Importin α/β Mediates Nuclear Transport of a Mammalian Circadian Clock Component, mCRY2, Together with mPER2, through a Bipartite Nuclear Localization Signal

Sakakida, Yoko; Miyamoto, Yoichi; Nagoshi, Emi; Akashi, Makoto; Nakamura, Takahiro J.; Mamine, Takayoshi; Kasahara, Megumi; Minami, Yasuhiro; Yoneda, Yoshihiro; Takumi, Toru

doi:10.1074/jbc.m413236200

Cited by 37 publications

(34 citation statements)

References 54 publications

(67 reference statements)

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“…This differential effect may represent a biochemical mechanism by which CRY2, but not CRY1, represses the CLOCK-BMAL1 complex. Until now, the only biochemical differences seen between CRY1 and CRY2 were subtle differences in the levels of potency of their repression and binding to some core clock components (9,22) and differential nuclear localization mechanisms in Xenopus and mammals (4,20,31). Our data support the notion that the opposing period length phenotypes in Cry1 Ϫ/Ϫ and Cry2…”

Section: Random Mutagenesis and Cell-based Functional Screen Ofsupporting

confidence: 79%

“…As a result of our random mutagenesis screen, we generated many novel mutant Cry alleles. The residue changes corresponding with defects in transcriptional repression were confined to the PHR, and no changes were observed in the Cterminal tail, which has been shown to be important for nuclear localization in mammals (4,20) and Xenopus (31). This is in line with data from Chaves et al (4), who reported that interaction of mCRY1 with BMAL1 via the coiled-coil domain in its PHR could facilitate CRY's nuclear localization, even in the event that its NLSs are mutated.…”

Section: Random Mutagenesis and Cell-based Functional Screen Ofmentioning

confidence: 99%

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Generation of a Novel Allelic Series of Cryptochrome Mutants via Mutagenesis Reveals Residues Involved in Protein-Protein Interaction and CRY2-Specific Repression

McCarthy

Baggs

Geskes

et al. 2009

Molecular and Cellular Biology

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CRYPTOCHOME proteins are necessary for mammalian circadian rhythms and have many well-established biochemical roles within the molecular clock. While studies examining the effect of null Cry alleles have been informative, they have failed to dissect out the relative importance of, and the molecular mechanisms behind, the many roles of the CRY1 and CRY2 proteins. To address this, we created an allelic series of Cry mutants through random mutagenesis, followed by a cell-based screen to isolate mutants with aberrant repression of CLOCK-BMAL1. We identified 22 mutants with mutations resulting in single amino acid substitutions which cause a variety of deficiencies in different CRY functions. To illustrate the breadth and value of these new tools, we present an in-depth analysis of two of these mutants, CRY2G354D and CRY2G351D; the former shows deficiency in clock protein binding and is required for repression by both CRYs, while in contrast, the latter displays normal binding function but exhibits a CRY2-specific repression phenotype. Further, while overexpression of CRY2 in NIH 3T3 cells caused a dose-dependent decrease in rhythm amplitude, overexpression of CRY2G351D abolished rhythmicity. In summary, characterization of these unique alleles provides new opportunities for more-sophisticated insight into the multifaceted functions of the CRY proteins in circadian rhythms.Organisms from cyanobacteria to humans have evolved circadian rhythms to anticipate the dramatic changes in environmental conditions that occur on a daily basis. The driving force behind the 24-h periodicity of these endogenous rhythms is a cell-autonomous molecular oscillator, composed of transcriptional/translational feedback loops (reviewed in reference 1).In mammals, the Cryptochrome (CRY) proteins CRY1 and CRY2 play an integral role in the circadian clock by closing the core negative-feedback loop (9,12,28,29). In 1999, two groups showed that mice lacking both Crys demonstrate a complete loss of rhythm in circadian wheel-running behavior when put in constant darkness (26,28), suggesting an essential role for murine CRY proteins (mCRYs) in the core molecular mechanism. This year, it was shown that both CRY1 and CRY2 repressed CLOCK-BMAL1-mediated transcription much more potently than the mammalian PERIOD (PER) proteins in transiently transfected cells (9, 12). While it is known that CRYs are essential for circadian rhythms at both the behavioral (28, 29) and molecular (21) levels, many questions concerning how the various known actions of the CRY proteins contribute to their essential roles in the molecular mechanism of the circadian clock remain unanswered.Mammalian CRY1 and CRY2 have several well-established biochemical roles in the molecular oscillator in addition to their role in repressing the CLOCK-BMAL1 heterodimer. Both CRY1 and CRY2 bind the PER proteins (9, 12), and this interaction affects PER's stability (30) and localization (12). While the CRY interaction domain on PER has been described, the regions important for PER binding on C...

