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. 2004 Jan 1;18(1):35–47. doi: 10.1101/gad.1159204

Figure 4.

Figure 4.

Rcn1 is phosphorylated by Mck1 and dephosphorylated by calcineurin. (A) The highly conserved central domain of RCNs contains an absolutely conserved GSK-3 consensus phosphorylation site (underscored). (B) Western blot analysis of epitope-tagged Rcn1 or Rcn1S113A obtained from either tcn1 mutants or mck1 tcn1 double mutants after 4 h of growth in YPDS with or without 100 mM CaCl2. Doublet migration of Rcn1 was abolished after disruption of Mck1 or substitution of serine 113 with alanine in the GSK-3 consensus phosphorylation site. (C) Purified recombinant GST-Rcn1 or GST-Rcn1S113A was incubated with [γ-32P]ATP and no kinase, p42 MAP kinase, or purified Mck1, and analyzed by SDS-PAGE and autoradiography. Preincubation of the proteins with nonradioactive ATP plus p42 MAP kinase followed by extensive washing was necessary for phosphorylation of GST-Rcn1 by Mck1 and [γ-32P]ATP. (D) Mck1-phosphorylated GST-Rcn1 was incubated with 0, 5, or 10 units of purified calcineurin plus calmodulin or 20 units of calf intestinal phosphatases and analyzed as in C.