Cytoskeletal Activation of a Checkpoint Kinase Jessie Hanrahan, Michael Snyder  Molecular Cell  Volume 12, Issue 3, Pages 663-673 (September 2003) DOI: 10.1016/j.molcel.2003.08.006

Figure 1 The N-terminal Kinase Domain of Hsl1 Interacts with the Noncatalytic Domain of Hsl1 (A) Positive two-hybrid interactions between the Hsl1 kinase domain and AD::Hsl1 constructs. Samples were spotted on SC medium lacking Ura, Leu, and His and assayed for growth. (B) Summary of the fragments of Hsl1 tested for two-hybrid interaction with the kinase domain of Hsl1 (aa 1–372). Black boxes indicate a positive interaction; plus and minus signs indicate strength of interaction. (C) A C-terminal fragment of Hsl1 (aa 987–1100) coimmunoprecipitates with the kinase domain of Hsl1 (aa 1–554). Lysates containing Hsl1 987-1100-GST and either untagged Hsl1, Hsl1::HA, Hsl1Kinase::HA, or Hsl1nonkinase::HA were immunoprecipitated with anti-HA antibodies and probed with both anti-HA and anti-GST antibodies. The total cellular lysate was probed with anti-GST antibodies. Asterisks indicate endogenous protein that reacts with the antibody. Molecular Cell 2003 12, 663-673DOI: (10.1016/j.molcel.2003.08.006)

Figure 2 Hsl1 987-1100 Is Inhibitory to Hsl1 Function In Vivo (A) Morphology of cells with GST overexpressed fragments of Hsl1 in a wild-type background as well as the hsl1Δ cells. (B) Summary of the regions of Hsl1 fused to GST and overexpressed in a wild-type background. “R” indicates a round cellular morphology and “L” indicates an elongated morphology. The percent of elongated cells is indicated (n > 200). ND, not quantified. (C) Morphology of cdc12-1 and cdc12-1 hsl1Δ987-1100 at room temperature and 37°C. Molecular Cell 2003 12, 663-673DOI: (10.1016/j.molcel.2003.08.006)

Figure 3 The Noncatalytic Domain of Hsl1 Is Inhibitory to Hsl1 Kinase Activity (A) Immunoblot analysis of Hsl1 immunoprecipitates. Hsl1kinase::HA and untagged Hsl1 were immunoprecipitated with anti-HA antibodies from strains containing GST or Hsl1-987-1518-GST. The blot was probed with anti-HA antibodies and the lysate was probed with anti-GST antibodies. (B) Autophosphorylation and MBP phosphorylation assays of strains from (A). The immunoprecipitated Hsl1::HA strains containing either GST or Hsl1 987-1518-GST were incubated at room temperature for 30 min with 32P-ATP and 0.1 mg/ml of MBP. Reactions were stopped by the addition of sample loading buffer, and the reactions were run out on a 15% polyacrylamide gel. The phosphorylated products were analyzed using a 15% polyacrylamide gel; the kinase domain and full-length protein exhibit similar mobility in this gel. Bar graphs represent the intensity of the bands; the IDVs (integrated density value) for autophosphorylation and MBP assays were determined. (C) Immunoblot of purified GST, Hsl1-1000-1282-GST, or Hsl1-987-1518-0GST probed with anti-GST antibodies as well as an immunoblot of Hsl1kinase::HA immunoprecipitated with anti-HA antibodies and probed with anti-HA antibodies. The GST Standard is commercially available GST used as a control (Sigma). (D) MBP phosphorylation assay. Purified GST, Hsl1-1000-1282-GST, or Hsl1-987-1518-GST were added to the immunoprecipitated Hsl1kinase::HA and then Hsl1kinase::HA was assayed for the ability to phosphorylate MBP in the presence of 32P-ATP. Bar graph represents IDVs for the MBP assay. Molecular Cell 2003 12, 663-673DOI: (10.1016/j.molcel.2003.08.006)

Figure 4 Cdc11 and Cdc12 Bind the C Terminus of Hsl1 (A) Two-hybrid interactions between Cdc11 and Cdc12 and AD::Hsl1 constructs. Samples were spotted on SC medium lacking Ura, Leu, and His and assayed for growth. (B) Summary of the fragments of Hsl1 tested for two-hybrid interaction with Cdc3, Cdc11, and Cdc12. Black boxes indicate a positive Cdc12 interaction, dashed boxes indicate a positive Cdc11 interaction, and plus and minus signs indicate strength of interaction. Molecular Cell 2003 12, 663-673DOI: (10.1016/j.molcel.2003.08.006)

Figure 5 Cdc11 and Cdc12 Relieve the Autoinhibition Imposed by the Inhibitory Domain In Vitro (A) Immunoblot of purified GST, Cdc11-GST, and Cdc12-GST probed with anti-GST antibodies, and an immunoblot of Hsl1kinase::HA immunoprecipitated with anti-HA antibodies and probed with anti-HA antibodies. (B) MBP phosphorylation assay. Purified GST, Cdc11-GST, or Cdc12-GST were added to a reaction containing the immunoprecipitated Hsl1kinase::HA as well as Hsl1-1000-1282-GST and MBP. Hsl1kinase::HA was assayed for the ability to phosphorylate MBP in the presence of 32P-ATP. A bar graph represents the IDVs for the MBP phosphorylation assay. Molecular Cell 2003 12, 663-673DOI: (10.1016/j.molcel.2003.08.006)

Figure 6 Cdc11 and Cdc12 Binding Domains Are Required for Hsl1 Function In Vivo (A) Morphology of the HA-tagged septin binding domain deletion Hsl1 strains: Hsl1::HA, hsl1Δ, hsl1Δ987-1100::HA, hsl1Δ1100-1282::HA, and hsl1Δ1282-1400::HA. hsl1Δ1100-1282::HA, and hsl1Δ1282-1400::HA have an elongated cellular morphology similar to that of hsl1Δ cells. (B) Localization of the following HA-tagged Hsl1 strains: Hsl1::HA, Hsl1Δ987-1100::HA, Hsl1Δ1100-1282::HA, and Hsl1Δ1282-1400::HA. Hsl1::HA, Hsl1Δ987-1100::HA, and Hsl1Δ1100-1282::HA are able to localize properly as a ring on the bud side of the mother-bud neck, but Hsl1Δ1100-1282::HA is not able to localize; instead, Hsl1Δ1282-1400::HA localizes as punctate dots throughout the mother and bud. Molecular Cell 2003 12, 663-673DOI: (10.1016/j.molcel.2003.08.006)

Figure 7 Model for Hsl1 Activation (A) Domains of Hsl1 defined by two-hybrid analyses. Kinase, kinase domain; ID, inhibitory domain; Cdc11 BD, Cdc11 binding domain; Cdc12 BDI, Cdc12 binding domain I; Cdc12 BDII, Cdc12 binding domain II. (B) Hsl1 is in an inactive conformation when the C-terminal ID (aa 987–1100) is bound to the N-terminal kinase domain. Upon binding of Cdc11 and Cdc12 to their respective binding domains, the autoinhibition imposed by the ID is relieved and the kinase domain of Hsl1 becomes activated and is then able to phosphorylate its substrates. Molecular Cell 2003 12, 663-673DOI: (10.1016/j.molcel.2003.08.006)