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Section: Random Mutagenesis and Cell-based Functional Screen Ofsupporting

confidence: 79%

Section: Random Mutagenesis and Cell-based Functional Screen Ofmentioning

confidence: 99%

Generation of a Novel Allelic Series of Cryptochrome Mutants via Mutagenesis Reveals Residues Involved in Protein-Protein Interaction and CRY2-Specific Repression

McCarthy

Baggs

Geskes

et al. 2009

Molecular and Cellular Biology

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“…5, right lower alignment). The C-tail of amCRY does not contain a second putative NLS found on the C-tails of mCRY2 and xCRY2b (Zhu et al 2003;Sakakida et al 2005;Chaves et al 2006). These analyses show that amCRY does not contain sequences thought to be necessary for dCRY photoreceptor function.…”

Section: Genome Research 1355mentioning

confidence: 99%

Molecular and phylogenetic analyses reveal mammalian-like clockwork in the honey bee (Apis mellifera) and shed new light on the molecular evolution of the circadian clock

et al. 2006

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The circadian clock of the honey bee is implicated in ecologically relevant complex behaviors. These include time sensing, time-compensated sun-compass navigation, and social behaviors such as coordination of activity, dance language communication, and division of labor. The molecular underpinnings of the bee circadian clock are largely unknown. We show that clock gene structure and expression pattern in the honey bee are more similar to the mouse than to Drosophila. The honey bee genome does not encode an ortholog of Drosophila Timeless (Tim1), has only the mammalian type Cryptochrome (Cry-m), and has a single ortholog for each of the other canonical "clock genes." In foragers that typically have strong circadian rhythms, brain mRNA levels of amCry, but not amTim as in Drosophila, consistently oscillate with strong amplitude and a phase similar to amPeriod (amPer) under both light-dark and constant darkness illumination regimes. In contrast to Drosophila, the honey bee amCYC protein contains a transactivation domain and its brain transcript levels oscillate at virtually an anti-phase to amPer, as it does in the mouse. Phylogenetic analyses indicate that the basal insect lineage had both the mammalian and Drosophila types of Cry and Tim. Our results suggest that during evolution, Drosophila diverged from the ancestral insect clock and specialized in using a set of clock gene orthologs that was lost by both mammals and bees, which in turn converged and specialized in the other set. These findings illustrate a previously unappreciated diversity of insect clockwork and raise critical questions concerning the evolution and functional significance of species-specific variation in molecular clockwork.

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“…Importins α and β are nuclear transport proteins that interact with CRY2 through this NLS and consequently facilitate nuclear entry of CRY2 and PER2 expressed with CRY2. When the CRY2 NLS sequence is mutated, it can no longer facilitate the nuclear translocation of PER2 in cell culture 47 . In addition, PER2 nuclear localization is enhanced by CRY1 and is dependent on CRY1 binding 48,49 .…”

Section: Q7 Q7 Q8 Q8mentioning

confidence: 99%

“…CRY2 has a functional NLS in the C-terminal region, but this region   is not conserved in CRY1 (ref. 47). Importins α and β are nuclear transport proteins that interact with CRY2 through this NLS and consequently facilitate nuclear entry of CRY2 and PER2 expressed with CRY2.…”

Section: Q7 Q7 Q8 Q8mentioning

confidence: 99%

The intricate dance of post-translational modifications in the rhythm of life

Hirano

Ptáček

2016

Nat Struct Mol Biol

150

136

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Importin α/β Mediates Nuclear Transport of a Mammalian Circadian Clock Component, mCRY2, Together with mPER2, through a Bipartite Nuclear Localization Signal

Cited by 37 publications

References 54 publications

Generation of a Novel Allelic Series of Cryptochrome Mutants via Mutagenesis Reveals Residues Involved in Protein-Protein Interaction and CRY2-Specific Repression

Generation of a Novel Allelic Series of Cryptochrome Mutants via Mutagenesis Reveals Residues Involved in Protein-Protein Interaction and CRY2-Specific Repression

Molecular and phylogenetic analyses reveal mammalian-like clockwork in the honey bee (Apis mellifera) and shed new light on the molecular evolution of the circadian clock

The intricate dance of post-translational modifications in the rhythm of life

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