No abstract

Plastids contain sigma factors, i.e. gene-regulatory proteins for promoter binding and transcription initiation. Despite the physical and functional similarity shared with their prokaryotic counterparts, the plant sigma factors have distinguishing features: most notably the existence of a variable extra sequence comprising their N-terminal portions. This distinct architecture is reflected by functional differences, including phosphorylation control by organellar protein kinase(s) closely related to nucleocytosolic, rather than bacterial-type, enzymes. In particular, cpCK2, a nuclear-coded plastid-targeted casein kinase 2, has been implicated as a key component in plant sigma factor phosphorylation and transcriptional regulation (Eur. J. Biochem. 269, 2002, 3329; Planta, 219, 2004, 298). Although this notion is based mainly on biochemical evidence and in vitro systems, the recent availability of Arabidopsis sigma knock-out lines for complementation by intact and mutant sigma cDNAs has opened up new strategies for the study of transcription regulatory mechanisms in vivo. Using Arabidopsis sigma factor 6 (AtSIG6) as a paradigm, we present data suggesting that: (i) this factor is a substrate for regulatory phosphorylation by cpCK2 both in vitro and in vivo; (ii) cpCK2 phosphorylation of SIG6 occurs at multiple sites, which can widely differ in their effect on the visual and/or molecular phenotype; (iii) in vivo usage of the perhaps most critical cpCK2 site defined by Ser174 requires (pre-)phosphorylation at the n + 3 serine residue Ser177, pointing to ‘pathfinder’ kinase activity capable of generating a functional cpCK2 substrate site.

A plastidic serine ⁄ threonine protein kinase, initially named plastid transcription kinase (PTK) has been implicated in phosphorylation and redox control of chloroplast transcription. This kinase was later renamed as chloroplast casein kinase 2 (cpCK2) because of its physical and functional similarity to nucleocytosolic casein kinase 2 (ncCK2). It shares all four of its cysteine residues with ncCK2 from land plants, while only three of these residues are conserved in algal CK2-type sequences, and just two in animals. Using bacterial overexpression of cpCK2 from Arabidopsis thaliana, here we show the principal features of this enzyme and assign functional determinants of its role as a transcriptional regulator in vitro. The recombinant protein is capable of using various plant sigma transcription factors as phosphorylation substrates. Electrophoretic mobility shift DNA-binding assays reveal differential effects of sigma phosphorylation, depending on the factor and the promoter used. Treatment of the kinase with redoxactive reagents indicate a critical involvement of thiol groups in both its enzymatic activity and interaction capabilities. Mutational exchanges of cysteine to serine residues, in combination with in vitro assays, have provided clues to the possible role of individual cysteines. For instance, while Cys4 but not Cys2 is essential for activity, the latter seems to be involved in the formation of intermolecular (regulatory) disulfide bonds.Structured digital abstract l cpCK2 phosphorylates SaSig2 by protein kinase assay (View interaction) l cpCK2 phosphorylates AtSig6 by protein kinase assay (View interaction) l cpCK2 and cpCK2 bind by comigration in gel electrophoresis (View interaction) l cpCK2 phosphorylates AtSig1 by protein kinase assay (View interaction) l cpCK2 phosphorylates SaSig1 by protein kinase assay (View interaction)