Submitted to: Plant Signaling and Behavior
Publication Type: Peer reviewed journal
Publication Acceptance Date: 12/27/2013
Publication Date: 12/31/2013
Citation: Oh, M., Wu, X., Huber, S.C. 2013. Impact of Ca2+ on structure of soybean CDPKß and accessibility of the Tyr-24 autophosphorylation site. Plant Signaling and Behavior. 8(12):e27671-1-5. Interpretive Summary: The calcium-dependent protein kinases (CDPKs) are unique to terrestrial plants, green algae, and certain unicellular organisms and are known to play important roles in control of plant growth and development and response to biotic and abiotic stresses. However, much remains to be learned about these important protein kinases. In the present study, we confirm the recent observation that CDPKs are actually dual-specificity kinases, meaning that they can phosphorylate serine and threonine residues either on themselves (autophosphorylation) or other proteins (transphosphorylation). Previously, the CDPKs were classified as serine/threonine protein kinases. We confirm the novel ability of soybean CDPKß to autophosphorylate itself on the tyrosine residue at the beginning of the kinase domain of the protein. This residue, referred to as tyrosine-24, was confirmed to be a site of autophosphorylation using custom antibodies and then used these antibodies to specifically determine whether this residue was accessible to protein phosphatases. This question arose because recent structural models suggest that the tyrosine-24 region of the protein may be physically covered by calcium-binding domain of the protein when fully loaded with calcium. However, a specific protein tyrosine phosphatase known as PTP1B was able to effectively remove the phosphate from tyrosine-24 regardless of the presence or absence of calcium. These results suggest that CDPKs tertiary structure may involve considerable breathing and flexing such that sites of autophosphorylation are fully accessible to protein phosphatases.
Technical Abstract: Several plant CDPKs were recently shown to be dual specificity kinases rather than Ser/Thr kinases as traditionally classified by sequence analysis. In the present study we confirm the autophosphorylation of recombinant soybean His6-GmCDPK-Beta at the Tyr-24 site using sequence- and modification specific antibodies. Homology modeling of soybean CDPK-Beta based on recent structures determined for several apicomplexan CDPKs suggested that phosphotyrosine-24 may be inaccessible to phosphatases. However, we report that dephosphorylation of CDPK-Beta by the phosphotyrosine protein phosphatase, PTP1B, was not restricted in the presence of calcium. Thus, despite conformational changes likely associated with calcium binding to the CDPKs, phosphotyrosine sites remain fully accessible to dephosphorylation suggesting the possibility of conformational breathing and flexing.