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Function of CikA in the cyanobacterial circadian system: the pseudo-receiver domain of CikA regulates the circadian input pathwayZhang, Xiaofan 30 October 2006 (has links)
The circadian input kinase gene (cikA) was first identified from a Tn5 mutant
of Synechococcus elongatus PCC 7942. A cikA null strain shows a striking
phenotype related to circadian gene regulation: all sampled loci show a
shortened circadian period and reduced amplitude of oscillation and a failure to
exhibit a wild-type resetting of the phase of the rhythm after an environmental
signal. This global defect in response to the environment suggests a key role for
CikA in the circadian input pathways. Bioinformatics results classify CikA as a
divergent member of the bacteriophytochrome family, suggesting a role in light
signal transduction. In vitro analysis previously showed that CikA is a bona fide
histidine protein kinase (HPK), and its kinase activity is regulated by the
presence of other domains. Its own pseudo-receiver (PsR) domain is not the
cognate receiver domain of its kinase HPK domain, and its GAF domain does
not likely bind a bilin chromophore as do photoreceptive phytochromes. Recent
results suggested that CikA may function as a redox-sensor. In this study, we examined the function of each domain of CikA using
different mutant cikA alleles, and determined their phenotypes with respect to
complementation of a null mutant and overexpression in both wild type and cikA
null strains. All domains except the featureless N-terminus were required for
CikA function. Overexpression of all mutant alleles that encoded the PsR
domain, whether or not the HPK was functional, caused a dominant arrhythmia
phenotype. In the absence of PsR, overexpressed variants did not cause
arrhythmia, but affected the amplitude and period of oscillation. The results
suggest a model in which the PsR domain regulates kinase activity and
mediates interaction with other input pathway components to allow CikA to reach
the correct cellular position to fulfill its function. Cellular localization assays
showed CikA can interact with a complex and showed a polar localization
pattern, whereas its variant without PsR showed uniform distribution in the cell.
In summary, CikA is an autoregulated kinase in which the PsR domain
regulates activity of the HPK domain and also serves as an interaction module to
lead the CikA to a specific cellular position.
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Genetic and biochemical analyses of hypothetical protein 1: an interacting partner of CikA in Synechococcus elongatus PCC 7942Guo, Haitao 17 September 2007 (has links)
Synechococcus elongatus PCC 7942 is a model organism used to study the circadian
rhythm, a process that is driven by an endogenous biological clock that can be modulated
by external cues such as light and temperature. Some proteins have been identified that
are involved in circadian signal transduction in S. elongatus. Of them, KaiA, KaiB and
KaiC comprise the central oscillator components, which are essential for internal
timekeeping. SasA is an important protein in the output pathway, which passes the
information from central oscillator to downstream components, and thus controls
metabolic and behavioral processes. CikA is a major component in the input pathway,
which maintains synchrony of the oscillator with the environment. CikA is an unusual
phytochrome-like histidine protein kinase. It has a pseudo receiver domain which can not
accept a phosphoryl group. CikA is thought to be located at the poles of the cell through
interaction between PsR and some protein or protein complex that is also localized at the
poles. One of the potential CikA-interacting proteins identified through a yeast two
hybrid screen is called hypothetical protein 1. It specifically recognizes a PsR bait in a
yeast two hybrid assay. A bioinformatics analysis showed that there are predicted signal
peptide and transmembrane domains at the N-terminal and a cytochrome C homolog
domain at the C-terminal of Hyp1. Elucidating the features and function of Hyp1 will
provide us with useful information to understand the function and working mechanism of
CikA, and therefore will help us to clarify the signal transduction in the clock. In this
research, I used genetic, cell biological and biochemical approaches to study the features
and function of this newly identified clock component Hyp1.
To confirm the interaction between PsR and Hyp1 and complement the yeast two
hybrid data, I truncated Hyp1 (Thyp1) and purified soluble Thyp1. At the same time, I
obtained purified PsR. I tried to copurify the PsR and 6-histidine-tagged Hyp1 on a nickel affinity column. However, PsR non-specifically bound to the column, which eliminated
the utility of this approach to study their interaction.
In addition to using a biochemical approach to study Hyp1, I constructed three
hyp1 overexpression alleles for genetic analysis and two hyp1-yfp overexpression fusion
alleles for subcellular localization studies. All of them will help us to understand the
features and function of Hyp1.
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