Introduction

1. Prediction of complex regulatory networks in Saccharomyces cerevisiae Nguyen AN1, Handfield LF1, Claridge C1, Moses AM1. 1Department of Cell & Systems Biology, University of Toronto, Willcocks Street, Toronto, Canada Can CDK1-regulated nucleocytoplasmic shuttling be predicted from protein primary sequence alone? CDC28 clusters Introduction NLStradamus NLStradamus • Prediction of phosphorylation site clusters - targets • Statistical framework (Hidden Markov Model and probabilistic enumeration) • Applicable to multiple models • Prediction of nuclear localization signals (NLSs) • Statistical framework (Hidden Markov Model) • www.moseslab.csb.utoronto.ca/NLStradamus Regulation of protein function by phosphorylation is ubiquitous in living organisms due to the extensive addition of local negative charges on the protein. This allows the protein to modify its structure or to modulate its interactions with other proteins [1]. It has been observed that certain proteins have clusters of phosphorylation sites that regulate the function of the protein [2] and one hypothesis concerning these clusters is that they serve to regulate other regulatory motifs nearby [3,4]. We focus our attention here on the regulation of nuclear localization signals (NLSs) by clusters of phosphorylation sites where the addition of phosphate groups may mask or uncover the signal. This clustering effect for NLS regulation has been observed on a few proteins such as SWI5 [5] where multiple CDC28 phosphorylation sites near the NLS regulate localization of the protein through the cell cycle. Here we sought to test if this hypothesis could be computationally predicted by combining multiple motif predictors and using a statistical method. Example Case : MCM3 Example Case : MCM3 Protein Sequence Protein Sequence a Background a b Protein NLS Nucleus Phosphorylation Figure 2. NLStradamus nuclear localization signal prediction of MCM3 Posterior trace of Mcm3p, a characterized NLS, using our two-state model. The peak corresponds to the true characterized NLS. b Figure 1. Example of known cases of regulated nuclear import a) MCM3 localizes to the nucleus in a cell cycle regulated manner. Picture shows MCM2-GFP, part of the MCM complex. b) Schematic of our hypothesis derived from known cases. Results b c d a c Ndd1p Yox1p Msa2p Figure 3. CDC28 clustering model pipeline a) Posterior trace of the putative CDC28 sites on the protein. b) Statistical analysis of the site positions and probability. c) Posterior trace of the clustering modelling from the various phosphorylation sites. Figure 4. Results from the model a) Posterior trace of the model on the example case MCM3. b) Localization of NDD1-GFP, a candidate protein, on unsynchronized cells. c) Localization of YOX1-GFP, a candidate protein, on unsynchronized cells. d) Localization of MSA2-GFP, a candidate protein, on unsynchronized cells. Acknowledgements We thank members of Nicholas Provart’s lab for providing us with valuable inputs during the beginning of the project, as well as providing us the web server for NLStradamus. We also thank Yolanda Chong in Brenda Andrews’ lab for providing us with the GFP images of their array. • References • 1) Holmberg CI, Tran SE, Eriksson JE, Sistonen L. Multisite phosphorylation provides sophisticated regulation of transcription factors. Trends Biochem Sci. 2002 Dec;27(12):619-27. 2) Traven A, Heierhorst J. SQ/TQ cluster domains: concentrated ATM/ATR kinase phosphorylation site regions in DNA-damage-response proteins. Bioessays. 2005 Apr;27(4):397-407. 3) JansDA, Hübner S. Regulation of protein transport to the nucleus: central role of phosphorylation. Physiol Rev. 1996 Jul;76(3):651-85.4) HarremanMT, Kline TM, Milford HG, Harben MB, Hodel AE, Corbett AH. Regulation of nuclear import by phosphorylation adjacent to nuclear localization signals.J Biol Chem. 2004 May 14;279(20):20613-21. Epub 2004 Mar 3. 5) JansDA, Moll T, Nasmyth K, Jans P. Cyclin-dependent kinase site-regulated signal-dependent nuclear localization of the SW15 yeast transcription factor in mammalian cells. J Biol Chem. 1995 Jul 21;270(29):17064-7.