PMC

ksg1.12 cells are hypersensitive to staurosporine. WT (SPB173), ksg1.12 (S12.4) pck1⁻ (SPB231), and pck2⁻ (SPB229) cells were plated with the indicated amounts of staurosporine. Photographs were taken after 5 days incubation.

ksg1.12 cells exhibit pleiotropic phenotypes. (A) ksg1.12 cells are sensitive to high temperature. Cells were plated for single colonies and incubated at 25° or at 36° for 3 days. At that time, the plate incubated at 36°, which showed no growth, was transferred to 25°. Incubation was continued for 3 days prior to photography. (B) A growth curve of wild-type (WT; SPB173) and ksg1.12 (S12.4) cells grown in complete medium at 25°.

(A) A schematic of PDK1-related proteins. The solid box indicates the conserved catalytic domain sequences. The open box shows conserved PH domain sequences. (B) Sequence alignment of the activation loop of putative Ksg1 substrates in fission yeast. (+) identical amino acids; (:) similar amino acids. The accession numbers for the indicated proteins are Pka1, BAA04891 (); Sck1, S55694 (); Sck2, CAA93901 (); Pck1, P36582 (); and Pck2, P36583 ().

Sexual development phenotypes of ksg1.12 cells. (A) Conjugation and sporulation frequencies for wild-type (WT) and ksg1.12 cells. Cells were examined microscopically following incubation at 25°. Conjugation was scored by examination of h⁹⁰ ksg1.12 cells following incubation of SPA medium to determine the number that had formed zygotes or asci. Sporulation was measured by counting asci formation in h⁺ ksg1.12/h⁻ ksg1.12 diploid cells incubated on SPA medium. (B) Flow cytometery of nitrogen-starved cells at 25°. (C) Quantitative PCR analysis of mei2 mRNA from nitrogen-starved cells. Strains are WT (SPB193) and ksg1.12 (S12.5). Cells were grown at 25° to midlog growth and then shifted to nitrogen-free medium and incubated for the indicated times.

Loss of ksg1 reverses phenotypes imparted by loss of the PKA regulatory subunit. (A) Photograph of WT (SPB193), cgs1::ura4 (SPB330), ksg1.12 (S12.17), ksg1.12 cgs1::ura4 (S12.32), or pka1⁻ (git6.261;SPB54) cells following growth at 25° on EMM/glucose or EMM/gluconate medium. (B and C) Quantitative PCR analysis of mei2 mRNA from nitrogen-starved (top) or growing (bottom) cells. Strains and conditions correspond to those used in A. (D) Photomicrographs of the indicated cells (described in A) grown to late log phase and stained with Hoechst 33324. (E) Cells were grown to stationary phase (1 × 10⁸ cells/ml) at 25°. Incubation was continued for the indicated number of days, and at each time point, a portion of the culture was removed, counted, and plated onto complete medium to determine the number of viable cells.

Activation loop phosphorylation of PKA is dependent on Ksg1. (A) Cell extracts were prepared from control cells (lane 1) or cells containing FLAG-tagged Pka1 (lanes 2 and 3), FLAG-Pka-T356D (lane 4), FLAG-Pka-T356A (lane 5), FLAG-Pka-T356E (lane 6), or FLAG-tagged Pka1 and ksg1.12 (lane 7). Cells were grown at 25°. The immunoprecipitated sample shown in lane 3 was treated with protein phosphatase I prior to SDS-PAGE. Epitope-tagged Pka1 proteins were visualized using a Western blot developed with anti-FLAG antibodies. The partial amino acid sequence of fission yeast Pka1 and human PKA is shown below to indicate the threonine phosphorylated by Ksg1/PDK. (B) Conjugation and sporulation was measured in wild-type h⁹⁰ cells or h⁹⁰ cells containing alanine (A), aspartic acid (D), or glutamic acid (E) substitutions for the threonine at position 356. Cells were grown to the indicated density in YEA and conjugating pairs or asci were quantitated using a microscope. (C) Spore germination was determined as described in materials and methods. The relevant yeast strains are WT (SP870), Pka1⁻ (SPB56), Pka1 T356A (SP870b), Pka1 T356D (SP870d), and Pka1 T356E (SP870c).