Faiqa Amin MSc Student, Monaghan Lab Phosphorylation of the Arabidopsis thaliana E3 ubiquitin ligase ATL6 by the Ca2+-dependent protein kinase CPK4 Post-translational modifications (PTMs) such as reversible phosphorylation integrate signalling and gene expression with cellular metabolic networks and represent some of the earliest responses of plant cells to (a)biotic stress that rapidly controls the functions of many proteins. Ca2+ -dependent protein kinases (CPKs) transduce Ca2+ signals via the catalytic activity of their kinase domain to phosphorylate specific residues of target proteins. Our research discovered that the Arabidopsis CPK isozyme CPK4 phosphorylates the E3 ubiquitin ligase ARABIDOPSIS TÓXICOS EN LEVADURA 6 (ATL6) at multiple residues including Ser278. ATL6 is a positive regulator of immune signalling as it polyubiquitinates CPK28, leading to its proteasomal degradation and subsequent activation of pathogen defense responses. Rabbit polyclonal antibodies were raised against purified, heterologously expressed ATL6 (anti-ATL6), whereas anti-(phosphoSer278-specific) antiserum (anti-pSer278) was raised against a synthetic ATL6 phosphopeptide. These antibodies will augment various biochemical and genetic tools that are being integrated to investigate the functional relationship of CPK4 and ATL6 during Arabidopsis stress signaling. For example, immunoblot time-course assays using anti-pSer278 have established the ATP- & Ca2+ -dependent phosphorylation of ATL6 at Ser278 by CPK4. In vitro E3 ubiquitin ligase assays are being developed to test the hypothesis that CPK4-mediated phosphorylation enhances ATL6’s ability to polyubiquitinate CPK28. Overall, This work will provide an insightful understanding of the role of AtATL6 in biotic or (a)biotic stress responses.
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