Cka1 or Gst
Cka2 kinases phosphorylate only the most C-terminal serines in Cdc34 and tm
Cdc34. (A). Scheme of Cdc34. Red: the six most C-terminal serines. Residue 244 represents the C-terminal end in Cdc34244 that includes the E2 catalytic core (a.a. 1 to 170) with the active site cysteine C95, the E3 RING domain-interacting fragment, and the 39 a.a. C-terminal fragment (a.a. 171-209) also implicated in binding to SCF. (B). In a physiological range of protein concentrations, yeast GstCka1 phosphorylates only the six most C-terminal serines within Cdc34. 1 hour assays included 10 μM ATP with 0.1 μ Ci of [γ-32P]ATP (4500 Ci/mmol), 1 pmol of the indicated Cdc34 constructs and 0.5 or 2.5 pmol of GstCka1. (C). GstCka2 phosphorylates the S207 and S216 most C-terminal serines in Cdc34244 and tmCdc34244. Assays were performed for 1 hour (or as indicated), with 5 pmols (or as indicated) of the indicated constructs, 1pmol of GstCka1 kinase and 10 μM ATP with 5 μCi [32P] ATP (4500Ci/mmol), leading to ~50-fold higher sensitivity of [32P] detection than in B. Graph: quantitation of the [32P] signal of the proteins at different autoradiogram exposure times. CB: Coomassie blue.