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. Author manuscript; available in PMC: 2009 May 6.
Published in final edited form as: Nat Cell Biol. 2008 May 25;10(6):665–675. doi: 10.1038/ncb1729

Figure 4.

Figure 4

Phosphorylation of Cdh1 by Cdc28 and Cdc5. (a) Cdc28–Clb2–HA3 and Cdc5–HA3 immunoprecipitated from nocodazole-treated wild-type (WT) cells (US1165, US3259) were used in kinase reactions with GST or GST–Cdh1 as substrates. For the two-step kinase reaction (lanes 6 and 7; see Methods), ‘1’ denotes the first kinase used and ‘2’ denotes the second kinase used after removal of the first kinase. To demonstrate substrate specificities, Cdc28–Clb2 and Cdc5 kinase activities were assayed using histone H1 and casein as substrates. (b) Phosphorylation of GST–Cdh1, GST–Cdh1–m4A and GST–Cdh1–mSps4A by immunoprecipitated Cdc28–Clb2–HA3 and Cdc5–HA3. Designations ‘1’ and ‘2’ in lanes 3, 6 and 9 represent sequential exposure to the two kinases, as described in a. (c) Schematic representation of putative Cdc5 phosphorylation sites in Cdh1. (d) The blot shows sequential phosphorylation of GST–Cdh1, GST–Cdh1–S125A, GST–Cdh1–S259A and GST–Cdh1–S125A S259A by Cdc28–Clb2–HA3 and Cdc5–HA3 kinases.