Figure 4.

Mek1-T318 and -T322 are necessary for the full kinase activity of Mek1 and meiotic recombination. (A) Schematic diagram of S. pombe Mek1 shows the locations of the Activation Loop (AL; shaded box) and four fragments (black and gray boxes) used as substrates for the in vitro kinase assay. Black and gray colors indicate phosphorylated and non-phosphorylated fragments, respectively. Mek1 fragment amino acid numbers are shown at the left of each box. (B) Alignment of the primary structure around the conserved T318 and T322 residues (highlighted) of S. pombe (Sp) Mek1 with other related protein fragments. Asterisks or colons signify identical or almost identical amino acids among the denoted proteins. (C–E) Mek1 kinase (Mek1-9Myc immunoprecipitate) phosphorylates the Mek1 fragments indicated above each lane. Upper parts, incorporation of ³²P; lower parts, the amount of loaded protein as assessed by CBB staining. Replacements of the S83, H86, T318 and T322 residues by alanine (A) are denoted. Asterisks indicate putative degradation products. (F) Detection of the kinase activity of Mek1 mutants in vitro. WT and Mek1 mutant proteins were prepared by immunoprecipitation, and their kinase activities were measured using the AL fragment as a substrate. The amount of immunoprecipitated Mek1 kinases assessed by western analysis with the anti-Myc antibody, the incorporation of ³²P into the tested substrate, and the amount of loaded protein assessed by CBB staining are shown in the upper, middle and lower parts, respectively. The lower bands in the lower part are degradation products (asterisk). (G) Intragenic recombination between the ade6-M26 and ade6-469 alleles on chromosome III in WT (TT230 × TT231), mek1-T318A (TT677 × TT678), mek1-T322A (TT679 × TT680), mek1-T318AT322A (TT681 × TT682) or mek1-D218A (TT591 × TT592) strains. The positions of the ade6⁺ locus and centromeres (gray balls) on chromosome III are illustrated in the inset. The data show the averages and standard deviation of triplicate experiments.