Biochemistry; Biotechnology; Hypertension; Molecular Biology; Phosphoproteins; Proteomics
Cellular & Molecular Physiology: Rinehart Lab
Our laboratory is focused on the discovery and characterization of novel sites of phosphorylation in kinases and regulatory networks of proteins that control electrolyte homeostasis. Our long term goal is to understand and “decode” complex signaling networks in physiological systems. Our research integrates cutting edge proteomics with mainstay techniques of molecular biology and physiology, to study signaling networks and provide critical new insight into regulated ion transport. We are focusing on mechanisms of cellular signaling transduction that involve protein phosphorylation. Many of the cellular signaling mechanisms that are disrupted in disease states, and are assumed to be fundamental for normal cellular physiology, are largely uncharacterized and hold a vast amount of therapeutic potential. Understanding the human phosphoproteome is a major challenge as research continues ever farther into the post genome era. We therefore employ established quantitative proteomic techniques, and aim to develop novel methods, in an effort to decode new signaling pathways. We aim to find new roles for kinases, phosphatases, their substrates, and protein-protein interactions on a system-wide level. Collectively, we hope these efforts will identify molecular mechanisms important for both healthy and disease states in humans.
Tens of thousands of phosphorylation sites in diverse eukaryotic proteins have been identified through large scale mass spectrometry studies. For the great majority of these sites, however, the responsible kinase is unknown, and the functional role of phosphorylation is not understood. We have recently made substantial progress towards narrowing this critical gap in knowledge with a new technology that enables site-specific incorporation of phosphoserine into proteins. This technology utilizes an E. coli strain with an expanded genetic code and contains a dedicated sense codon for phosphoserine. We use this breakthrough technology to synthesize human phosphoproteins and accelerate our efforts in “decoding” the human phosphoproteome.
Specialized Terms: Physiological Systems; Protein Phosphorylation; Cell Signaling; Phosphoproteomics; Protein Engineering; Ion Transport; Synthetic Biology; Translational Research
Learn more at http://rinehart.commons.yale.edu/
Evolution of translation machinery in recoded bacteria enables multi-site incorporation of nonstandard amino acids.
Amiram M, Haimovich AD, Fan C, Wang Y-S, Aerni H-R, Ioanna N, Moonan DW, Ma NJ, Rovner AJ, Hong SH, Kelleher NL, Goodman AL, Jewett MC, Soll D,Rinehart J, Isaacs FJ. Nature Biotechnology. Published online 16 November 2015. doi:10.1038/nbt.3372. 2015
A flexible codon in genomically recoded Escherichia coli permits programmable protein phosphorylation.
Pirman NL, Barber KW, Ma NJ, Haimovich AD, Rogulina S, Isaacs FJ, and Rinehart J. Nature Communications. 2015. 6, 8130. 2015
Robust Production of Recombinant Phosphoproteins Using Cell-Free Protein Synthesis.
Oza JP, Aerni HR, Pirman NL, Rogulina S, ter Haar CM, Isaacs FJ, Rinehart J, and Jewett MC. Nature Communications 2015. 6, 8168 2015
The PINK1-PARKIN Mitochondrial Ubiquitylation Pathway Drives a Program of OPTN/NDP52 Recruitment and TBK1 Activation to Promote Mitophagy.
Heo JM, Ordureau A, Paulo JA, Rinehart J, Harper JW. Molecular Cell. 2015. Sep 9. pii: S1097-2765(15)00662-0. doi: 10.1016/j.molcel.2015.08.016. [Epub ahead of print]. 2015
Genomically recoded organisms expand biological functions.
Lajoie MJ, Rovner AJ, Goodman DB, Aerni HR, Haimovich AD, Kuznetsov G, Mercer JA, Wang HH, Carr PA, Mosberg JA, Rohland N, Schultz PG, Jacobson JM, Rinehart J, Church GM, Isaacs FJ. Genomically recoded organisms expand biological functions. Science (New York, N.Y.) 2013, 342:357-60. 2013
Expanding the genetic code of Escherichia coli with phosphoserine.
Park HS, Hohn MJ, Umehara T, Guo LT, Osborne EM, Benner J, Noren CJ, Rinehart J, Söll D. Expanding the genetic code of Escherichia coli with phosphoserine. Science (New York, N.Y.) 2011, 333:1151-4. 2011
Sites of regulated phosphorylation that control K-Cl cotransporter activity.
