.gsuppressed the G2/M accumulation of the mutant cells (Fig. 5b, middle and bottom rows). Upon GLC7 over-expression, the cell cycle distribution of shp1-7 (46 G1/S, 53 G2/M) and shp1-a1 cells (42 G1/S, 57 G2/M) approached that of wild-type cells without GLC7 over-expression (43 G1/S, 54 G2/M). Unbalanced Ipl1 and Glc7 activities give rise to chromosome segregation defects [50,53,59], suggesting that shp1 mutants may be impaired in chromosome segregation as well. Indeed, yeast cells depleted of Shp1 were recently shown to exhibit defective chromosome bi-orientation [31]. Using strains containing a lacO array integrated at the LEU2 locus of chromosome III and expressing GFPLacI and the spindle pole body marker Spc42Mars, we analyzed chromosome segregation in wild-type and shp1 mutants by live-cell fluorescence microscopy (Fig. 5cd). Compared to wild-type, cultures of shp1-7 and shp1-a1 contained significantly more large budded cells with a short spindle and unseparated chromosomes III, and significantly less cells with a long spindle and two separated chromosomes III (Fig. 5c). This finding is fully consistent with the metaphase to anaphase delay described above. Of note, shp1-7 and shp1-a1 also showed a significant increase in cells with chromosome segregation defects (15?0 of largebudded cells in comparison to 3 in the wild-type), as well as some aberrant spindles, confirming that Shp1 is required for faithful chromosome segregation. Importantly, and in line with the FACS data shown in Fig. 5b, over-expression of GLC7 in the shp1 mutants suppressed both the metaphase to anaphase delay and the chromosome segregation defects. Taken together, these results demonstrate for the first time that nuclear Glc7 activity is reduced in shp1 and that the mitotic phenotype of shp1 results from limiting Glc7 activity.Dam1 hyper-phosphorylation causes growth defects of shp1 mutantsThe phosphorylation state of the kinetochore protein Dam1 is critical for proper microtubule attachments during mitosis [55,78?80]. Since Dam1 has been identified as a common substrate of Ipl1 kinase and Glc7 phosphatase activities [54?6,81,82], we next analyzed the phosphorylation state of Dam1 in shp1 mutants. To this end, shp1, glc7 and ipl1 mutants were shifted to 35uC, and phosphorylation of Dam1 was analyzed by Western blot (Fig. 6a). Compared to wild-type cells, Dam1 was indeed hyper-phosphorylated in shp1-7, as judged by the reduction of the faster migrating non-phosphorylated form and the relative increase of the slower migrating phosphorylated form of Dam1. Of note, the increase of Dam1 phosphorylation in shp1 was comparable to that observed in glc7-129 cells. As expected, ipl1-321 cells exhibited strongly reduced Dam1 phosphorylation under these conditions. It has previously been shown that the hypo-phosphorylation of Dam1 in ipl1.

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