Figure 5: Functional interaction of recombinant FRQ and CK1a in vitro.

(a) _His6CK1a phosphorylates recombinant FRQ only to a limited extend. Purified recombinant FRQ (100 ng) and _His6CK1a (5 μg) were incubated with γ-³²P ATP for 1 h at 30 °C. The upper panel shows a western blot analysis of FRQ. The lower panels are autoradiograms showing incorporation of ³²P into recombinant FRQ, _His6CK1a (autophosphorylation) and β-casein (CK1a activity control). (b) Hyperphosphorylation of recombinant FRQ by _His6CK1a is dependent on priming kinases supplied by cytosol of Neurospora crassa (N.c.) or S. cerevisiae (S.c.). Recombinant FRQ (100 ng) was incubated in the absence or presence of _His6CK1a (5 μg) with and without cytosol (400 μg) of a N.c. frq¹⁰ (Δfrq) strain or yeast cytosol when indicated. The samples were incubated 1 h at 30 °C. (c) CK1a present in Neurospora cytosol supports hyperphosphorylation of recombinant FRQ at 4 °C but not at 30 °C. Purified FRQ (100 mg) was incubated with frq¹⁰ cytosol for the indicated time periods. (d) CK1a is activated by recruitment to FRQ. _His6CK1a (100 ng) was pre-incubated for 30 min at 4 °C with a four-fold molar excess of recombinant FRQ (1 μg) to allow stable complex formation or with 1 μg BSA to balance the total protein content. Subsequently, the phosphopeptide Ac-KRRRALS(pS)VASLdP(Sox)G-NH (2 μg) (Life Technologies) was incubated at 4 °C with γ-³²P ATP (25 μM) and with either the preformed _His6CK1a-FRQ complex or the corresponding amount of free _His6CK1a (+BSA). Reactions were terminated after the indicated time periods and analysed by native-PAGE and autoradiography (upper panel). FRQ and the peptide were phosphorylated. BSA was not phosphorylated and autophosphorylation of CK1a is not visible at the shown exposure. Lower panel: phosphorylation of the peptide was quantified by densitometry. Error bars indicate s.d. (n=3).