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Multistep regulation of protein kinase A in its localization, phosphorylation and binding with a regulatory subunit in fission yeast

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Abstract

The cAMP–PKA is the major glucose-sensing pathway that controls sexual differentiation in Schizosaccharomyces pombe. Sequencing from the pka1 locus of recessive sam mutants, in which cells are highly inclined to sexual differentiation, led to the identification of mutations in the pka1 locus in sam5 (pka1-G441E) and sam7 (pka1-G441R). Rst2 and Ste11 proteins were induced and localized to the nucleus of sam5 and sam7 mutants even under rich glucose conditions, indicating that the function of Pka1 was completely abolished by mutations. Pka1-G441E and Pka1-G441R mutant proteins reside in the cytoplasm, even under glucose-rich conditions, while wild-type Pka1 resides in the nucleus, indicating that the functionality of Pka1 is important for its nuclear localization. This is supported by the observation that the Pka1-T356A mutant, which partially lacks Pka1 function, was localized to both the cytoplasm and the nucleus, but an active phosphomimetic Pka1-T356D mutant prtotein was localized to the nucleus under glucose-rich conditions. In addition to the basal phosphorylation of Pka1 at T356, hyperphosphorylation of Pka1 was observed under glucose-starved conditions, and such hyperphosphorylation was not observed in pka1-G441E, pka1-G441R, pka1-T356A or pka1-T356D mutants. As these mutant proteins failed to interact with a regulatory subunit Cgs1, hyperphosphorylation of Pka1 mutant proteins was considered to be dependent on Cgs1 interaction. Consistent with a role for Cgs1 in Pka1 phosphorylation, we detected the formation of a Cgs1–Pka1 complex prior to Pka1 hyperphosphorylation. Together, these results indicate that nuclear localization of Pka1 depends on its activity and hyperphosphorylation of Pka1 depends on Cgs1 interaction.

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Acknowledgments

This study was partly supported by the Sasakawa Scientific Research Grant from The Japan Science Society. We thank Dr. T. Kaino, Dr. T Nakagawa, K. Tanae and Y. Matsuo for helpful discussions and experimental support.

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Authors and Affiliations

  1. Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue, 690-8504, Japan

    Dipali Rani Gupta, Swapan Kumar Paul, Yasuo Oowatari, Yasuhiro Matsuo & Makoto Kawamukai

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  1. Dipali Rani Gupta
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  2. Swapan Kumar Paul
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  3. Yasuo Oowatari
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  4. Yasuhiro Matsuo
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  5. Makoto Kawamukai
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Correspondence to Makoto Kawamukai.

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Communicated by C. Hoffman.

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Gupta, D.R., Paul, S.K., Oowatari, Y. et al. Multistep regulation of protein kinase A in its localization, phosphorylation and binding with a regulatory subunit in fission yeast. Curr Genet 57, 353–365 (2011). https://doi.org/10.1007/s00294-011-0354-2

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  • DOI: https://doi.org/10.1007/s00294-011-0354-2

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