Modeling, Synthesis and Characterization of Phospho–Penta–Peptide derived from PKA RII Sub Unit: A Candidate Substrate for CaN Assay

  • Sarojini R. Bulbule
  • P. Aravind
  • N. Hemalatha
  • K. S. DevarajuEmail author


Calcineurin (CaN) plays an important role in many pathological conditions like cancer, metabolic aberrations and neurodegenerative disorders. Till date contemporary methods to assay CaN uses p-nitrophenylphosphate (p-NPP) and specific protein phosphatases 2B inhibitor or use of phospho-peptide substrate derived from protein kinase A (PKA) regulatory sub unit type PKARII. Here we have modeled a series of small phospho peptides derived from PKARII and were docked by Molegro Virtual Docking software to find out the binding free enegy and to determine the affinity with modeled Calcineurin catalytic subunit A (CaN A). Further the candidate phospho–penta–peptide (PPP) derived from PKA regulatory sub unit type II was synthesized in house by employing Fmoc chemistry by solution phase method, purified over HPLC and characterized by mass spectrometry. This PPP was employed to assay CaN activity by Malachite Green method found to be a candidate substrate for CaN.


Calcineurin (CaN) Protein kinase-A (PKA) Docking Peptide Enzyme assay 



Authors acknowledge the financial support DST-SERB, Government of India (Grant No. SB/EMEQ-238/2013).

Compliance with Ethical Standards

Conflict of interest

Authors contributed to the writing of the manuscript and approved the final version and declare that they have no conflict of interest.

Research Involving Human and Animal Rights

This article does not contain studies with human or animal subjects performed by any of the authors that should be approved by Ethics Committee. The article does not contain any studies in patients by any of the authors.

Informed Consent

The article does not contain any studies in patients by any of the authors.


