Getting a good night's sleep is on everyone's 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.

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.

Biological clocks with a period of Ϸ24 h (circadian) exist in most organisms and time a variety of functions, including sleep-wake cycles, hormone release, bioluminescence, and core body temperature fluctuations. Much of our understanding of the clock mechanism comes from the identification of specific mutations that affect circadian behavior. A widely studied mutation in casein kinase I (CKI), the CKI tau mutant, has been shown to cause a loss of kinase function in vitro, but it has been difficult to reconcile this loss of function with the current model of circadian clock function. Here we show that mathematical modeling predicts the opposite, that the kinase mutant CKI tau increases kinase activity, and we verify this prediction experimentally. CKI tau is a highly specific gain-of-function mutation that increases the in vivo phosphorylation and degradation of the circadian regulators PER1 and PER2. These findings experimentally validate a mathematical modeling approach to a complex biological function, clarify the role of CKI in the clock, and demonstrate that a specific mutation can be both a gain and a loss of function depending on the substrate.kinase ͉ systems biology ͉ phosphorylation ͉ PER ͉ degradation C ircadian rhythms govern key physiologic processes including sleep-wake cycles; glucose, lipid, and drug metabolism; heart rate; stress and growth hormones; and immunity, as well as basic cellular processes such as timing of the cell division cycle (1-6). The disruption of circadian rhythm causes significant physiologic stress, is frequently experienced in jet lag and night-shift work, and has been linked to bipolar disorder (7). Thus, circadian regulation of physiology has important consequences for health. A detailed quantitative model that makes clear, testable, and accurate predictions about the clock and how we may manipulate it can therefore have benefits for human health.Much of our understanding of clock components and their interactions began with the identification of mutations that affect circadian behavior (8, 9). In mammals, the original and most extensively studied circadian rhythm mutation is the semidominant tau, first described in 1988. Hamsters with this mutation show phase-advanced activity and have a circadian period of 20 h when homozygous mutant animals are isolated from time cues (9). This tau mutation has been identified as a missense mutation within the substrate recognition site of casein kinase I (denoted CKI tau ) (10). CKI and the closely related CKI␦ are widely expressed serine-threonine protein kinases implicated in development, circadian rhythms, and DNA metabolism (11). When tested in vitro on multiple substrates, CKI tau was shown to have a much reduced overall catalytic activity (10,12,13). This partial loss-of-function mutation and its phenotype have been difficult to reconcile with our current understanding of the molecular feedback loop that governs timing in mammalian cells (13) and recent empirical observations on clock function (14-16). For example, Dey et al. (1...

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In this work we have combined biochemical and electrophysiological approaches to explore the modulation of rat ventricular transient outward K(+) current (I(to)) by calmodulin kinase II (CaMKII). Intracellular application of CaMKII inhibitors KN93, calmidazolium, and autocamtide-2-related inhibitory peptide II (ARIP-II) accelerated the inactivation of I(to), even at low [Ca(2+)]. In the same conditions, CaMKII coimmunoprecipitated with Kv4.3 channels, suggesting that phosphorylation of Kv4.3 channels modulate inactivation of I(to). Because channels underlying I(to) are heteromultimers of Kv4.2 and Kv4.3, we have explored the effect of CaMKII on human embryonic kidney (HEK) cells transfected with either of those Kvalpha-subunits. Whereas Kv4.3 inactivated faster upon inhibition of CaMKII, Kv4.2 inactivation was insensitive to CaMKII inhibitors. However, Kv4.2 inactivation became slower when high Ca(2+) was used in the pipette or when intracellular [Ca(2+)] ([Ca(2+)](i)) was transiently increased. This effect was inhibited by KN93, and Western blot analysis demonstrated Ca(2+)-dependent phosphorylation of Kv4.2 channels. On the contrary, CaMKII coimmunoprecipitated with Kv4.3 channels without a previous Ca(2+) increase, and the association was inhibited by KN93. These results suggest that both channels underlying I(to) are substrates of CaMKII, although with different sensitivities; Kv4.2 remain unphosphorylated unless [Ca(2+)](i) increases, whereas Kv4.3 are phosphorylated at rest. In addition to the functional impact that phosphorylation of Kv4 channels could cause on the shape of action potential, association of CaMKII with Kv4.3 provides a new role of Kv4.3 subunits as molecular scaffolds for concentrating CaMKII in the membrane, allowing Ca(2+)-dependent modulation by this enzyme of the associated Kv4.2 channels.

