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CELL GROWTH AND DEVELOPMENT

DBF2 Protein Kinase Binds to and Acts through the Cell Cycle-Regulated MOB1 Protein

Svetlana I. Komarnitsky, Yueh-Chin Chiang, Francis C. Luca, Junji Chen, Jeremy H. Toyn, Mark Winey, Leland H. Johnston, Clyde L. Denis
Svetlana I. Komarnitsky
Department of Biochemistry and Molecular Biology, University of New Hampshire, Durham, New Hampshire 03824;
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Yueh-Chin Chiang
Department of Biochemistry and Molecular Biology, University of New Hampshire, Durham, New Hampshire 03824;
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Francis C. Luca
Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado 80309-0347
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Junji Chen
Department of Biochemistry and Molecular Biology, University of New Hampshire, Durham, New Hampshire 03824;
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Jeremy H. Toyn
Division of Yeast Genetics, National Institute for Medical Research, London NW7 1AA, United Kingdom; and
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Mark Winey
Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado 80309-0347
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Leland H. Johnston
Division of Yeast Genetics, National Institute for Medical Research, London NW7 1AA, United Kingdom; and
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Clyde L. Denis
Department of Biochemistry and Molecular Biology, University of New Hampshire, Durham, New Hampshire 03824;
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DOI: 10.1128/MCB.18.4.2100
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  • Fig. 1.
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    Fig. 1.

    Coimmunoprecipitation of LexA-MOB1 with B42-DBF2. Crude extracts (Cr. Ex.) from strain EGY188 containing either B42 and LexA-MOB1 (lanes 1, 4, and 7), B42-DBF2 and LexA (lanes 2, 5, and 8), or B42-DBF2 and LexA-MOB1 (lanes 3, 6, and 9) were incubated with either anti-LexA antibody (lanes 4 to 6) or anti-HA1 antibody (lanes 7 to 9), and the resulting immunoprecipitates (Ip) were subjected to electrophoresis on an SDS–10% polyacrylamide gel. Western analysis was conducted as described previously (8), and the blot was probed with HA1 antibody. Lanes 1 to 3 have crude extracts containing B42 (lane 1) or B42-DBF2 (lanes 2 and 3). The same extracts were immunoprecipitated and analyzed as described above. The blot was probed with LexA antibody. Lanes 1 and 3 contain LexA-MOB1 from crude extracts, and lane 2 has crude extract containing LexA. LexA-MOB1 was capable of being immunoprecipitated with anti-LexA antibody from a strain containing B42 and LexA-MOB1 (data is not shown). Molecular masses are as follows: B42, 10 kDa; B42-DBF2, 72 kDa; LexA, 22 kDa; and LexA-MOB1, 54 kDa.

  • Fig. 2.
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    Fig. 2.

    GST-MOB1 binds B42-DBF2 and B42-CAF1 from crude extracts. The GST and GST-MOB1 proteins expressed in E. coliwere bound to glutathione-agarose beads and then incubated with crude extracts (Cr. Ex.) from the EGY191 strain containing either B42 (lane 1), B42-DBF2 (lane 2), or B42-CAF1 (lane 3). The beads were then boiled with SDS sample buffer, the eluted protein was loaded on an SDS–10% polyacrylamide gel. HA1-containing proteins were detected by Western analysis as previously described (8). Lanes: 1 to 3, crude extracts; 4 to 6, GST incubated with crude extracts from EGY191/B42, EGY191/B42-DBF2, and EGY191/B42-CAF1, respectively; 7 to 9, same as 4 to 6, respectively, except crude extracts were incubated with GST-MOB1. The molecular mass of B42-CAF1 is 54 kDa.

  • Fig. 3.
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    Fig. 3.

    DBF2 binds to MOB1 at physiological concentrations. Extracts from strains S7-4A (wt) and S7-4A-c-myc were incubated with either preimmune MOB1 serum (pI) (lanes 3 and 4), anti-MOB1 antibody (lanes 5 and 6), or anti-c-myc antibody (lanes 7 and 8), and resulting immunoprecipitates (Ip) were subjected to electrophoresis on an SDS–8% polyacrylamide gel. Western analysis was conducted as described previously (8). The upper portion of the blot was probed with c-myc antibody (Ab), and the lower portion was probed with MOB1 antibody. Lanes 1 and 2, crude extracts (CE) containing DBF2-c-myc (lane 2) and/or MOB1 (lanes 1 and 2). DBF2-c-myc is 62 kDa and MOB1 is 34 kDa in size.

  • Fig. 4.
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    Fig. 4.