Rinehart J, Maksimova YD, Tanis JE, Stone KL, Hodson CA, Zhang J, Risinger M, Pan W, Wu D, Colangelo CM, Forbush B, Joiner CH, Gulcicek EE, Gallagher PG, Lifton RP. Sites of regulated phosphorylation that control K-Cl cotransporter activity. Cell 2009, 138:525-36. 2009
Mineralocorticoid receptor phosphorylation regulates ligand binding and renal response to volume depletion and hyperkalemia.
Shibata S, Rinehart J, Zhang J, Moeckel G, Castañeda-Bueno M, Stiegler AL, Boggon TJ, Gamba G, Lifton RP. Mineralocorticoid receptor phosphorylation regulates ligand binding and renal response to volume depletion and hyperkalemia. Cell Metabolism 2013, 18:660-71. 2013
Full List of PubMed Publications
- Ma NJ, Hemez CF, Barber KW, Rinehart J, Isaacs FJ: Organisms with alternative genetic codes resolve unassigned codons via mistranslation and ribosomal rescue. Elife. 2018 Oct 30; 2018 Oct 30. PMID: 30375330
- Gassaway BM, Petersen MC, Surovtseva YV, Barber KW, Sheetz JB, Aerni HR, Merkel JS, Samuel VT, Shulman GI, Rinehart J: PKCε contributes to lipid-induced insulin resistance through cross talk with p70S6K and through previously unknown regulators of insulin signaling. Proc Natl Acad Sci U S A. 2018 Sep 18; 2018 Sep 4. PMID: 30181290
- Barber KW, Miller CJ, Jun JW, Lou HJ, Turk BE, Rinehart J: Kinase Substrate Profiling Using a Proteome-wide Serine-Oriented Human Peptide Library. Biochemistry. 2018 Aug 7; 2018 Jun 19. PMID: 29920078
- Barber KW, Muir P, Szeligowski RV, Rogulina S, Gerstein M, Sampson JR, Isaacs FJ, Rinehart J: Encoding human serine phosphopeptides in bacteria for proteome-wide identification of phosphorylation-dependent interactions. Nat Biotechnol. 2018 Aug; 2018 Jun 11. PMID: 29889213
- Thomas Y, Scott DC, Kristariyanto YA, Rinehart J, Clark K, Cohen P, Kurz T: The NEDD8 E3 ligase DCNL5 is phosphorylated by IKK alpha during Toll-like receptor activation. PLoS One. 2018; 2018 Jun 29. PMID: 29958295
- Barber KW, Rinehart J: The ABCs of PTMs. Nat Chem Biol. 2018 Feb 14. PMID: 29443972
- Mohler K, Aerni HR, Gassaway B, Ling J, Ibba M, Rinehart J: MS-READ: Quantitative measurement of amino acid incorporation. Biochim Biophys Acta Gen Subj. 2017 Nov; 2017 Jan 24. PMID: 28130155
- D'Lima NG, Khitun A, Rosenbloom AD, Yuan P, Gassaway BM, Barber KW, Rinehart J, Slavoff SA: Comparative Proteomics Enables Identification of Nonannotated Cold Shock Proteins in E. coli. J Proteome Res. 2017 Oct 6; 2017 Sep 19. PMID: 28861998
- Fan Y, Evans CR, Barber KW, Banerjee K, Weiss KJ, Margolin W, Igoshin OA, Rinehart J, Ling J: Heterogeneity of Stop Codon Readthrough in Single Bacterial Cells and Implications for Population Fitness. Mol Cell. 2017 Sep 7; 2017 Aug 3. PMID: 28781237
- Mohler K, Mann R, Bullwinkle TJ, Hopkins K, Hwang L, Reynolds NM, Gassaway B, Aerni HR, Rinehart J, Polymenis M, Faull K, Ibba M: Editing of misaminoacylated tRNA controls the sensitivity of amino acid stress responses in Saccharomyces cerevisiae. Nucleic Acids Res. 2017 Apr 20. PMID: 28168297
- Park HB, Perez CE, Barber KW, Rinehart J, Crawford JM: Genome mining unearths a hybrid nonribosomal peptide synthetase-like-pteridine synthase biosynthetic gene cluster. Elife. 2017 Mar 15; 2017 Mar 15. PMID: 28431213