The electrocardiogram of diabetic patients shows a series of deviations from normal patterns, most of which involve the QT interval and the T wave. The QT interval is longer in patients with diabetes mellitus than in normal people [1]. Diabetic patients show a corrected QT interval (QTc), measured using Bazett's formula, longer than 0.44 s [2,3]. Using Holter ECG monitoring, the appearance of ventricular late potentials is more frequent in patients with Type I (insulin-dependent) diabetes mellitus than in healthy people, and even more frequent in patients with Type II (non-insulin-dependent) diabetes mellitus [4,5]. Nearly all these changes are related to repolarisation. The QRS complex lasts longer in diabetic patients than in control subjects, suggesting intracardiac conduction disorders, but this type of change has only been described in children [6].Various studies report a lengthening of the duration of the rat ventricular action potential (APD) stemming from diabetic cardiomyopathy as a possible cause of these changes [7±9]. None took into account, however, the regional variability in the duration of the action potential under control conditions [10,11]. Various reports pointed to a lowering of the transient outward current, I to [9,12,13] and, to a lesser extent, the delayed rectifying outward current, I K [9,12], as the main cause of the lengthening of APD. Initial studies did not take into account regional variations in the distribution of the repolarising outward Diabetologia (2000) Abstract Aims/hypothesis. To identify the possible causes of the lengthening of the action potential duration described in patients affected by diabetes mellitus. Methods. We studied the effects of streptozotocininduced diabetes on the current density of the repolarising potassium currents I to , I K , I ss and I K1 in enzymatically isolated myocytes from three different regions of rat heart: total right ventricle, subepicardium at the apex of the left ventricle and subendocardium at the base of the left ventricle. Results. No changes in I K1 were found due to diabetes, but there was a uniform decrease in I to (50 %) and I ss (40 %) current densities in the three regions.In contrast, I K diminished unevenly, with the greatest decrease in the subendocardium at the base of the left ventricle (48 %), followed by the subepicardium at the apex of the left ventricle (32 %) and right ventricle (10 %). Conclusion/interpretation. These findings suggest the existence of regional differences in ion channel expression associated with diabetes. The decrease of these repolarising currents could account for the lengthening of action potential and the consequent change in the Q-T interval of the ECG observed in diabetic rats. [Diabetologia (2000)

The circadian clock is regulated by a transcription/translation negative feedback loop. A key negative regulator of circadian rhythm in mammals is the PER2 (mammalian PERIOD 2) protein. Its daily degradation at the end of the night accompanies de-repression of transcription. CKI (casein kinase I ) has been identified as the kinase that phosphorylates PER2, targeting it for ubiquitin-mediated proteasomal degradation. We now report that PER2 degradation is also negatively regulated by PP1 (protein phosphatase 1)-mediated dephosphorylation. In Xenopus egg extract, PP1 inhibition by Inhibitor-2 accelerated mPER2 degradation. Co-immunoprecipitation experiments showed that PER2 bound to PP1c in transfected HEK-293 cells. PP1 immunoprecipitated from HEK-293 cells, mouse liver and mouse brain, dephosphorylated CKI-phosphorylated PER2, showing that PER2 is a substrate for mammalian endogenous PP1. Moreover, over-expression of the dominant negative form of PP1c, the D95N mutant, accelerated ubiquitin and proteasome-mediated degradation of PER2, and shortened the PER2 half-life in HEK-293 cells. Over-expression of the PP1 inhibitors, protein phosphatase 1 holoenzyme inhibitor-1 and Inhibitor-2, confirmed these results. Thus PP1 regulates PER2 stability and is therefore a candidate to regulate mammalian circadian rhythms.

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