    Binding of MOB1 to DBF2. (A) Coomassie-stained GST, GST-MOB1, and GST-Vpu. GST fusions were induced as described elsewhere (9), bound to glutathione-agarose beads, eluted from the beads by boiling, and fractionated on an SDS–8% polyacrylamide gel. (B) T7 fusion proteins were translated in vitro with [35S]methionine as described in Materials and Methods. One milliliter of each radioactive protein was separated by SDS-polyacrylamide gel electrophoresis and identified following fluorography. Ten milliliters of each in vitro-translated protein was incubated with 50 mg of a GST fusion and, after washing, eluted by boiling. Molecular masses (in kilodaltons) are indicated on the left.

  • Fig. 5.
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    Fig. 5.

    MOB1 is expressed under cell cycle control. A culture of strain CG378 was synchronized by use of α-factor, and samples were taken for RNA hybridization analysis to determine the levels of the MOB1 (□) and DBF2 (▵) transcripts (12). Actin transcript levels were also determined as a control and used to normalize the MOB1 andDBF2 levels in the graph. The percentages of buds in the synchronized population are also shown as an indication of culture synchrony (○).

  • Fig. 6.
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    Fig. 6.

    Ability of B42-DBF2 and B42-MOB1 to suppress themob1 defect. Strain FLY59 mob1(pRS316-MOB1) was transformed with five different plasmids, expressing B42 alone, B42-DBF2, B42-MOB1(145–314), B42-MOB1(79–314), or B42-MOB1(9–314) as indicated. Transformants were grown overnight in medium lacking uracil, and about 15 × 104 cells of each strain, including the FLY59 strain without any B42 plasmids (−), were plated on medium containing fluoroorotic acid. The picture was taken after 72 h of incubation at 30°C.

  • Fig. 7.
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    Fig. 7.

    Coimmunoprecipitation of LexA-DBF20 with B42-MOB1 and B42-mob1 temperature-sensitive fusions. Extracts from diploid strain EGY188/EGY191 containing LexA-DBF20 (full length) and either B42-MOB1(79–314) (lane 1), B42–mob1-95 (lane 2), B42–mob1-77 (lane 3), or B42–mob1-55 (lane 4) were incubated with LexA antibody, and the resulting immunoprecipitates (Ip) were subjected to electrophoresis on an SDS–10% polyacrylamide gel. Western analysis was conducted as described previously (8), and the blot was probed with HA1 antibody. The positions of molecular mass markers (in kilodaltons) are indicated on the left.

Tables

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  • Table 1.

    Yeast strains used in this study

    StrainGenotype
    EGY188 MAT a ura3 his3 trp1 LexA-LEU2
    EGY188-c1Isogenic to EGY188 except caf1::URA3
    EGY188-1Isogenic to 188 except ccr4::URA3
    EGY191 MATα ura3 his3 trp1 LexA-LEU2
    991-1-1b MAT a ura3 his3 trp1 leu2 dbf2::URA3
    1045-2b MATα ura3 his3 trp1 leu2 dbf20::TRP1
    1300-1a MAT a ura3 his3 trp1 leu2 mob1-77
    CG378 MAT a ura3 leu2 trp1
    FLY30 MAT a ura3 his3 trp1 leu2 mob1-77
    FLY59 MAT a ura3 his3 trp1 leu2 mob1::HIS3; contains plasmid pRS316-MOB1 (URA3)
    S7-4A MAT a dbf2::URA3 ura3 leu2 ade5 trp1 his7
    S7-4A-c-mycIsogenic to S7-4A excepttrp1::c-myc-DBF2-TRP1
  • Table 2.

    Two-hybrid interaction of MOB1 with DBF2 and CAF1a

    LexA fusionB42 fusionβ-gal (U/mg)b
    LexA-DBF2(1–561)B42-MOB1(9–314)260
    LexA-DBF2(1–561)B42-MOB1(79–314)530
    LexA-DBF2(1–561)B42-MOB1(145–314)66
    LexA-DBF2(1–561)B42<1
    LexAB42-MOB1(9–314)<1
    LexA-DBF2(1–220)B42-MOB1(9–314)59
    LexA-DBF2(1–220)B42-MOB1(145–314)120
    LexA-DBF2(1–220)B42<1
    LexA-MOB1(9–314)B42-DBF2(1–561)6,700
    LexA-MOB1(9–314)B42-DBF2(205–561)350
    LexA-MOB1(9–314)B42-CAF1300
    LexA-MOB1(9–314)B4281
    • ↵a Strains were grown on minimal medium lacking uracil, histidine, and tryptophan and supplemented with 2% raffinose and 2% galactose as previously described (8). All assays were done in EGY188/EGY191 diploids containing the p34 reporter (eight LexA operators upstream of the GAL1-lacZ promoter). B42-DBF2(1–561) and B42-DBF2(205–561) were expressed to comparable levels as determined by Western analysis (data not shown).

    • ↵b β-Galactosidase (β-Gal) activities represent the average of determinations for at least three separate transformants. The standard error of the mean was less than 20% in each case.

