Summary
Pulses of some Ca2+ channel blockers (dantrolene, Co2+, nifedipine) and calmodulin inhibitors (chlorpromazine) lead to medium (maximally 5–9 h) phase shifts of the circadian conidiation rhythm ofNeurospora crassa. Pulses of high Ca2+, or of low Ca2+, a Ca2+ ionophore (A23187) together with Ca2+, and other Ca2+ channel blockers (La3+, diltiazem), however, caused only minor phase shifts. The effect of these substances (A 23187) and of different temperatures on the Ca2+ release from isolated vacuoles was analyzed by using the fluorescent dye Fura-2. A 23187 and higher temperatures increased the release drastically, whereas dantrolene decreased the permeation of Ca2+ (Cornelius et al., 1989).
Pulses of 8-PCTP-cAMP, IBMX and of the cAMP antagonist RP-cAMPS, also caused medium (maximally 6–9 h) phase shifts of the conidiation rhythm. The phase response curve of the agonist was almost 180° out of phase with the antagonist PRC. In spite of some variability in the PRCs of these series of experiments all showed maximal shifts during ct 0–12. The variability of the response may be due to circadian changes in the activity of phosphodiesterases: After adding cAMP to mycelial extracts HPLC analysis of cAMP metabolites showed significant differences during a circadian period with a maximum at ct 0.
Protein phosphorylation was tested mainly in an in vitro phosphorylation system (with35S-thio γ-ATP). The results showed circadian rhythmic changes predominantly in proteins of 47/48 kDa. Substances and treatments causing phase-shifts of the conidiation rhythm also caused changes in the phosphorylation of these proteins: an increase was observed when Ca2+ or cAMP were added, whereas a decrease occurred upon addition of a calmodulin inhibitor (TFP) or pretreatment of the mycelia with higher (42° C) temperatures.
Altogether, the results indicate that Ca2+-calmodulin-dependent and cAMP-dependent processes play an important, but perhaps not essential, role in the clock mechanism ofNeurospora. Ca2+ calmodulin and the phosphorylation state of the 47/48-kDa proteins may have controlling or essential functions for this mechanism.
Similar content being viewed by others
References
Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of proteins utilizing the principle of protein dye binding. Anal Biochem 72:248–254
Braumann T, Jastorff B (1985) Physico-chemical characterization of cyclic nucleotides by reversed-phase high-performance liquid chromatography: quantitative determination of hydrophobicity. J Chromatogr 350:105–118
Braumann T, Jastorff B, Richter-Landsberg C (1986) Fate of cyclic nucleotides in PC12 cell cultures: uptake, metabolism and effects on nerve growth factor-induced neurite out-growth. J Neurochem 47:912–919
Cornelius G, Rensing L (1986) Circadian rhythm of heat shock protein synthesis ofNeurospora crassa. Europ J Cell Biol 40:130–132
Cornelius G, Gebauer G, Techel D (1989) Inositol trisphosphate induces calcium release fromNeurospora crassa vacuoles. Biochem Biophys Res Comm 162:852–856
Cornelius G, Nakashima K (1987) Vacuoles play a decisive role in calcium homeostatis inNeurospora crassa. J Gen Microbiol 133:2341–2347
Crynkiewicz G, Poenie M, Tsien RJ (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260:3340–3450
Davis RH, de Serres FJ (1970) Genetic and microbiological research techniques forNeurospora crassa. Meth Enzymol 17A:79–143
De Wit RJW, Hekstra D, Jastorff B, Stec WJ, Baraniak J, Van Driel R, Van Haastert PJM (1985) Inhibitory action of certain cyclophosphate derivatives of cAMP on cAMP-dependent protein kinases. Eur J Biochem 142:255–260
Dharmanada S, Feldman J (1979) Spatial distribution of circadian clock phase in aging cultures ofNeurospora crassa. Plant Physiol 63:1049–1054
Drescher K, Cornelius G, Rensing L (1982) Phase response curves obtained by perturbing different variables of a 24-h model oscillator based on translational control. J Theor Biol 94:345–353
Eskin A, Corrent G, Lin CY, McAdoo DJ (1982) Mechanism for shifting the phase of a circadian rhythm by serotonin: Involvement of cAMP. Proc Natl Acad Sci USA 79:660–664
Eskin A, Takahashi JS, Zatz M, Block GD (1984) Cyclic guanosine 3′,5′ monophosphate mimics the effects of light on a circadian pacemaker in the eye ofAplysia. J Neurosci 4:2466–2471
Feldman J (1975) Circadian periodicity inNeurospora crassa: Alteration by inhibitors of cyclic AMP phosphodiesterase. Science 190:789–790
Feldman JF, Gardner G, Denison R (1979) Genetic analysis of the circadian clock inNeurospora. In: Suda M, Hayishi O, Nakagawa H (eds) “Biological rhythms and their control mechanisms”, Amsterdam.
