Sgt1 dimerization is negatively regulated by protein kinase CK2-mediated phosphorylation at Ser361

Abstract

The kinetochore, which consists of centromere DNA and structural proteins, is essential for proper chromosome segregation in eukaryotes. In budding yeast, Sgt1 and Hsp90 are required for the binding of Skp1 to Ctf13 (a component of the core kinetochore complex CBF3) and therefore for the assembly of CBF3. We have previously shown that Sgt1 dimerization is important for this kinetochore assembly mechanism. In this study, we report that protein kinase CK2 phosphorylates Ser(361) on Sgt1, and this phosphorylation inhibits Sgt1 dimerization.

FIGURE 1.

Sgt1 is phosphorylated at serine 361. A (top), Sgt1 is phosphorylated at Ser³⁶¹ in vivo. The 3Myc-Sgt1 (Y1870) or 3Myc-sgt1-S361A (Y1871) was immunoprecipitated from cycling cells, and immunoprecipitates were treated with alkaline phosphatase (AP-treated). Immunoprecipitates were analyzed by two-dimensional gel electrophoresis. An anti-Myc antibody was used to detect Sgt1-Myc on the immunoblots. (bottom) The signal intensity was quantified, and the ratio of phosphorylated Sgt1 (toward low pH) to unphosphorylated Sgt1 (toward high pH) in untreated immunoprecipitates is shown (gray bars). B, recombinant human CK2 phosphorylates Sgt1 in vitro. Kinase activity was determined by assaying phosphorylation levels of GST-Sgt1 (lanes 1–3), GST only (lanes 4–6), or His-Sgt1 (lanes 8–10), incubated with [γ-³²P]ATP, as described under “Experimental Procedures.” The arrowhead indicates autophosphorylated CK2. C, CK2-mediated phosphorylation of Sgt1. His-Sgt1 (1.6 pmol) was mixed in the reaction buffer with CK2 (710 pmol) and incubated with 0.5 μCi of [γ-³²P]ATP for the indicated time periods at 30 °C. The protein was then analyzed by SDS-PAGE. Phosphorylated Sgt1 bands were quantified with a STORM 860 PhosphorImager and ImageQuant software (Amersham Biosciences), and data were converted into pmol units of phosphate relative to that of the control [γ-³²P]ATP. The level of Sgt1 phosphorylation was calculated by dividing the pmol units of phosphate by the pmol units of Sgt1 used in the reaction. Bottom, the phosphor image of phosphorylated Sgt1. D, kinetics of Sgt1 phosphorylation by CK2. Reactions were performed in 15 μl of solution containing 20 mm HEPES (pH 7.5), 50 mm NaCl, 10 mm MgCl₂, 0.2 mm ATP, 0.5 μCi of [γ-³²P]ATP, 1 mm dithiothreitol, 1 mm CaCl₂, 35 units of CK2, and varying concentrations of recombinant His-Sgt1 at 30 °C. Reactions were stopped after 10 min with SDS buffer, and proteins were resolved by SDS-PAGE. Phosphorylated Sgt1 was quantified using a STORM 860 PhosphorImager and ImageQuant software (Amersham Biosciences). Bottom, the kinetic properties V_max and K_m of Sgt1 phosphorylation were determined from Michaelis-Menten plots using GraFit (Erithacus Software Ltd.).

FIGURE 2.

A, CK2 phosphorylates Sgt1 at S361 in vitro. Top, 100 ng of recombinant GST-Sgt1 and GST-sgt1-S361A mutant proteins were used for the in vitro kinase assay (left, a phosphor image; right, a SYPRO Ruby-stained image). Bottom, phosphorylation signals were quantified by a PhosphorImager. Input Sgt1 (top bands on the gel) in the reaction was quantified from the SYPRO Ruby-stained gel image, and the phosphorylation signals were normalized. GST-Sgt1 was given the arbitrary value of 1. Numbers on the y axis are arbitrary. Signals were quantified from three independent experiments. C, a negative control without substrates. B, Sgt1 Ser³⁶¹ is conserved from yeast to humans. A short peptide sequence around Ser³⁶¹ in yeast Sgt1 is aligned with the amino acid sequence of the corresponding region in Sgt1 homologs of several other organisms, as indicated: barley (HvSgt1), rice (OsSgt1), Arabidopsis (AtSgt1), S. cerevisiae (ScSgt1), mouse (mSgt1), and human (HsSgt1). C, human SGT1A is phosphorylated by CK2 in vitro. Recombinant GST-hSGT1A, GST-hSGT1A-S299A (hSGT1A-S299 corresponds to yeast Sgt1-S361), and GST alone were used as substrate for the CK2-dependent in vitro kinase assay. WT, wild type.

FIGURE 3.

