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ARS Home » Midwest Area » Urbana, Illinois » Global Change and Photosynthesis Research » Research » Publications at this Location » Publication #283513
Title: CDPKs are dual-specificity protein kinases and tyrosine autophosphorylation attenuates kinase activity

Author
item OH, MAN-HO - University Of Illinois
item WU, XIA - University Of Illinois
item KIM, HYOUNG SEOK - University Of Illinois
item HARPER, JEFFREY - University Of Nevada
item ZIELINSKI, RAYMOND - University Of Illinois
item CLOUSE, STEVEN - North Carolina State University
item Huber, Steven

Submitted to: FEBS Letters
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/1/2012
Publication Date: 11/30/2012
Citation: Oh, M., Wu, X., Kim, H., Harper, J., Zielinski, R., Clouse, S.D., Huber, S.C. 2012. CDPKs are dual-specificity protein kinases and tyrosine autophosphorylation attenuates kinase activity. FEBS Letters. 586(23):4070-4075.

Interpretive Summary: Calcium signaling is firmly established as an essential second messenger controlling key aspects of plant growth and development and many specific mechanisms are involved in transducing those Ca2+ signals. One important mechanism involves the regulation of protein phosphorylation by Ca2+ and this is accomplished in part by the family of calcium-dependent protein kinases (CDPKs) that are found in terrestrial plants, green algae, and certain unicellular organisms. However, much remains to be learned about these important protein kinases that control plant growth and development and response to biotic and abiotic stresses. For example, the CDPKs are classified as serine/threonine protein kinases, meaning that they phosphorylate serine and threonine residues either on themselves (autophosphorylation) or other proteins (transphosphorylation). Here we report that several CDPKs can autophosphorylate not only on serine and threonine residues but also tyrosine residues, which suggests that at least some CDPKs are dual specificity kinases. In more detailed studies with soybean CDPKß, several sites of autophosphorylation were identified including tyrosine-24, which is the first residue of the kinase domain. Autophosphorylation of this site is shown to inhibit kinase activity, but the magnitude of the effect varied depending on the particular substrate used. Collectively, these results indicate that autophosphorylation is an important mechanism regulating CDPK activity and that phosphorylation of tyrosine residues also plays an important role. Identifying specific sites of autophosphorylation provides new targets for manipulation to engineer signaling pathways in plants.

Technical Abstract: Calcium-dependent protein kinases (CDPKs or CPKs) are classified as serine/threonine protein kinases but we made the surprising observation that soybean CDPK' and several Arabidopsis isoforms (AtCPK4 and AtCPK34) could also autophosphorylate on tyrosine residues. In studies with His6-GmCDPK', we identified Ser-8 and Thr-17 (in the N-terminal domain), Tyr-24 (kinase domain), Ser-304 (in the autoinhibitory junction domain) and Ser-358 (between EF hand 1 and 2 in the calmodulin-like domain; CLD) as autophosphorylation sites. In general, overall autophosphorylation increased kinase activity with synthetic peptides, but different phosphosites have opposing effects on kinase activity. In particular, autophosphorylation of Tyr-24 appears to attenuate kinase activity. Interestingly, the ability of the recombinant phosphotyrosine protein phosphatase, PTP1B, to remove phosphotyrosine from CDPK' was inhibited by calcium, suggesting a conformational change that renders phosphotyrosine site(s) inaccessible to the phosphatase. It is conceivable that the phosphorylated tyrosine residue(s) play a role in this overall conformational change. While many things remain to be determined, it is clear that several CDPK isoforms are dual-specificity protein kinases, which raises the possibility that phosphotyrosine signaling functions in Ca2+/CDPK-mediated processes in planta.