David M. Virshup

Post-translational modifications regulate the ticking of the circadian clock

Getting a good nights sleep is on everyones to-do list. So is, no doubt, staying awake during late afternoon seminars. Our internal clocks control these and many more workings of the body, and disruptions of the circadian clocks predispose individuals to depression, obesity and cancer. Mutations in kinases and phosphatases in hamsters, flies, fungi and humans highlight how our timepieces are regulated and provide clues as to how we might be able to manipulate them.

From Promiscuity to Precision: Protein Phosphatases Get a Makeover

The control of biological events requires strict regulation using complex protein phosphorylation and dephosphorylation strategies. The bulk of serine-threonine dephosphorylations are catalyzed by a handful of phosphatase catalytic subunits, giving rise to the misconception that these phosphatases are promiscuous and unregulated enzymes in vivo. The reality is much more nuanced: PP1 and PP2A, the most abundant serine-threonine phosphatases, are, in fact, families of hundreds of protein serine/threonine phosphatases, assembled from a few catalytic subunits in combination with a highly diverse array of regulators. As recent publications illustrate, these regulatory subunits confer specificity, selectivity, localization, and regulation on these important enzymes.

Control of Mammalian Circadian Rhythm by CKIε-Regulated Proteasome-Mediated PER2 Degradation

ABSTRACT The mammalian circadian regulatory proteins PER1 and PER2 undergo a daily cycle of accumulation followed by phosphorylation and degradation. Although phosphorylation-regulated proteolysis of these inhibitors is postulated to be essential for the function of the clock, inhibition of this process has not yet been shown to alter mammalian circadian rhythm. We have developed a cell-based model of PER2 degradation. Murine PER2 (mPER2) hyperphosphorylation induced by the cell-permeable protein phosphatase inhibitor calyculin A is rapidly followed by ubiquitination and degradation by the 26S proteasome. Proteasome-mediated degradation is critically important in the circadian clock, as proteasome inhibitors cause a significant lengthening of the circadian period in Rat-1 cells. CKIε (casein kinase Iε) has been postulated to prime PER2 for degradation. Supporting this idea, CKIε inhibition also causes a significant lengthening of circadian period in synchronized Rat-1 cells. CKIε inhibition also slows the degradation of PER2 in cells. CKIε-mediated phosphorylation of PER2 recruits the ubiquitin ligase adapter protein β-TrCP to a specific site, and dominant negative β-TrCP blocks phosphorylation-dependent degradation of mPER2. These results provide a biochemical mechanism and functional relevance for the observed phosphorylation-degradation cycle of mammalian PER2. Cell culture-based biochemical assays combined with measurement of cell-based rhythm complement genetic studies to elucidate basic mechanisms controlling the mammalian clock.

Protein phosphatase 2A: a panoply of enzymes.

Protein phosphatase 2A describes an extended family of intracellular protein serine/threonine phosphatases sharing a common catalytic subunit that regulates a variety of processes by means of diverse regulatory subunits. During the past year, studies have shown that protein phosphatase 2A influences events ranging from the initiation of DNA replication to vertebrate axis formation to apoptosis.

The Circadian Regulatory Proteins BMAL1 and Cryptochromes Are Substrates of Casein Kinase Iε

The serine/threonine protein kinase casein kinase I ε (CKIε) is a key regulator of metazoan circadian rhythm. Genetic and biochemical data suggest that CKIε binds to and phosphorylates the PERIOD proteins. However, the PERIOD proteins interact with a variety of circadian regulators, suggesting the possibility that CKIε may interact with and phosphorylate additional clock components as well. We find that CRY1 and BMAL1 are phosphoproteins in cultured cells. Mammalian PERIOD proteins act as a scaffold with distinct domains that simultaneously bind CKIε and mCRY1 and mCRY2 (mCRY). mCRY is phosphorylated by CKIε only when both proteins are bound to mammalian PERIOD proteins. BMAL1 is also a substrate for CKIεin vitro, and CKIε kinase activity positively regulates BMAL1-dependent transcription from circadian promoters in reporter assays. We conclude that CKIε phosphorylates multiple circadian substrates and may exert its effects on circadian rhythm in part by a direct effect on BMAL1-dependent transcription.

Nuclear entry of the circadian regulator mPER1 is controlled by mammalian casein kinase I epsilon

ABSTRACT The molecular oscillator that keeps circadian time is generated by a negative feedback loop. Nuclear entry of circadian regulatory proteins that inhibit transcription from E-box-containing promoters appears to be a critical component of this loop in bothDrosophila and mammals. The Drosophila double-time gene product, a casein kinase I ɛ (CKIɛ) homolog, has been reported to interact with dPER and regulate circadian cycle length. We find that mammalian CKIɛ binds to and phosphorylates the murine circadian regulator mPER1. Unlike both dPER and mPER2, mPER1 expressed alone in HEK 293 cells is predominantly a nuclear protein. Two distinct mechanisms appear to retard mPER1 nuclear entry. First, coexpression of mPER2 leads to mPER1-mPER2 heterodimer formation and cytoplasmic colocalization. Second, coexpression of CKIɛ leads to masking of the mPER1 nuclear localization signal and phosphorylation-dependent cytoplasmic retention of both proteins. CKIɛ may regulate mammalian circadian rhythm by controlling the rate at which mPER1 enters the nucleus.