- Castañeda-Bueno M, Arroyo JP, Zhang J, Puthumana J, Yarborough O 3rd, Shibata S, Rojas-Vega L, Gamba G, Rinehart J, Lifton RP: Phosphorylation by PKC and PKA regulate the kinase activity and downstream signaling of WNK4. Proc Natl Acad Sci U S A. 2017 Jan 31; 2017 Jan 17. PMID: 28096417
- Padovano V, Kuo IY, Stavola LK, Aerni HR, Flaherty BJ, Chapin HC, Ma M, Somlo S, Boletta A, Ehrlich BE, Rinehart J, Caplan MJ: The polycystins are modulated by cellular oxygen-sensing pathways and regulate mitochondrial function. Mol Biol Cell. 2017 Jan 15; 2016 Nov 23. PMID: 27881662
- Barber KW, Rinehart J: Expression of Recombinant Phosphoproteins for Signal Transduction Studies. Methods Mol Biol. 2017. PMID: 28730473
- Ferdaus MZ, Barber KW, López-Cayuqueo KI, Terker AS, Argaiz ER, Gassaway BM, Chambrey R, Gamba G, Rinehart J, McCormick JA: SPAK and OSR1 play essential roles in potassium homeostasis through actions on the distal convoluted tubule. J Physiol. 2016 Sep 1; 2016 May 29. PMID: 27068441
- Amiram M, Haimovich AD, Fan C, Wang YS, Aerni HR, Ntai I, Moonan DW, Ma NJ, Rovner AJ, Hong SH, Kelleher NL, Goodman AL, Jewett MC, Söll D, Rinehart J, Isaacs FJ: Evolution of translation machinery in recoded bacteria enables multi-site incorporation of nonstandard amino acids. Nat Biotechnol. 2015 Dec; 2015 Nov 16. PMID: 26571098
- Heo JM, Ordureau A, Paulo JA, Rinehart J, Harper JW: The PINK1-PARKIN Mitochondrial Ubiquitylation Pathway Drives a Program of OPTN/NDP52 Recruitment and TBK1 Activation to Promote Mitophagy. Mol Cell. 2015 Oct 1; 2015 Sep 10. PMID: 26365381
- Oza JP, Aerni HR, Pirman NL, Barber KW, ter Haar CM, Rogulina S, Amrofell MB, Isaacs FJ, Rinehart J, Jewett MC: Robust production of recombinant phosphoproteins using cell-free protein synthesis. Nat Commun. 2015 Sep 9; 2015 Sep 9. PMID: 26350765
- Pirman NL, Barber KW, Aerni HR, Ma NJ, Haimovich AD, Rogulina S, Isaacs FJ, Rinehart J: A flexible codon in genomically recoded Escherichia coli permits programmable protein phosphorylation. Nat Commun. 2015 Sep 9; 2015 Sep 9. PMID: 26350500
- Hoesl MG, Oehm S, Durkin P, Darmon E, Peil L, Aerni HR, Rappsilber J, Rinehart J, Leach D, Söll D, Budisa N: Chemical Evolution of a Bacterial Proteome. Angew Chem Int Ed Engl. 2015 Aug 17; 2015 Jul 1. PMID: 26136259
- Ordureau A, Heo JM, Duda DM, Paulo JA, Olszewski JL, Yanishevski D, Rinehart J, Schulman BA, Harper JW: Defining roles of PARKIN and ubiquitin phosphorylation by PINK1 in mitochondrial quality control using a ubiquitin replacement strategy. Proc Natl Acad Sci U S A. 2015 May 26; 2015 May 12. PMID: 25969509
- Lin DH, Yue P, Yarborough O 3rd, Scholl UI, Giebisch G, Lifton RP, Rinehart J, Wang WH: Src-family protein tyrosine kinase phosphorylates WNK4 and modulates its inhibitory effect on KCNJ1 (ROMK). Proc Natl Acad Sci U S A. 2015 Apr 7; 2015 Mar 24. PMID: 25805816
- Colangelo CM, Ivosev G, Chung L, Abbott T, Shifman M, Sakaue F, Cox D, Kitchen RR, Burton L, Tate SA, Gulcicek E, Bonner R, Rinehart J, Nairn AC, Williams KR: Development of a highly automated and multiplexed targeted proteome pipeline and assay for 112 rat brain synaptic proteins. Proteomics. 2015 Apr; 2015 Feb 12. PMID: 25476245
