On the role of Ca2(+)-calmodulin-dependent and cAMP-dependent protein phosphorylation in the circadian rhythm of Neurospora crassa.

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 of Neurospora 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 degrees 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 (with 35S-thio gamma-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 degrees 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 of Neurospora. Ca2+ calmodulin and the phosphorylation state of the 47/48-kDa proteins may have controlling or essential functions for this mechanism.