Mps1p regulates assembly of Spc42p | Castillo et al. 459 Figure 8. Spc42p is a substrate in vitro for Mps1p, and Spc42p phosphorylation is dependent on Mps1p in vivo. A kinase assay was performed in vitro using Mps1p-myc immunoprecipitated (as described in Materials and methods) from an asynchronously growing strain (SBY650). Recombinant Spc42p purified from baculovirus-infected insect cells (a gift from Danni Vinh and Trisha Davis, University of Washington, Seattle, WA) was used as a substrate in this assay. Kinase reactions were resolved using SDS-PAGE, and the gel was transferred to nitrocellulose. A fluorescence-based imaging system allowed us to use the same nitrocellulose blot to assess 32P incorporation and for Western analysis (as described in Materials and methods) to detect Mps1p-myc and Spc42p. (A) Signal corresponding to autophosphorylation of Mps1p-myc and phosphorylation of Spc42p was observed in the reaction where both Mps1p-myc and Spc42p were present (arrows). No signal was observed when Mps1p-myc was not included in the reaction. (B) Western blotting with an anti-myc antibody shows that Mps1p-myc is present in lane 1, and anti-Spc42p antibody shows that Spc42p is present in both reactions (C). (D–F) Two-dimensional gel analysis (as described in Materials and methods) of whole cell extracts prepared from MPS1 GAL-SPC42-myc (ACY122-1c) and mps1-1 GAL-SPC42-myc (ACY123-10a) strains, that were released from an -factor arrest into inducing media at the mps1-1 nonpermissive temperature (D), indicates that Spc42p phosphorylation is dependent on Mps1p. Only 6 of the 11 spots corresponding to Spc42p-myc in the MPS1 strain (E) were present in the mps1-1 strain (D). When extract from MPS1 GAL-SPC42-myc was treated with calf alkaline phosphatase before two-dimensional gel analysis, seven spots were detected (F). Five of these spots correspond to those detected in the mps1-1 strain (D and F). The other two (F, arrow) are in a new position, relative to those identified in the MPS1 strain, migrating faster and more basic as might be expected for less phosphorylated forms of Spc42p-myc. The two dashed lines under spots (D–F) are for orientation. in vitro. A band corresponding to Spc42p was detected by autoradiography (Fig. 8, A, lane 1) and the presence of this band was dependent on the addition of Mps1p-myc (Fig. 8 A, lane 2). Additionally, we observed autophosphorylation of Mps1p-myc (Fig. 8 A), which has been reported previ- ously (Lauze et al., 1995). We subsequently assessed whether phosphorylation of Spc42p was dependent on Mps1p function in vivo. We were unable to detect differences in the phosphorylation state of Spc42p-myc isolated from MPS1 and mps1-1 strains using one-dimensional SDS-PAGE (unpublished data). However, differences in Spc42p phosphorylation were revealed using two-dimensional gel electrophoresis (Fig. 8, D–F). For this assay, MPS1 and mps1-1 strains containing SPC42-myc un- der the control of the galactose promoter were arrested in G1 using -factor and then released into inducing media at the mps1-1 restrictive temperature for 2 h. Extracts were pre- pared from these samples and resolved by two-dimensional gel electrophoresis and blotted to PVDF membranes. 11 spots corresponding to Spc42p-myc were identified on the two-dimensional blot prepared using the MPS1 extract (Fig. 8 E). Only six of these spots were detected on the blot pre- pared using the mps1-1 extract (Fig. 8 D). We believe that the absence of these forms of Spc42p-myc in the mps1-1 strain is due to the inability of Mps1-1p to phosphorylate Spc42p-myc at the mps1-1 restrictive temperature. Indeed,