The Functional Interplay between Protein Kinase CK2 and CCA1 Transcriptional Activity Is Essential for Clock Temperature Compensation in Arabidopsis Sergi Portole´ s, Paloma Ma´ s* Consortium CSIC-IRTA-UAB, Centre for Research in Agricultural Genomics (CRAG), Department of Plant Molecular Genetics, Barcelona, Spain Abstract Circadian rhythms are daily biological oscillations driven by an endogenous mechanism known as circadian clock. The protein kinase CK2 is one of the few clock components that is evolutionary conserved among different taxonomic groups. CK2 regulates the stability and nuclear localization of essential clock proteins in mammals, fungi, and insects. Two CK2 regulatory subunits, CKB3 and CKB4, have been also linked with the Arabidopsis thaliana circadian system. However, the biological relevance and the precise mechanisms of CK2 function within the plant clockwork are not known. By using ChIP and Double–ChIP experiments together with in vivo luminescence assays at different temperatures, we were able to identify a temperature-dependent function for CK2 modulating circadian period length. Our study uncovers a previously unpredicted mechanism for CK2 antagonizing the key clock regulator CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1). CK2 activity does not alter protein accumulation or subcellular localization but interferes with CCA1 binding affinity to the promoters of the oscillator genes. High temperatures enhance the CCA1 binding activity, which is precisely counterbalanced by the CK2 opposing function. Altering this balance by over-expression, mutation, or pharmacological inhibition affects the temperature compensation profile, providing a mechanism by which plants regulate circadian period at changing temperatures. Therefore, our study establishes a new model demonstrating that two opposing and temperature-dependent activities (CCA1-CK2) are essential for clock temperature compensation in Arabidopsis. Citation: Portole´s S, Ma´s P (2010) The Functional Interplay between Protein Kinase CK2 and CCA1 Transcriptional Activity Is Essential for Clock Temperature Compensation in Arabidopsis. PLoS Genet 6(11): e1001201. doi:10.1371/journal.pgen.1001201 Editor: Gregory P. Copenhaver, The University of North Carolina at Chapel Hill, United States of America Received June 18, 2010; Accepted October 7, 2010; Published November 4, 2010 Copyright: 2010 Portole´s, Ma´s. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants to PM from the Spanish Ministry of Science and Education (MEC) (BIO2007-66068), from the EMBO YIP program, and from EUROHORCS (European Heads Of Research Councils) and European Science Foundation (ESF) through the EURYI Award. SP is a recipient of the FPI pre- doctoral fellowship (MEC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: paloma.mas@cid.csic.es Introduction Circadian rhythms are daily biological oscillations driven by an endogenous mechanism known as circadian clock. The phase of the rhythms is synchronized by environmental cues, mostly changes in light and temperature, that occur during the 24-hour day/night cycle. Synchronization ensures adequate timing and allows the rhythmic activities to occur at the most appropriate phase relationships with the environment [1–5]. In many organisms, the reciprocal regulation among key clock genes and proteins sustains molecular oscillations that are translated into metabolic and behavioral rhythms [6–8]. Additional mechanisms involving chromatin remodeling [9,10] and post-translational regulation of clock components [11,12] also contribute to circadian rhythmicity. Despite the conservation of clock mechanisms, the actual molecular components responsible for circadian function are not conserved among phylogenetic kingdoms. A remarkable exception is the protein kinase CK2 (formerly casein kinase 2) with an important function within the plant, fungi, insect and mammalian circadian systems [13]. CK2 is an evolutionarily conserved serine/threonine protein kinase involved in the regulation of key cellular events including tumorigenesis, cell viability and proliferation [14]. CK2 achieves its function by regulating more than 300 putative substrates [15]. The CK2 holoenzyme consists of two catalytic a-subunits and two regulatory b-subunits forming a hetero-tetrameric (a2b2) structure [16]. On a broad sense, the regulatory CK2b subunits provide the substrate selectivity and increase the overall catalytic activity [17,18]. Regarding the circadian function, CK2 has emerged as a conserved molecular component modulating the subcellular localization and stability of key clock proteins [13]. For example, CK2 regulates the nuclear localization of the mammalian clock component BMAL1 [19] and the protein stability of PERIOD2 (PER2) [20,21]. These findings are consistent with studies in Drosophila melanogaster [22,23] showing that CK2 regulates the subcellular distribution and stability of the core components PERIOD (PER) and TIMELESS (TIM) [22–24]. In plants, two of the four members of the Arabidopsis family of CK2 regulatory subunits, CKB3 [25–27] and CKB4 [28,29] have been also functionally linked with the plant circadian clock. CK2 phosphorylates the Arabidopsis central clock compo- nents CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) [26]. Furthermore, the CCA1 phosphorylation was proposed to be important for CCA1 clock function [25]. Over-expression of CKB3 or CKB4 leads to period shortening [27,28] and altered day-length-dependent regu- lation of developmental outputs [27,28]. The pervasive alterations of many clock outputs and the changes in oscillator expression suggested that CK2 might be closely regulating the oscillator function. PLoS Genetics | www.plosgenetics.org 1 November 2010 | Volume 6 | Issue 11 | e1001201