Phosphorylation of S344 in the calmodulin-binding domain negatively affects CCaMK function during bacterial and fungal symbioses

Authors

  • Pratyush Routray,

    1. Graduate Program in Molecular Plant Sciences, Washington State University, Pullman, WA, USA
    2. Department of Horticulture, Washington State University, Pullman, WA, USA
    Current affiliation:
    1. Department of Biochemistry, Cellular and Molecular Biology, University of Tennessee, Knoxville, TN, USA
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    • These authors contributed equally to this work.
  • J. Benjamin Miller,

    1. Department of Cell & Developmental Biology, John Innes Centre, Norwich, UK
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    • These authors contributed equally to this work.
  • Liqun Du,

    1. Graduate Program in Molecular Plant Sciences, Washington State University, Pullman, WA, USA
    2. Department of Horticulture, Washington State University, Pullman, WA, USA
    3. College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, Zhejiang, China
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    • These authors contributed equally to this work.
  • Giles Oldroyd,

    1. Department of Cell & Developmental Biology, John Innes Centre, Norwich, UK
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  • B. W. Poovaiah

    Corresponding author
    1. Department of Horticulture, Washington State University, Pullman, WA, USA
    • Graduate Program in Molecular Plant Sciences, Washington State University, Pullman, WA, USA
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For correspondence (e-mail poovaiah@wsu.edu).

Summary

Calcium and Ca2+/calmodulin-dependent protein kinase (CCaMK) plays a critical role in the signaling pathway that establishes root nodule symbiosis and arbuscular mycorrhizal symbiosis. Calcium-dependent autophosphorylation is central to the regulation of CCaMK, and this has been shown to promote calmodulin binding. Here, we report a regulatory mechanism of Medicago truncatula CCaMK (MtCCaMK) through autophosphorylation of S344 in the calmodulin-binding/autoinhibitory domain. The phospho-ablative mutation S344A did not have significant effect on its kinase activities, and supports root nodule symbiosis and arbuscular mycorrhizal symbiosis, indicating that phosphorylation at this position is not required for establishment of symbioses. The phospho-mimic mutation S344D show drastically reduced calmodulin-stimulated substrate phosphorylation, and this coincides with a compromised interaction with calmodulin and its interacting partner, IPD3. Functional complementation tests revealed that the S344D mutation blocked root nodule symbiosis and reduced the mycorrhizal association. Furthermore, S344D was shown to suppress the spontaneous nodulation associated with a gain-of-function mutant of MtCCaMK (T271A), revealing that phosphorylation at S344 of MtCCaMK is adequate for shutting down its activity, and is epistatic over previously identified T271 autophosphorylation. These results reveal a mechanism that enables CCaMK to ‘turn off’ its function through autophosphorylation.

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