Phenotypes of the sgt1-S361D mutant. A, mimic of Sgt1 phosphorylation at Ser³⁶¹ inhibits Sgt1-Sgt1 and Sgt1-Skp1 binding. Sgt1-S361S (wild type), Sgt1-S361A, or sgt1-S361D was expressed as HA-tagged and Myc-tagged proteins from two different plasmids (pRS416-HA and pRS414-Myc). Sgt1-S361S was expressed in the Y1681 strain, Sgt1-S361A in the Y1682 strain, and sgt1-S361D in the Y1684 strain. (All three strains were derived from the YPH499 strain.) TheYPH499 strain carrying only pRS416-HA-SGT1-S361S was used as a negative control (leftmost lane). B, mimic of Sgt1 phosphorylation at Ser³⁶¹ is lethal. Yeast cells that were haploid for SGT1 (Y26), sgt1-S361A (Y1686), or sgt1-S361D carrying SGT1 on a URA/CEN plasmid (Y1687) were streaked onto plates that contained Sc+ 5-fluoroorotic acid (5-FOA) or Sc-Leu. Plates were incubated at 25 °C for 3 days. C (right), the addition of 0.5 mm CuSO₄ to strain CDY26 (N-degron-Sgt1) induced the proteolysis of N-degron-4HA-Sgt1 after either a 4- or 8-h incubation with CuSO₄, as determined by immunoblotting with anti-HA antibodies, but Myc-tagged Sgt1 or sgt1-S361D was not affected. Left, protein extracts of the indicated strains were collected at the indicated time points after adding CuSO₄. Cell extracts (40 μg) were subjected to the band shift assay to examine the CBF3 assembly activity. Competitor CEN DNA (100 or 200 μg) or mutant CEN DNA (CCG motif at CDEIII region is mutated to CCC) was added to CuSO₄-untreated extracts of each cell type. D, survival of cells after a 4- or 8-h incubation with 0.5 mm CuSO₄, as determined by a dilution-spotting assay. The numbers of cells that were spotted onto the indicated plates were ∼1.25 × 10⁶, 2.5 × 10⁵, 5 × 10⁴, 1 × 10⁴, and 2 × 10³. Plates were incubated at 30 °C for 2 days. IP, immunoprecipitation.

FIGURE 4.

CK2 phosphorylates Sgt1 in vivo. A, Cka1, a subunit of CK2, associates with Sgt1. Cell lysates obtained from cycling untagged wild-type (untag; strain YPH499) and Cka1-Myc (strain Y1734) cells were immunoprecipitated by using anti-Myc antibodies. Sgt1 was detected with anti-Sgt1 antibodies, and Cka1-Myc was detected with anti-Myc antibodies. An untagged strain was used as a negative control. B, phosphorylated Sgt1 diminished in cka1Δ cells. 3Myc-Sgt1 was immunoprecipitated from wild-type (WT; Y1872) cells or cka1Δ cells (cka1Δ; Y1873) harboring 3Myc-Sgt1. Immunoprecipitates were analyzed by two-dimensional gel electrophoresis, and immunoblotting was performed by using anti-Myc antibodies. C (top left panels), Sgt1 dimerization increases in cka1Δ cells. Immunoprecipitation was carried out on cell lysates from wild-type (WT; Y1681) cells or cka1Δ cells (cka1Δ; Y1736) harboring 3HA-Sgt1 and 3Myc-Sgt1. Bottom left panels, Sgt1 dimerization increases in cka1Δ cells, which is dependent on serine 361. Data are from experiments similar to those above, except that cell lysates were prepared from cells expressing HA-sgt1-S361A and Myc-sgt1-S361A in wild-type (Y1682) and cka1Δ cells (cka1Δ; Y1874). HA-tagged Sgt1 (wild type or mutant) was identified by anti-HA antibodies, and Myc-tagged Sgt1 (wild type or mutant) was identified by anti-Myc antibodies. Right panels, signals of HA-Sgt1 and Skp1 in Myc-Sgt1 immunoprecipitates were quantified from two independent experiments. Quantification was performed by giving an arbitrary value of 1 to HA-Sgt1 or Skp1 in the wild-type cells. D, the benomyl sensitivity of the sgt1–3 mutant was suppressed by the deletion of cka1. The indicated strains (wild type (YPH499), sgt1–3 (YKK54), cka1Δ (Y1773), and sgt1–3 cka1Δ (Y1775)) were grown on yeast extract-peptone-dextrose plates containing 10 μg/ml benomyl or DMSO. The numbers of cells that were spotted onto each plate (left to right) were ∼1.25 × 10⁶, 2.5 × 10⁵, 5 × 10⁴, 1 × 10⁴, 2 × 10³, and 4 × 10². Plates were incubated at 30 °C for 3 days. IP, immunoprecipitation.