Feldman J, Dunlap JC (1983)Neurospora crassa: A unique system for studying circadian rhythms. Photochem Photobiol Rev 7:319–368
Genieser H-G, Dostmann W, Botteri U, Butt E, Jastorff B (1988) Synthesis of nucleoside 3′,5′-cyclic phosphorothioates by cyclophosphorylation of unprotected nucleosides. Tetrahedron Lett 29(23):2803–2804
Genieser H-G, Butt E, Botteri U, Dostmann W, Jastorff B (1989) Synthesis of the 3′,5′-cyclic phosphates from unprotected nucleosides. Synthesis 1:53–54
Goto K, Laval-Martin D, Edmunds LN Jr (1985) Biochemical modeling of an antonomously oscillatory circadian clock inEuglena. Science 228:1284–1288
Hall JC, Rosbash M (1987) Genes and biological rhythms. Trends Genet 3:185–191
Hall JC, Rosbash M (1987) Genes and biological rhythms. Trends Genet 3:185–191
Hasunuma L, Funadera K, Shinohara Y, Furukawa K, Watanabe M (1987) Circadian oscillation and light-induced changes in the concentration of cyclic nucleotides inNeurospora. Curr Genet 12:127–133
Johnson A (1986) Clinical Pharmacology St. Bartholomeus Hospital London EC 1A 7BE.
Kippert F (1987) Endocytobiotic coordination, intracellular calcium signaling, and the origin of endogenous rhythms. Ann NY Acad Sci 503:476–495
Klee CB, Vanaman TC (1982) Calmodulin. Adv Prot Chem 35:213–321
Laemmli HK (1970) Cleavage of structural proteins during the assembling of the head of bacteriophage T4. Nature 227:680–685
Landry J, Crête P, Lamarche S, Chrétien P (1988) Activation of Ca2+-dependent processes during heat shock: Role in cell thermoresistance. Radiation Res 113:426–436
Lettvin JY, Pickard WF, Culloch WS, Pitts W (1964) Theory of passive ion flux through axon membrane. Nature 202:1338–1339
Lonergan TA (1986) A possible second role for calmodulin in biological clock-controlled processes inEuglend. Plant Physiol 82:226–229
McCormack JG, Denton RM (1986) Calcium as second messenger in mitochondria. Trends in Biochem Sci II:258–262
Mine T, Kojima J, Kimura S, Ogata E (1987) Assessment of the role of Ca2+ mobilization from intracellular pool(s), using dantrolene in the glycogenolytic action of and α-adrenergic stimulation in perfused rat liver. Biochim Biophys Acta 927:229–234
Nakashima H (1981) A liquid culture method for the biochemical analysis of the circadian clock inNeurospora crassa. Plant Physiol 69:619–623
Nakashima H (1984) Calcium inhibits phase shifting of the circadian conidiation rhythm ofNeurospora crassa by the calcium ionophore A23187. Plant Physiol 74:268–271
Nakashima J (1986) Phase shifting of the circadian conidiation rhythm inNeurospora crassa by calmodulin antagonists. J Biol Rhythms I:163–169
Ohnishi ST (1987) Effects of halothane, caffeine, dantrolene and tetracaine on the calcium permeability of sceletal sarcoplasmatic reticulum of malignant hyperthermic pigs. Biochim Biophys Acta 897:261–268
Parker Botelho LH, Rothermel JD, Coombs RV, Jastorff B (1988) cAMP analog antagonists of cAMP aciton. Meth Enzymol 159:159–172
