Figure 4: Phosphorylation of FRQ in cell-free extracts.

(a) CK1a-dependent hyperphosphorylation of FRQ in a cell-free system. Native protein extract of light-grown Neurospora expressing heterogeneously phosphorylated FRQ (lane 1) was incubated for 1 h at 30 °C with the indicated amounts of recombinant _His6CK1a. The phosphorylation state of FRQ was dependent on the _His6CK1a concentration. (b) Temperature-dependent interaction of CK1a and FRQ. Native cytosol was prepared from WT and CK1a was immunoprecipitated at 4 and 30 °C. Aliquots of IP and supernatant (S) were analysed by western blot. FRQ and CK1a signals were quantified by densitometry. Error bars indicate s.d. (n=3). FRQ co-immunoprecipitated with higher efficiency at 4 °C than at 30 °C. (c) Temperature dependence of _His6CK1a activity. Recombinant _His6CK1a and β-casein were incubated with γ-³²P ATP for 1 h at 4 and 30 °C, respectively. Phosphorylation of β-casein and autophosphorylation of CK1a was detected by autoradiography and quantified. Error bars indicate s.d. (n=3). (d) Hyperphosphorylation of FRQ in vitro is dependent on CK1a interaction domains (FCDs) even at high _His6CK1a concentration. Protein extracts from cultures expressing WT FRQ and FRQΔFCD1 and FRQΔFCD2, respectively, were incubated overnight at 4 °C in the absence or presence of ATP and purified recombinant _His6CK1a as indicated. The phosphorylation state of FRQ was analysed by immunoblotting. (e) Hyperphosphorylation of FRQ reduces its affinity for CK1a. CK1a was immunoprecipitated overnight at 4 °C from WT cytosol (containing endogenous FRQ and CK1a) in the absence or presence of ATP. Without ATP, FRQ remained heterogeneously phosphorylated and co-immunoprecipitated efficiently with CK1a. With ATP, FRQ was hyperphosphorylated and its association with CK1a was reduced. Total protein extract before incubation (T), the IP and the supernatant (S) are shown. A three-fold excess of IP and S were loaded with respect to T.