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Peer-reviewed

Non-radioactive LATS in vitro Kinase Assay

非放射法体外测定LATs激酶活性

AH Audrey W. Hong
KG Kun-Liang Guan

发布: 2017年07月20日第7卷第14期 DOI: 10.21769/BioProtoc.2391 浏览次数: 11697

评审: Ralph BottcherHsin-Yi ChangAnonymous reviewer(s)

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Abstract

This protocol describes a method to directly measure LATS activity by an in vitro kinase assay using YAP as a substrate.

Keywords: LATS (LATS（大肿瘤抑制因子）) search Phosphorylation (磷酸化) search in vitro kinase assay (体外激酶活性检测) search Hippo pathway (Hippo信号通路) search YAP (YAP) search

Background

Large tumor suppressor 1/2 (LATS1/2) are protein kinases and core components of the Hippo pathway, which regulates organ size and tissue homeostasis. LATS kinase is activated by phosphorylation on its hydrophobic motif (HM, Thr 1079 for LATS1 and Thr 1041 for LATS2). As a result, Western blotting with phosphoantibody recognizing LATS at HM provides an indirect way to assess LATS kinase activity (Data analysis, Figure 1). In addition, active LATS phosphorylates and inhibits the transcription co-activator Yes-associated protein (YAP) at Ser 127, leading to YAP binding to 14-3-3 and cytoplasmic retention (Zhao et al., 2007). Using YAP as a substrate in LATS in vitro kinase assay provides a method to directly assess LATS kinase activity. Through this assay, we were able to show that serum starvation and sorbitol-induced osmotic activate LATS (Yu et al., 2012; Hong et al., 2017) and further lead to YAP Ser 127 phosphorylation (Data analysis, Figure 2).

Materials and Reagents

  1. Pipette tips
  2. 10 cm plates
  3. 1.5 ml Eppendorf tube
  4. Dialyzer (EMD Millipore, catalog number: 71507-3 )
  5. pGEX-KG-GST-YAP plasmid (Addgene, catalog number: 33052 )
  6. BL21(DE3) competent cells (Agilent Technologies, catalog number: 230134 )
  7. HEK293A cells
  8. LB broth (Fisher Scientific, catalog number: BP1426-2 )
  9. Carbenicillin disodium salt (Sigma-Aldrich, catalog number: 205805-250MG )
  10. Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Sigma-Aldrich, catalog number: I5502 )
  11. Phosphate buffered saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010049 )
  12. Protease inhibitor cocktail tablet (Roche Diagnostics, catalog number: 11873580001 )
  13. Phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626-5G )
  14. Dithiothreitol (DTT) (Bio-Rad Laboratories, catalog number: 1610611 )
  15. Triton X-100 (Sigma-Aldrich, catalog number: T9284 )
  16. Glutathione Sepharose 4B (GE Healthcare, catalog number: 17075601 )
  17. Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, GibcoTM, catalog number: 11965092 )
  18. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10437028 )
  19. Phosphatase inhibitor mini tablet (Thermo Fisher Scientific, Thermo ScientificTM , catalog number: 88667 )
  20. Protein A/G magnetic beads (Thermo Fisher Scientific, Thermo ScientificTM , catalog number: 88802 )
  21. Kinase buffer (New England Biolabs, catalog number: B6022S )
  22. Cold ATP (Sigma-Aldrich, catalog number: A2383 )
  23. Sorbitol (Fisher Scientific, catalog number: S459-500 )
  24. Antibodies
    1. Lats1 antibody (Cell Signaling Technology, catalog number: 3477S )
    2. pYAP S127 antibody (Cell Signaling Technology, catalog number: 4911S )
    3. pLATS-HM antibody (Cell Signaling Technology, catalog number: 8654S )
    4. GAPDH antibody (Santa Cruz Biotechnology, catalog number: sc-25778 )
    5. GST antibody (Sigma-Aldrich, catalog number: SAB4200237 )
  25. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A3294 )
  26. Tris-base (Fisher Scientific, catalog number: BP152-10 )
  27. L-glutathione reduced (Sigma-Aldrich, catalog number: G4251 )
  28. β-mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
  29. Sodium chloride (NaCl) (Fisher Scientific, catalog number: S271-10 )
  30. Glycerol (Fisher Scientific, catalog number: G33-1 )
  31. Sodium lauryl sulfate (SDS) (Fisher Scientific, catalog number: S529-500 )
  32. Bromophenol blue (Bio-Rad Laboratories, catalog number: 1610404 )
  33. Sodium fluoride (NaF) (Acros Organics, catalog number: 424325000 )
  34. EDTA (Mediatech, catalog number: 46-034-CI )
  35. NP-40 substitute (Sigma-Aldrich, catalog number: 74385 )
  36. Elution buffer (see Recipes)
  37. Dialysis buffer (see Recipes)
  38. 4x SDS sample buffer (see Recipes)
  39. Mild lysis buffer (see Recipes)
  40. TBS buffer (see Recipes)

Equipment

  1. Pipettes
  2. Spectrophotometer (Biochrom, model: Biochrom WPA Biowave II )
  3. Centrifuge (Thermo Fisher Scientific, Thermo ScientificTM , model: SorvallTM RC 6 Plus)
  4. Sonic dismembrator (Sonicator) (Fisher Scientific, model: Model 505 )
  5. Bench-top centrifuge (Denville Scientific, model: Denville 300D , catalog number: C0265-24)
  6. Magnetic rack (Thermo Fisher Scientific, catalog number: 12321D )
  7. Shaker (Eppendorf, New BrunswickTM, model: Excella® E24 )
  8. Heat block

Procedure

English
中文翻译

文章信息

版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Hong, A. W. and Guan, K. (2017). Non-radioactive LATS in vitro Kinase Assay. Bio-protocol 7(14): e2391. DOI: 10.21769/BioProtoc.2391.

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分类

癌症生物学 > 癌症生物化学 > 蛋白质

生物化学 > 蛋白质 > 活性

细胞生物学 > 细胞信号传导 > 磷酸化

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Thank you for the protocol. I am having trouble detecting YAP...
edit 0 Answer 5 Views Aug 2, 2018
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