NON-CDK PHOSPHORYLATION OF THE APC ACTIVATOR CDH1

Mark C. Hall*, Erin Warren, and Christoph H. Borchers

Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, CB# 7260 Mary Ellen Jones Building, Chapel Hill, NC 27599

*mark_hall@med.unc.edu

INTRODUCTION.  Cdh1 recruits substrates to the anaphase-promoting complex (APC), an E3 ubiquitin ligase that regulates key cell cycle transitions (1,2).  APC^Cdh1 is active from the end of mitosis through G₁ of the following cell cycle (3).  The activity of APC^Cdh1 is regulated by phosphorylation of Cdh1 by cyclin-dependent kinase (CDK), which inhibits binding of Cdh1 to APC (3,4).  Thus, when CDK activity is high during S and early M, APC^Cdh1 is inactive.  Removal of phosphates from Cdh1 at the end of mitosis allows interaction with APC and results in degradation of cyclins and other proteins to promote mitotic exit.  We identified in vivo phosphorylation sites on yeast Cdh1 by mass spectrometry (MS) and found that additional kinases are involved in its phosphoregulation.

METHODS.  An epitope-tagged Cdh1 was expressed from the ADH1 promoter on a single copy plasmid in a cdc15-2 yeast strain to obtain sufficient protein for MS analysis.  Cdh1 was immunoaffinity purified in the presence of phosphatase inhibitors from cells arrested in late M by temperature shift to 37 °C, in S by addition of 10 mg/ml hydroxyurea, or in G₁ by addition of 50 mg/L a-factor.  Cdh1 was further purified by reverse phase HPLC, and digested extensively with trypsin.  Tryptic peptides were fractionated by HPLC and analyzed by MALDI TOF MS to identify potential Cdh1 phosphopeptides by virtue of an 80 Da mass increase.  Potential phosphopeptides were then sequenced by tandem MS to identify phosphorylation sites.

RESULTS.  Treatment of purified Cdh1 from M-phase yeast with phosphatase and analysis by western blot and MS of full-length protein confirmed the existence of extensive phosphorylation.  MS analysis of Cdh1 tryptic peptides revealed at least 16 phosphorylation sites.  Thirteen phosphorylated amino acids have been positively identified by tandem MS.  The majority of phosphopeptides were detected in Cdh1 from S- and M-arrested yeast.  Several expected CDK consensus sites were phosphorylated.  Unexpectedly, there were numerous non-CDK sites phosphorylated as well.  The phosphorylation sites were clustered in the amino terminal half of the protein, although two phosphorylation sites were identified near the C-terminal end of Cdh1.  Current work is aimed at defining the biological role of the non-CDK phosphorylation sites using site-specific mutants and testing for effects on cellular localization and interaction with APC.

DISCUSSION.  Our results reveal several new insights into the phosphoregulation of Cdh1.  First, phosphorylation of Cdh1 was not limited to consensus CDK sequences (S/T-P-x-K/R or S/T-P).  This suggests that at least one additional kinase is involved in regulating Cdh1 activity in vivo.  The sheer number of phosphorylation sites revealed that Cdh1 is hyperphosphorylated to a greater extent than previously thought, and suggests that a dramatic charge reversal on Cdh1 may act to repel the APC.  Finally, previous work demonstrating that mutation of the consensus CDK sites in Cdh1 eliminates phosphorylation (3,4) implies that CDK phosphorylation may be a prerequisite for phosphorylation by one or more other kinase(s).

REFERENCES.

1. Pfleger, C.M., Lee, E., and Kirschner, M.W. (2001) Genes Dev. 15, 2396-2407.
2. Schwab, M., Neutzer, M., Mocker, D., and Seufert, W. (2001) EMBO J 20, 5165-5175. 
3. Zachariae, W., Schwab, M., Nasmyth, K., Seufert, W. (1998) Science 282, 1721-1724.
4. Jaspersen, S.L., Charles, J.F., and Morgan, D.O. (1999) Curr Biol 9, 227-236.