  • Table 3.

    Suppression analysis of MOB1 and DBF2

    GenotypeSuppression with plasmida:
    B42-MOB1B42-DBF2B42
    37°CCaffeine37°CCaffeine37°CCaffeine
    mob1-77b ++++−−
    dbf2c ++++−−
    • ↵a Growth was monitored on yeast extract-peptone agar plates supplemented with 2% each galactose and raffinose and incubated at 37°C and on the same plates additionally supplemented with 8 mM caffeine but incubated at 30°C. B42-MOB1 refers to MOB1(9–314), although B42-MOB1(79–314) complemented as well. Similar results were obtained for a mob1-95 allele or for a dbf2 temperature-sensitive allele. +, suppression evident; −, no suppression evident.

    • ↵b Strain 1300-1a.

    • ↵c Strain 991-1-1b.

  • Table 4.

    Two-hybrid interactions of wild-type and mutated forms of MOB1 with DBF2, MPS1, and DBF20

    LexA fusionaB42 fusionbβ-gal activity (U/mg)c
    LexA-DBF2B42-MOB1530
    LexA-DBF2B42–MOB1-55 (T85P, Q167R, Y183H)70
    LexA-DBF2B42–MOB1-77 (E151K)d 40
    LexA-DBF2B42–MOB1-95 (L157P, A158I)20
    LexA-MPS1B42-MOB1100
    LexA-MPS1B42–MOB1-55 (T85P, Q167R, Y183H)40
    LexA-MPS1B42–MOB1-77 (E151K)d 120
    LexA-MPS1B42–MOB1-99 (L157P, A158I)120
    LexA-DBF20B42-MOB17.7
    LexA-DBF20B42–MOB1-55 (T85P, Q167R, Y183H)<1
    LexA-DBF20B42–MOB1-77 (E151K)d <1
    LexA-DBF20B42–MOB1-95 (L157P, A158I)<1
    • ↵a LexA-DBF2 contains full-length DBF2(1–561), LexA-MPS1 contains full-length MPS1(1–764), LexA-DBF20 contains full-length DBF20(1–564), and B42-MOB1 fusions contain residues 79 to 314 of MOB1.

    • ↵b Specific mutations associated with the MOB moieties of the constructs are given in parentheses; e.g., T85P indicates a substitution of a proline for the threonine at position 85.

    • ↵c β-Galactosidase (β-Gal) activities represent the average of three to six separate determinations. The standard error of the mean was less than 15% in each case. Assays were conducted as described in Table 2. All B42-MOB1 fusions were found to be expressed to comparable levels, as determined by Western analysis, and the LexA fusions were comparably expressed as well (data not shown). Assays were conducted in EGY188/EGY191 diploids containing the p34 reporter.

    • ↵d The original mob1-77temperature-sensitive mutant contained an additional mutation, N65I, but the E151K alteration is sufficient to make MOB1 unable to fully complement a mob1 defect (data not shown).

  • Table 5.

    Transactivation effects of LexA-MOB1 variants

    LexA fusionaβ-Gal (U/mg)b
    LexA-MOB1230
    LexA–MOB1-55 (T85P, Q167R, Y183H)100
    LexA–MOB1-77 (E151K)440
    LexA–MOB1-95 (L157P, A158I)36
    • ↵a Assays were conducted as described in Table 2. All LexA-MOB1 fusions were expressed to comparable levels as determined by Western analysis (data not shown). LexA-MOB1 fusions contain residues 79 to 314 of MOB1 and were expressed in EGY188 containing the p34 reporter.

    • ↵b β-Galactosidase (β-Gal) activities represent the average of the determinations for at least three transformants. The standard error of the mean was less than 10% in each case except for LexA–MOB1-95, in which it was 21%.

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DBF2 Protein Kinase Binds to and Acts through the Cell Cycle-Regulated MOB1 Protein
Svetlana I. Komarnitsky, Yueh-Chin Chiang, Francis C. Luca, Junji Chen, Jeremy H. Toyn, Mark Winey, Leland H. Johnston, Clyde L. Denis
Molecular and Cellular Biology Apr 1998, 18 (4) 2100-2107; DOI: 10.1128/MCB.18.4.2100

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DBF2 Protein Kinase Binds to and Acts through the Cell Cycle-Regulated MOB1 Protein
Svetlana I. Komarnitsky, Yueh-Chin Chiang, Francis C. Luca, Junji Chen, Jeremy H. Toyn, Mark Winey, Leland H. Johnston, Clyde L. Denis
Molecular and Cellular Biology Apr 1998, 18 (4) 2100-2107; DOI: 10.1128/MCB.18.4.2100
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KEYWORDS

cell cycle
Cell Cycle Proteins
Fungal Proteins
Phosphoproteins
protein kinases
Saccharomyces cerevisiae Proteins

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