Protein phosphorylation is a ubiquitous post-translational modification used to control central functions of life including cell division, cell growth and cell death. There are 518 human protein kinases and thousands of protein phosphorylation sites identified to date in the human proteome. Multiple complementary methods exist for forward mapping of kinase signaling pathways starting with a specific kinase and searching for downstream substrates. No methods exist for determining which kinase is responsible for a specific phosphorylation event. We term this approach mapping kinase pathways in reverse. The absence of a method to address this challenge has created a fundamental gap in our understanding of kinase networks. How many different upstream kinases are capable of phosphorylating a given serine? What degree of crosstalk exists at the level of a single phosphorylation site? In this proposal we focus on the development of a chemical method which will allow for the identification of the kinase or kinases responsible for specific serine or threonine phosphorylation events in mammalian cells. We propose to identify the kinases which phosphorylate Glycogen Synthase Kinase- 32 (GSK-32) at it's inhibitory Ser-9 residue, a subject of much study and debate in multiple biological contexts from metabolism (insulin signaling) to neuropsychiatric disorders. Recent studies have revealed the presence of hypo-phosphorylated GSK-32 in schizophrenic patients and in animal models. Furthermore, several drugs which are used to treat schizophrenia cause an increase in phosphorylation of Ser9 of GSK-32, by unknown kinases. We propose to identify the kinases responsible for phosphorylation of GSK-32 under different cell stimulation conditions through development of a kinase-substrate selective crosslinking reaction capable of covalently crosslinking GSK-32 to its upstream kinases allowing their identification using traditional affinity isolation and mass spectrometry. The crosslinking reaction we propose involves a three-component chemical reaction between a catalytically essential lysine residue conserved in all kinases, a cysteine engineered at Ser-9 of GSK-32, and an ATP analog bearing a functional group, o-pthalaldehyde, capable of chemoselectively reacting with lysine and cysteine moieties. The proposal first focuses on chemical design and optimization of the crosslinking reaction and then its application to identification of the kinases activated by diverse cellular stimuli which cause phosphorylation of Ser- 9 of GSK-32.

Public Health Relevance

A chemical method for mapping protein kinase substrate interactions in a fundamentally new way from current approaches is proposed. The method will be applied to identification of the kinases responsible for phosphorylation and inhibition of glycogen synthase kinase 32, (GSK-32) an enzyme which is hyperactivated in a number of important human disorders including schizophrenia and bi- polar disorder. These diseases affect approximately 5.7 million American adults or about 2.6 percent of the population age 18 and older. A method for understanding the signaling defects in these diseases will potentially lead to new drugs which are safer and have fewer side effects than current therapies.

Agency
National Institute of Health (NIH)
Institute
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Type
Research Project (R01)
Project #
2R01EB001987-14
Application #
7623784
Study Section
Special Emphasis Panel (ZRG1-BCMB-B (90))
Program Officer
Hunziker, Rosemarie
Project Start
1996-05-01
Project End
2013-03-31
Budget Start
2009-05-01
Budget End
2010-04-30
Support Year
14
Fiscal Year
2009
Total Cost
$278,100
Indirect Cost
Name
University of California San Francisco
Department
Pharmacology
Type
Schools of Medicine
DUNS #
094878337
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Ultanir, Sila K; Yadav, Smita; Hertz, Nicholas T et al. (2014) MST3 kinase phosphorylates TAO1/2 to enable Myosin Va function in promoting spine synapse development. Neuron 84:968-82
Riel-Mehan, Megan M; Shokat, Kevan M (2014) A crosslinker based on a tethered electrophile for mapping kinase-substrate networks. Chem Biol 21:585-90
Ducker, G S; Atreya, C E; Simko, J P et al. (2014) Incomplete inhibition of phosphorylation of 4E-BP1 as a mechanism of primary resistance to ATP-competitive mTOR inhibitors. Oncogene 33:1590-600
Tan, Ying Xim; Manz, Boryana N; Freedman, Tanya S et al. (2014) Inhibition of the kinase Csk in thymocytes reveals a requirement for actin remodeling in the initiation of full TCR signaling. Nat Immunol 15:186-94
Warkentin, Alexander A; Lopez, Michael S; Lasater, Elisabeth A et al. (2014) Overcoming myelosuppression due to synthetic lethal toxicity for FLT3-targeted acute myeloid leukemia therapy. Elife 3:
Zhang, Chao; Lopez, Michael S; Dar, Arvin C et al. (2013) Structure-guided inhibitor design expands the scope of analog-sensitive kinase technology. ACS Chem Biol 8:1931-8
Kaasik, Krista; Kivimäe, Saul; Allen, Jasmina J et al. (2013) Glucose sensor O-GlcNAcylation coordinates with phosphorylation to regulate circadian clock. Cell Metab 17:291-302
Lopez, Michael S; Choy, Jonathan W; Peters, Ulf et al. (2013) Staurosporine-derived inhibitors broaden the scope of analog-sensitive kinase technology. J Am Chem Soc 135:18153-9
Schachter, Miriam Merzel; Merrick, Karl A; Larochelle, Stephane et al. (2013) A Cdk7-Cdk4 T-loop phosphorylation cascade promotes G1 progression. Mol Cell 50:250-60
Hertz, Nicholas T; Berthet, Amandine; Sos, Martin L et al. (2013) A neo-substrate that amplifies catalytic activity of parkinson's-disease-related kinase PINK1. Cell 154:737-47

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