Perez RO, Tuinen D van, Marme D, Turian G (1983) Calmodulin-stimulated cyclic nucleotide phosphodiesterase fromNeurospora crassa. Biochim Biophys Acta 758:84–87
Perlman J, Nakashima H, Feldman J (1981) Assay and characteristics of circadian rhythmicity in liquid cultures ofNeurospora crassa. Plant Physiol 67:404–407
Putney JW, Binachi CP (1974) Site of action of dantrolene in frog satorius muscle. J Pharmacol Exper Therap 180:202–212
Raeburn D (1987) Calcium entry blocking drugs: Their classification and sites of action in smooth muscle cells. Medical Biol 65:178–180
Randerath K (1975) Dünnschichtchromotographie. Verlag Chemie, Weinheim.
Rensing L, Schill W (1985) Perturbation by single and double pulses as analytical tools for analyzing oscillatory mechanisms. In: Rensing L, Jaeger N (eds) Temporal order. Springer, Heidelberg
Rensing L, Schill W (1987) Perturbation of cellular circadian rhythms by light and temperature. In: Rensing L, An der Heiden U, Mackey J (eds) Temporal disorder in human oscillatory systems. Springer, Berlin Heidelberg New York
Rensing L, Techel D, Schröder-Lorenz A (1987a) Protein phosphorylation and circadian clock mechanism. In: Hildebrandt G, Moog R, Raschke F (eds) Chronobiology and chronomedicine. P. Lang, Frankfurt
Rensing L, Bos A, Kroeger J, Cornelius G (1987b). Possible links between circadian rhythm and heat shock response inNeurospora crassa. Chronobiol Intern 4:543–549
Richter-Landsberg CH, Jastorff B (1985) In vitro phosphorylation of mictrotubule-associated proteins: differential effects of cyclic AMP analogues. J Neurochem 45:1218–1222
Schröder-Lorenz A, Rensing L (1987) Circadian changes in protein synthesis rate and protein phosphorylation in cell-free extracts ofGonyaulax polyedra. Planta 170:7–13
Schulz R, Pilatus U, Rensing L (1985) On the role of energy metabolism inNeurospora circadian clock function. Chronobiol Intern 2:223–233
Tolbert NE (1974) Isolation of subcellular organelles and metabolites on isopyconic sucrose gradients. Meth Enzymol 31:734–746
Tuinen D van, Perez RO, Marme D, Turian G (1984) Calcium, calmodulin-dependent protein phosphorylation inNeurospora crassa. FEBS Letters 176:317–320
Vaghy PL, Johnson JD, Matlib MA, Wang T, Schwartz A (1982) Selective inhibition of Na2+-induced Ca2+ release from heart mitochondria by diltiazem and certain other Ca2+ antagonist drugs. J Biol Chem 257:6000–6002
Van Haastert PJM, Van Driel R, Jastorff B, Baraniak J, Stec WJ, De Wit RJW (1984) Competitive cAMP antagonists for cAMP-receptor proteins. J Biol Chem 259:10020–10024
Winfree AT (1980) The geometry of biological time. Springer, Berlin Heidelberg New York
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Techel, D., Gebauer, G., Kohler, W. et al. On the role of Ca2+-calmodulin-dependent and cAMP-dependent protein phosphorylation in the circadian rhythm ofNeurospora crassa . J Comp Physiol B 159, 695–706 (1990). https://doi.org/10.1007/BF00691715
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00691715