鑑定絲氨酸269磷酸化的第二型水通道蛋白質PDZ Motif和Sipa1l1 Domain之交互作用

Characterization of the Interaction between Serine 269 Phosphorylated Aquaporin-2 PDZ Motif and Sipa1l1 Domain

10.6342/NTU201603085

劉劭萱

細胞頂膜 ； 第二型水通道蛋白質serine 269 磷酸化 ； PDZ domain ； apical plasma membrane ； AQP2 serine 269 phosphorylation ； PDZ domain

臺灣大學生物化學暨分子生物學研究所學位論文

2016年

碩士

余明俊

英文

在腎臟及尿管細胞(collecting duct cells)，抗利尿激素 (vasopressin) 藉由調控第二型水通道蛋白(aquaporin-2, AQP2) 在細胞頂膜 (apical plasma membrane) 上的量來調節身體的水分平衡。先前的研究指出，vasopressin的刺激會促使AQP2的絲氨酸269 (serine 269) 被磷酸化，使得serine 269磷酸化的AQP2大量增加並停留在頂膜上。我們在先前的研究中鑑定了一個會調控AQP2 停留在細胞頂膜的PDZ domain蛋白質─Sipa1l1。Sipa1l1 和serine 269 磷酸化的AQP2 PDZ motif交互作用減少，使serine 269 磷酸化的AQP2 停留在細胞頂膜。為了直接測量和AQP2 PDZ motif間的交互作用，我們將Sipa1l1 PDZ domain 接上glutathione-S-transferase、thioredoxin、6x His以及maltose binding protein進行表現並純化。表面電漿共振 (surface plasmon resonance) 結合實驗結果顯示，和serine 269非磷酸化的AQP2合成胜肽相比，serine 269 磷酸化的AQP2 合成胜肽對Sipa1l1 PDZ domain有較弱結合。Sipa1l1 PDZ domain 與AQP2 PDZ motif結合的模型顯示serine 269 磷酸化造成立體障礙而降低Sipa1l1 PDZ domain與AQP2 PDZ motif之間的交互作用。

Vasopressin regulates the amount of the water channel protein aquaporin-2 (AQP2) in the apical plasma membrane via enhancing exocytosis and/or reducing endocytosis of AQP2-containing vesicles to and from the apical plasma membrane of the kidney collecting duct cells. Previous studies suggest that vasopressin induces AQP2 phosphorylation at serine 269 (S269), which enhances retention of AQP2 in the apical plasma membrane. We previously identified Sipa1l1, a PDZ domain-containing protein that regulates apical retention of serine 269-phosphorylated AQP2. Sipa1l1 reduces its interaction with the serine 269 phosphorylated AQP2 PDZ motif. To directly measure this PDZ interaction, we expressed and purified the Sipa1l1 PDZ domain with fusion tags glutathione-S-transferase, thioredoxin, 6x His, and maltose binding protein. Surface plasmon resonance binding experiments showed weaker binding of the serine 269 phosphorylated AQP2 peptide to the Sipa1l1 PDZ compared with the serine 269 non-phosphorylated AQP2 peptide. Models of the Sipa1l1 PDZ domain-AQP2 PDZ motif complex showed phosphorylation at serine 269 of the AQP2 PDZ domain reduces its interaction with the Sipa1l1 PDZ domain.