- Rovner AJ, Haimovich AD, Katz SR, Li Z, Grome MW, Gassaway BM, Amiram M, Patel JR, Gallagher RR, Rinehart J, Isaacs FJ: Recoded organisms engineered to depend on synthetic amino acids. Nature. 2015 Feb 5; 2015 Jan 21. PMID: 25607356
- Colangelo CM, Shifman M, Cheung KH, Stone KL, Carriero NJ, Gulcicek EE, Lam TT, Wu T, Bjornson RD, Bruce C, Nairn AC, Rinehart J, Miller PL, Williams KR: YPED: an integrated bioinformatics suite and database for mass spectrometry-based proteomics research. Genomics Proteomics Bioinformatics. 2015 Feb; 2015 Feb 21. PMID: 25712262
- Aerni HR, Shifman MA, Rogulina S, O'Donoghue P, Rinehart J: Revealing the amino acid composition of proteins within an expanded genetic code. Nucleic Acids Res. 2015 Jan; 2014 Nov 5. PMID: 25378305
- O'Donoghue P, Prat L, Kucklick M, Schäfer JG, Riedel K, Rinehart J, Söll D, Heinemann IU: Reducing the genetic code induces massive rearrangement of the proteome. Proc Natl Acad Sci U S A. 2014 Dec 2; 2014 Nov 17. PMID: 25404328
- Sawyer N, Gassaway BM, Haimovich AD, Isaacs FJ, Rinehart J, Regan L: Designed phosphoprotein recognition in Escherichia coli. ACS Chem Biol. 2014 Nov 21; 2014 Oct 6. PMID: 25272187
- Steinfeld JB, Aerni HR, Rogulina S, Liu Y, Rinehart J: Expanded cellular amino acid pools containing phosphoserine, phosphothreonine, and phosphotyrosine. ACS Chem Biol. 2014 May 16; 2014 Mar 27. PMID: 24646179
- Shibata S, Rinehart J, Zhang J, Moeckel G, Castañeda-Bueno M, Stiegler AL, Boggon TJ, Gamba G, Lifton RP: Mineralocorticoid receptor phosphorylation regulates ligand binding and renal response to volume depletion and hyperkalemia. Cell Metab. 2013 Nov 5. PMID: 24206662
- Prat L, Heinemann IU, Aerni HR, Rinehart J, O'Donoghue P, Söll D: Carbon source-dependent expansion of the genetic code in bacteria. Proc Natl Acad Sci U S A. 2012 Dec 18; 2012 Nov 26. PMID: 23185002
- Ling J, Cho C, Guo LT, Aerni HR, Rinehart J, Söll D: Protein aggregation caused by aminoglycoside action is prevented by a hydrogen peroxide scavenger. Mol Cell. 2012 Dec 14; 2012 Oct 30. PMID: 23122414
- Heinemann IU, Rovner AJ, Aerni HR, Rogulina S, Cheng L, Olds W, Fischer JT, Söll D, Isaacs FJ, Rinehart J: Enhanced phosphoserine insertion during Escherichia coli protein synthesis via partial UAG codon reassignment and release factor 1 deletion. FEBS Lett. 2012 Oct 19; 2012 Sep 13. PMID: 22982858
- Rinehart J, Vázquez N, Kahle KT, Hodson CA, Ring AM, Gulcicek EE, Louvi A, Bobadilla NA, Gamba G, Lifton RP: WNK2 kinase is a novel regulator of essential neuronal cation-chloride cotransporters. J Biol Chem. 2011 Aug 26; 2011 Jul 6. PMID: 21733846
- Park HS, Hohn MJ, Umehara T, Guo LT, Osborne EM, Benner J, Noren CJ, Rinehart J, Söll D: Expanding the genetic code of Escherichia coli with phosphoserine. Science. 2011 Aug 26. PMID: 21868676
- Zhang L, Jouret F, Rinehart J, Sfakianos J, Mellman I, Lifton RP, Young LH, Caplan MJ: AMP-activated protein kinase (AMPK) activation and glycogen synthase kinase-3β (GSK-3β) inhibition induce Ca2+-independent deposition of tight junction components at the plasma membrane. J Biol Chem. 2011 May 13; 2011 Mar 7. PMID: 21383016