Setting clock speed in mammals: the CK1 epsilon tau mutation in mice accelerates circadian pacemakers by selectively destabilizing PERIOD proteins.

The intrinsic period of circadian clocks is their defining adaptive property. To identify the biochemical mechanisms whereby casein kinase1 (CK1) determines circadian period in mammals, we created mouse null and tau mutants of Ck1 epsilon. Circadian period lengthened in CK1epsilon-/-, whereas CK1epsilon(tau/tau) shortened circadian period of behavior in vivo and suprachiasmatic nucleus firing rates in vitro, by accelerating PERIOD-dependent molecular feedback loops. CK1epsilon(tau/tau) also accelerated molecular oscillations in peripheral tissues, revealing its global role in circadian pacemaking. CK1epsilon(tau) acted by promoting degradation of both nuclear and cytoplasmic PERIOD, but not CRYPTOCHROME, proteins. Together, these whole-animal and biochemical studies explain how tau, as a gain-of-function mutation, acts at a specific circadian phase to promote degradation of PERIOD proteins and thereby accelerate the mammalian clockwork in brain and periphery.

Identification of a New Family of Protein Phosphatase 2A Regulatory Subunits

Protein phosphatase 2A (PP2A) is a major intracellular protein phosphatase that regulates multiple aspects of cell growth and metabolism. The ability of this widely distributed heterotrimeric enzyme to act on a diverse array of substrates is largely controlled by the nature of its regulatory B subunit. Only two gene families encoding endogenous B subunits have been cloned to date, although the existence of several additional regulatory subunits is likely. We have identified by two-hybrid interaction a new human gene family encoding PP2A B subunits. This family, denoted B56, contains three distinct genes, one of which is differentially spliced. B56 polypeptides co-immunoprecipitate with PP2A A and C subunits and with an okadaic acid-inhibitable, heparin-stimulated phosphatase activity. The three B56 family members are 70% identical to each other but share no obvious homology with previously identified B subunits. These phosphatase regulators are differentially expressed, with B56α and B56 highly expressed in heart and skeletal muscle and B56β highly expressed in brain. The identification of this novel phosphatase regulator gene family will facilitate future studies on the control of protein dephosphorylation and the role of PP2A in cellular function.

Casein kinase I phosphorylates and destabilizes the β-catenin degradation complex

Wnt signaling plays a key role in cell proliferation and development. Recently, casein kinase I (CKI) and protein phosphatase 2A (PP2A) have emerged as positive and negative regulators of the Wnt pathway, respectively. However, it is not clear how these two enzymes with opposing functions regulate Wnt signaling. Here we show that both CKIδ and CKIɛ interacted directly with Dvl-1, and that CKI phosphorylated multiple components of the Wnt-regulated β-catenin degradation complex in vitro, including Dvl-1, adenomatous polyposis coli (APC), axin, and β-catenin. Comparison of peptide maps from in vivo and in vitro phosphorylated β-catenin and axin suggests that CKI phosphorylates these proteins in vivo as well. CKI abrogated β-catenin degradation in Xenopus egg extracts. Notably, CKI decreased, whereas inhibition of CKI increased, the association of PP2A with the β-catenin degradation complex in vitro. Additionally, inhibition of CKI in vivo stabilized the β-catenin degradation complex, suggesting that CKI actively destabilizes the complex in vivo. The ability of CKI to induce secondary body axes in Xenopus embryos was reduced by the B56 regulatory subunit of PP2A, and kinase-dead CKIɛ acted synergistically with B56 in inhibiting Wnt signaling. The data suggest that CKI phosphorylates and destabilizes the β-catenin degradation complex, likely through the dissociation of PP2A, providing a mechanism by which CKI stabilizes β-catenin and propagates the Wnt signal.

A conserved docking motif for CK1 binding controls the nuclear localization of NFAT1.

ABSTRACT In resting cells, the NFAT1 transcription factor is kept inactive in the cytoplasm by phosphorylation on multiple serine residues. These phosphorylated residues are primarily contained within two types of serine-rich motifs, the SRR-1 and SP motifs, which are conserved within the NFAT family. Several different kinases have been proposed to regulate NFAT, but no single candidate displays the specificity required to fully phosphorylate both types of motifs; thus, the identity of the kinase that regulates NFAT activity remains unclear. Here we show that the NFAT1 serine motifs are regulated by distinct kinases that must coordinate to control NFAT1 activation. CK1 phosphorylates only the SRR-1 motif, the primary region required for NFAT1 nuclear import. CK1 exists with NFAT1 in a high-molecular-weight complex in resting T cells but dissociates upon activation. GSK3 does not phosphorylate the SRR-1 region but can target the NFAT1 SP-2 motif, and it synergizes with CK1 to regulate NFAT1 nuclear export. We identify a conserved docking site for CK1 in NFAT proteins and show that mutation of this site disrupts NFAT1-CK1 interaction and causes constitutive nuclear localization of NFAT1. The CK1 docking motif is present in proteins of the Wnt, Hedgehog, and circadian-rhythm pathways, which also integrate the activities of CK1 and GSK3.