1. 1. Nielsen, S., et al., Cellular and subcellular immunolocalization of vasopressin-regulated water channel in rat kidney. Proceedings of the National Academy of Sciences of the United States of America, 1993. 90(24): p. 11663-11667.
   連結：
2. 2. Pisitkun, T., et al., Akt and ERK1/2 pathways are components of the vasopressin signaling network in rat native IMCD. American Journal of Physiology - Renal Physiology, 2008. 295(4): p. F1030-F1043.
   連結：
3. 3. Star, R.A., et al., Calcium and cyclic adenosine monophosphate as second messengers for vasopressin in the rat inner medullary collecting duct. Journal of Clinical Investigation, 1988. 81(6): p. 1879-1888.
   連結：
4. 4. Katsura, T., et al., Protein kinase A phosphorylation is involved in regulated exocytosis of aquaporin-2 in transfected LLC-PK1 cells. American Journal of Physiology - Renal Physiology, 1997. 272(6): p. F817-F822.
   連結：
5. 5. Fushimi, K., S. Sasaki, and F. Marumo, Phosphorylation of Serine 256 Is Required for cAMP-dependent Regulatory Exocytosis of the Aquaporin-2 Water Channel. Journal of Biological Chemistry, 1997. 272(23): p. 14800-14804.
   連結：
6. 6. Xie, L., et al., Quantitative analysis of aquaporin-2 phosphorylation. American Journal of Physiology - Renal Physiology, 2010. 298(4): p. F1018-F1023.
   連結：
7. 7. Lu, H.J., et al., The phosphorylation state of serine 256 is dominant over that of serine 261 in the regulation of AQP2 trafficking in renal epithelial cells. American Journal of Physiology - Renal Physiology, 2008. 295(1): p. F290-F294.
   連結：
8. 8. Hoffert, J.D., et al., Dynamics of aquaporin-2 serine-261 phosphorylation in response to short-term vasopressin treatment in collecting duct. American Journal of Physiology - Renal Physiology, 2007. 292(2): p. F691-F700.
   連結：
9. 9. Hoffert, J.D., et al., Vasopressin-stimulated Increase in Phosphorylation at Ser(269) Potentiates Plasma Membrane Retention of Aquaporin-2. The Journal of Biological Chemistry, 2008. 283(36): p. 24617-24627.
   連結：
10. 10. Moeller, H.B., M.A. Knepper, and R.A. Fenton, Serine 269 phosphorylated aquaporin-2 is targeted to the apical membrane of collecting duct principal cells. Kidney international, 2009. 75(3): p. 295-303.
    連結：
11. 11. Moeller, H.B., et al., Phosphorylation of aquaporin-2 regulates its endocytosis and protein–protein interactions. Proceedings of the National Academy of Sciences, 2010. 107(1): p. 424-429.
    連結：
12. 12. Chetkovich, D.M., et al., Phosphorylation of the postsynaptic density-95 (PSD-95)/discs large/zona occludens-1 binding site of stargazin regulates binding to PSD-95 and synaptic targeting of AMPA receptors. J Neurosci, 2002. 22(14): p. 5791-6.
    連結：
13. 13. Cao, T.T., et al., A kinase-regulated PDZ-domain interaction controls endocytic sorting of the beta2-adrenergic receptor. Nature, 1999. 401(6750): p. 286-90.
    連結：
14. 14. Cohen, N.A., et al., Binding of the inward rectifier K+ channel Kir 2.3 to PSD-95 is regulated by protein kinase A phosphorylation. Neuron, 1996. 17(4): p. 759-67.
    連結：
15. 15. Noda, Y., et al., Aquaporin-2 trafficking is regulated by PDZ-domain containing protein SPA-1. FEBS Letters, 2004. 568(1–3): p. 139-145.
    連結：
16. 16. Brown, B.L., M. Hadley, and R. Page, Heterologous high-level E. coli expression, purification and biophysical characterization of the spine-associated RapGAP (SPAR) PDZ domain. Protein Expr Purif, 2008. 62(1): p. 9-14.
    連結：
17. 17. Sali, A. and T.L. Blundell, Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol, 1993. 234(3): p. 779-815.
    連結：
18. 18. Karthikeyan, S., T. Leung, and J.A.A. Ladias, Structural Determinants of the Na+/H+Exchanger Regulatory Factor Interaction with the β2Adrenergic and Platelet-derived Growth Factor Receptors. Journal of Biological Chemistry, 2002. 277(21): p. 18973-18978.
    連結：
19. 19. London, N., et al., Rosetta FlexPepDock web server--high resolution modeling of peptide-protein interactions. Nucleic Acids Res, 2011. 39(Web Server issue): p. W249-53.
    連結：
20. 20. Kaminski, G.A., et al., Evaluation and Reparametrization of the OPLS-AA Force Field for Proteins via Comparison with Accurate Quantum Chemical Calculations on Peptides. The Journal of Physical Chemistry B, 2001. 105(28): p. 6474-6487.
    連結：
21. 21. Laskowski, R.A., et al., PROCHECK: a program to check the stereochemical quality of protein structures. Journal of Applied Crystallography, 1993. 26(2): p. 283-291.
    連結：
22. 22. Biasini, M., et al., SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Research, 2014. 42(W1): p. W252-W258.
    連結：
23. 23. Kumar, T.A., CFSSP: Chou and Fasman Secondary Structure Prediction server. Wide Spectrum, 2013. 1(9): p. 15-19.
    連結：
24. 24. Jemth, P. and S. Gianni, PDZ Domains:  Folding and Binding. Biochemistry, 2007. 46(30): p. 8701-8708.
    連結：
25. 25. Novak, K.A.P., N. Fujii, and R.K. Guy, Investigation of the PDZ domain ligand binding site using chemically modified peptides. Bioorganic & Medicinal Chemistry Letters, 2002. 12(17): p. 2471-2474.
    連結：
26. 26. Saro, D., et al., Thermodynamic Analysis of a Hydrophobic Binding Site:  Probing the PDZ Domain with Nonproteinogenic Peptide Ligands. Organic Letters, 2004. 6(20): p. 3429-3432.
    連結：
27. 27. Doyle, D.A., et al., Crystal Structures of a Complexed and Peptide-Free Membrane Protein–Binding Domain: Molecular Basis of Peptide Recognition by PDZ. Cell, 1996. 85(7): p. 1067-1076.
    連結：
28. 28. Hoffert, J.D., et al., Quantitative phosphoproteomics of vasopressin-sensitive renal cells: regulation of aquaporin-2 phosphorylation at two sites. Proc Natl Acad Sci U S A, 2006. 103(18): p. 7159-64.
    連結：
29. 29. Nourry, C., S.G. Grant, and J.P. Borg, PDZ domain proteins: plug and play! Sci STKE, 2003. 2003(179): p. Re7.
    連結：