Scott Rusin Kettenbach Lab NERIC 2017 August 18th 2017 Identification of Candidate CK2 Substrates in Mitosis by Quantitative Phosphoproteomics Scott Rusin Kettenbach Lab NERIC 2017 August 18th 2017

P ¾ of human proteins are phosphorylated Sharma et al., 2014

~500 human protein kinases ¾ of human proteins are phosphorylated P Manning et al., 2002; Sharma et al., 2014

majority of dephosphorylation ~500 human protein kinases ¾ of human proteins are phosphorylated 7 phosphoprotein phosphatases Manning et al., 2002; Brautigan, 2012; Sharma et al., 2014

Kinases and Phosphatases in Mitosis CDK1 Plk1 Aurora A Aurora A/B Aurora B PP2A/PP1/PP6 PP2A PP1 PP1/ Prophase PP6 Prometaphase Interphase Add PP6 Metaphase Cytokinesis Anaphase Telophase

CK2 Rabalski et al, 2016

CK2 in Mitosis

CK2i Screen Experimental Design B B’ A A’ m/z

CK2i Screen Results Phosphopeptides identified: 30,345 Phosphoproteins identified: 5,060 Phosphopeptides quantified: 24,366 Phosphopeptides downregulated: 330 Phosphopeptides upregulated: 150

CK2i Screen Results

In vitro Kinase Assay Validation

Mitotic CK2 Substrate Network Analysis

Mitotic CK2 Interaction Analysis

Mitotic CK2 Interaction Network Analysis

PP6 Substrate Screen Results AURKA Consensus Motif ~ 400 p-sites increased Add legends for colors Rusin et al., 2015

Coupled CK2/PP6 IVKA/IVPA

Coupled CK2/PP6 IVKA/IVPA

CK2 Effects on Chromosome Condensation

CK2 Effects on Chromosome Condensation

Our Proposed Model Modified from Takemoto et al, EMBO, 2006

Conclusions Using mass spectrometry based phosphoproteomics we identified 330 potential substrates for CK2 in mitotic HeLa cells AP-MS revealed 315 interactors in mitotic HEK-293T cells across the 3 CK2 subunits Several substrates confirmed as both targets of CK2 and an opposing phosphatase PP6 in vitro We identify multiple potential CK2 sites on condensin I members, and show a role for CK2 activity as a negative regulator of chromosome condensation in human cells Could be substrates of pp6 regulates kinases

Acknowledgements Kettenbach Lab Thesis Committee Arminja Kettenbach Scott Lyons Brooke Brauer Isha Nasa Mark Adamo Jack Sears Gerber Lab Scott Gerber Katelyn Cassidy Andrew Grassetti Rufus Hards Thesis Committee Jamie Moseley Duane Compton R35 GM119455 NIGMS MIRA P20 GM113132 NIGMS COBRE V2016-022 V FoundationMCBD Training Grant #4T32GM008704-19 http://kettenbachlab.org/