We have discovered a family of novel secreted kinases that are key mediators of a variety of cellular processes including differentiation, mineralization, and extracellular signaling. These kinases share little sequence identity with canonical kinases and function extracellularly and/or in the lumen of the secretory pathway. We have determined that Fam20C is the authentic Golgi casein kinase that phosphorylates casein and other secreted proteins involved in mineralization within Ser-x-Glu (S-x-E) motifs. Phosphoproteomic studies have established that greater than 75% of secreted phosphoproteins are phosphorylated on S-x-E motifs, thus making Fam20C an important potential regulator of numerous physiological functions. This proposal is focused on understanding the substrate specificity, evolution, and structure/function of Fam20C.
Specific Aim 1 is an in depth biochemical characterization of Fam20C, including a detailed kinetic analysis and identification of novel substrates.
Specific Aim 2 addresses the evolution and functional conservation of Fam20 kinases in Caenorhabditis elegans and Drosophila melanogaster, two organisms lacking mineralized tissue. Using bioinformatics we have identified potential substrates for C. elegans and Drosophila Fam20 enzymes that will shed new light on the evolutionary origins of secreted protein phosphorylation. The goal of Specific Aim 3 is to solve the crystal structure of Fam20C. We have made significant progress toward obtaining the structure of the C. elegans Fam20C ortholog and have obtained crystals that diffract to 2.6. We propose to solve structures for C. elegans Fam20 complexed with its co-substrates. In addition, we will pursue the structure of Fam20B, a closely related family member that does not phosphorylate S-x-E, but utilizes a distinct proteoglycan substrate. Successful completion of these specific aims will result in a better understanding of this important family of novel secreted kinases.

Public Health Relevance

This project is focused on novel secreted kinases, which add phosphate residues to numerous secreted proteins. These modifications are critical for many biological processes, playing essential roles in development, kidney function, tooth enamel, and bone mineralization.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK018024-45
Application #
9535979
Study Section
Molecular and Integrative Signal Transduction Study Section (MIST)
Program Officer
Silva, Corinne M
Project Start
1991-10-01
Project End
2019-07-31
Budget Start
2018-08-01
Budget End
2019-07-31
Support Year
45
Fiscal Year
2018
Total Cost
Indirect Cost
Name
University of California, San Diego
Department
Pharmacology
Type
Schools of Medicine
DUNS #
804355790
City
La Jolla
State
CA
Country
United States
Zip Code
92093
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Zhang, Hui; Zhu, Qinyu; Cui, Jixin et al. (2018) Structure and evolution of the Fam20 kinases. Nat Commun 9:1218
Qiu, Yimin; Poppleton, Erik; Mekkat, Arya et al. (2018) Enzymatic Phosphorylation of Ser in a Type I Collagen Peptide. Biophys J 115:2327-2335
Pollak, Adam J; Haghighi, Kobra; Kunduri, Swati et al. (2017) Phosphorylation of serine96 of histidine-rich calcium-binding protein by the Fam20C kinase functions to prevent cardiac arrhythmia. Proc Natl Acad Sci U S A 114:9098-9103
Cui, Jixin; Zhu, Qinyu; Zhang, Hui et al. (2017) Structure of Fam20A reveals a pseudokinase featuring a unique disulfide pattern and inverted ATP-binding. Elife 6:
Nguyen, Kim B; Sreelatha, Anju; Durrant, Eric S et al. (2016) Phosphorylation of spore coat proteins by a family of atypical protein kinases. Proc Natl Acad Sci U S A 113:E3482-91
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Zhu, Qinyu; Venzke, David; Walimbe, Ameya S et al. (2016) Structure of protein O-mannose kinase reveals a unique active site architecture. Elife 5:
Guo, Xing; Dixon, Jack E (2016) The 26S proteasome: A cell cycle regulator regulated by cell cycle. Cell Cycle 15:875-6
Cui, Jixin; Xiao, Junyu; Tagliabracci, Vincent S et al. (2015) A secretory kinase complex regulates extracellular protein phosphorylation. Elife 4:e06120

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