- Monette MY, Rinehart J, Lifton RP, Forbush B: Rare mutations in the human Na-K-Cl cotransporter (NKCC2) associated with lower blood pressure exhibit impaired processing and transport function. Am J Physiol Renal Physiol. 2011 Apr; 2011 Jan 5. PMID: 21209010
- Rinehart J, Gulcicek EE, Joiner CH, Lifton RP, Gallagher PG: Determinants of erythrocyte hydration. Curr Opin Hematol. 2010 May. PMID: 20182354
- Rinehart J, Maksimova YD, Tanis JE, Stone KL, Hodson CA, Zhang J, Risinger M, Pan W, Wu D, Colangelo CM, Forbush B, Joiner CH, Gulcicek EE, Gallagher PG, Lifton RP: Sites of regulated phosphorylation that control K-Cl cotransporter activity. Cell. 2009 Aug 7. PMID: 19665974
- Ring AM, Leng Q, Rinehart J, Wilson FH, Kahle KT, Hebert SC, Lifton RP: An SGK1 site in WNK4 regulates Na+ channel and K+ channel activity and has implications for aldosterone signaling and K+ homeostasis. Proc Natl Acad Sci U S A. 2007 Mar 6; 2007 Feb 22. PMID: 17360471
- Ring AM, Cheng SX, Leng Q, Kahle KT, Rinehart J, Lalioti MD, Volkman HM, Wilson FH, Hebert SC, Lifton RP: WNK4 regulates activity of the epithelial Na+ channel in vitro and in vivo. Proc Natl Acad Sci U S A. 2007 Mar 6; 2007 Feb 26. PMID: 17360470
- Kahle KT, Rinehart J, Ring A, Gimenez I, Gamba G, Hebert SC, Lifton RP: WNK protein kinases modulate cellular Cl- flux by altering the phosphorylation state of the Na-K-Cl and K-Cl cotransporters. Physiology (Bethesda). 2006 Oct. PMID: 16990453
- Leng Q, Kahle KT, Rinehart J, MacGregor GG, Wilson FH, Canessa CM, Lifton RP, Hebert SC: WNK3, a kinase related to genes mutated in hereditary hypertension with hyperkalaemia, regulates the K+ channel ROMK1 (Kir1.1). J Physiol. 2006 Mar 1; 2005 Dec 15. PMID: 16357011
- Rinehart J, Kahle KT, de Los Heros P, Vazquez N, Meade P, Wilson FH, Hebert SC, Gimenez I, Gamba G, Lifton RP: WNK3 kinase is a positive regulator of NKCC2 and NCC, renal cation-Cl- cotransporters required for normal blood pressure homeostasis. Proc Natl Acad Sci U S A. 2005 Nov 15; 2005 Nov 7. PMID: 16275913
- Kahle KT, Rinehart J, de Los Heros P, Louvi A, Meade P, Vazquez N, Hebert SC, Gamba G, Gimenez I, Lifton RP: WNK3 modulates transport of Cl- in and out of cells: implications for control of cell volume and neuronal excitability. Proc Natl Acad Sci U S A. 2005 Nov 15; 2005 Nov 7. PMID: 16275911
- Rinehart J, Krett B, Rubio MA, Alfonzo JD, Söll D: Saccharomyces cerevisiae imports the cytosolic pathway for Gln-tRNA synthesis into the mitochondrion. Genes Dev. 2005 Mar 1; 2005 Feb 10. PMID: 15706032
- Rinehart J, Horn EK, Wei D, Soll D, Schneider A: Non-canonical eukaryotic glutaminyl- and glutamyl-tRNA synthetases form mitochondrial aminoacyl-tRNA in Trypanosoma brucei. J Biol Chem. 2004 Jan 9; 2003 Oct 16. PMID: 14563839
- Rosas-Sandoval G, Ambrogelly A, Rinehart J, Wei D, Cruz-Vera LR, Graham DE, Stetter KO, Guarneros G, Söll D: Orthologs of a novel archaeal and of the bacterial peptidyl-tRNA hydrolase are nonessential in yeast. Proc Natl Acad Sci U S A. 2002 Dec 24; 2002 Dec 10. PMID: 12475929