  1. Attila R, Mathew CG, Wendell AL (2006) Docking interactions in protein kinase and phosphatase networks. Curr Opin Struct Biol 6:676–685Google Scholar
  2. Bruce LM, David JR (1999) Effect of substitution inert metal complexes on CaN. Arch Biochem Biophys 366:168–176CrossRefGoogle Scholar
  3. Catherin JP, Jerry HW (1986) Stoichiometry and dynamic interaction of metal ion activators with CaN phosphatase. J Biol Chem 34:16115–16120Google Scholar
  4. Catherine JP, Wang JH (1983) Calmodulin-stimulated dephosphorylation of p-nitrophenyl phosphote and free phosphotyrosine by CaN. J Biol Chem 258:8550–8553Google Scholar
  5. Chernoff J, Sells M, Li HC (1984) Characterization of phosphotyrosyl-protein phosphatase activity associated with CaN. Biochem Biophys Res Commun 121:141–148CrossRefGoogle Scholar
  6. David WR (2003) Evaluation of protein docking predictions using Hex3.1 in CAPRI rounds 1–2. Proteins Struct Funct Genet 52:98–106CrossRefGoogle Scholar
  7. Devaraju KS, Basanagoud SP, Subramanyam JT, Babu S, Taranath Shetty SV, Vommina K, Suresh Babu V (2006) Synthesis of a modified peptide fragment analog Val-Tyr (P)-Val-Ala-Ala-OH of camp protein kinase regulatory subunit type II employing Fmoc-chemistry. Indian J Chem 45B:1747–1752Google Scholar
  8. Donella DA, Mac Gowan CH, Cohen P, Marchiori F, Meyer HE, Pinna LA (1990) An investigation of the substrate specificity of protein phosphatase 2C using synthetic peptide substrates; comparison with protein phosphatase 2A. Biochem Biophys Acta 1051(2):199–202CrossRefGoogle Scholar
  9. Donella DA, Mac GCH, Cohen P, Marchiori F, Meyer HE, Pinna LA (1994) Dephosphorylation of phosphopeptides by CaN (protein phosphatase 2B). Eur J Biochem 219:109–117CrossRefGoogle Scholar
  10. Gal M, Li S, Luna RE, Takeuchi K, Wagner G (2014) The LxVP and PxIxIT NFAT motifs bind jointly to overlapping epitopes on CaN’s catalytic domain distant to the regulatory domain. Structure 22(7):1016–1027CrossRefPubMedCentralGoogle Scholar
  11. Gracia CFJ, Okamura H, Aramburu JF, Shaw KT, Pelletier L, Showalter R. Villafranca E. Rao A (1998) Two-site interaction of nuclear factor of activated T cells with activated CaN. JBiolChem 273:23877–23883Google Scholar
  12. Grigoriu S, Bond R, Cossio P, Chen JA, Ly N, Hummer G, Page R, Cyert MS, Peti W (2013) The molecular mechanism of substrate engagement and immunosuppressant inhibition of calcineurin. PLoS Biol 11(2):e1001492CrossRefPubMedCentralGoogle Scholar
  13. Harish BM, Devaraju KS, Gopi A, Saraswathi R, Gupta A, Babu RL, Chidananda S (2013) In silico binding affinity study of CaN inhibitors to CaN and its close associates. Indian J Biotechnol 12:213–217Google Scholar
  14. Harish BM, Saraswathi R, Devaraju KS (2014) Molecular docking study of auto inhibitory domain fragments to CaN A. Int J Sci Eng Res 5:2095–2100Google Scholar
  15. Hosen MJ, Zubaer A, Thapa S, Khadka B, De Paepe A, Vanakker OM (2014) Molecular docking simulations provide insights in the substrate binding sites and possible substrates of the ABCC6 transporter. PLoS ONE 9:102779CrossRefGoogle Scholar
  16. Kathryn JS, Hubb HVT, Fred PH, Romijn T, Niceo PM, Smit. Johan WDF, Johannes VP (2006) Spectorophotometric assay for CaN activity in leukocytes isolated from human blood. Anal Biochem 358:104–110CrossRefGoogle Scholar
  17. Kennelly PJ, Krebs EG (1991) Consensus sequences as substrate specificity determinants for protein kinases and protein phosphatases. J Biol Chem 266:15555–15558PubMedGoogle Scholar
  18. Koichi S, Hao CC, Kuo PH (1995) Dephosphorylation of protein kinase C Substrate neurogranin, neuromodulin and MARCKS by CaN and protein phosphatases 1 and 2A. Arch Biochem Biophys 316:673–679CrossRefGoogle Scholar
  19. Luo C, Shaw KT, Raghavan A, Aramburu J, Garcia-Cozar F, Perrino BA, Hogan PG, Rao A (1996) Interaction of CaN with a domain of the transcription factor NFAT1 that controls nuclear import. Proc Natl Acad Sci USA. 93(17):8907–8912CrossRefGoogle Scholar
  20. Manalan AS, Klee CB (1983) Activation of CaN by limited proteolysis. Biochem Biophys Res Commun 114:955–961CrossRefGoogle Scholar
  21. Manjunath GS, Aravind P, Supreet G, Devaraju KS, Shrinivas DJ, Sheshagiri RD, Harish BM, Imtiyaz Ahmed MK (2018) In silico binding affinity studies of N-9 substituted 6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purine derivatives-Target for P70-S6K1 & PI3K-δ kinases. Beni Suef Univ J Basic Appl Sci 7(1):84–91Google Scholar
  22. Martin BL, Rhode DJ (1999) Effect of substitution inert metal complexes on CaN. Arch Biochem Biophys 366(1):168–176CrossRefGoogle Scholar
  23. Martin BL, Li B, Liao C, Rhode DJ (2000) Differences between Mg2 + and transition metal ions in the activation of CaN. Arch Biochem Biophys 380(1):71–77CrossRefGoogle Scholar
  24. Micheail P, Mariata MK (1987) Calcium- and Calmodulin- sensitive interactions of CaN with phospholipids. J Biol Chem 262:10109–10113Google Scholar
  25. Mulero MC, Aubareda A, Orzáez M, Messeguer J, Serrano-Candelas E, Martínez-Hoyer S, Messeguer À, Pérez Payá E, Pérez-Riba M (2009) Inhibiting the CaN-NFAT (nuclear factor of activated T cells) signaling pathway with a regulator of CaN-derived peptide without affecting general CaN phosphatase activity. J Biol Chem 284(14):9394–9401CrossRefPubMedCentralGoogle Scholar
  26. Pallen CJ, Wang JH (1983) Calmodulin-stimulated dephosphorylation of p-nitrophenyl phosphate and free phosphotyrosine by CaN. J Biol Chem 258(14):8550–8553PubMedGoogle Scholar
  27. Ping L, Ke Z, Bengoing X, Qun W (2004) Effect of metal ions on the activity of the catalytic domain of CaN. Biometals 2:157–165CrossRefGoogle Scholar
  28. Ritchie GJ, Kemp L (2000) Protein docking using spherical polar fourier correlation. PROTEINS: Struct Funct Genet 39:178–194CrossRefGoogle Scholar
  29. Rusnak F (2000) Manganese-activated phosphatases. Metal Ions Biol Syst 37:305–343Google Scholar
  30. Sara MM, Lali G, Antonio R, Alicia R, Eulalia S, Fellipe W, Maria DLM, Juan MR, Susana DL (2009) The RCAN carboxyl end mediates CaN docking-dependent inhibition via a site that dictates binding to substrates and regulators. PNAS. 106, 6117–6122CrossRefGoogle Scholar
  31. Simina G, Rachel B, Pilar C, Jennifer AC, Nina L, Gerhard H, Rebecca P, Martha SC, Wolfgang P (2013) The molecular mechanism of substrate engagement and immunosuppressant inhibition of CaN. PLoS Biol 11:e1001492CrossRefGoogle Scholar
  32. Stewart AA, Ingrebritsen TS, Manalan A, Klee CB, Cohen P (1983) Purification and Properties of a Ca2+ calmoddin-dependent protein phosphatase (2B) from rabbit skeletal muscle. Eur J Biochem 132:289–295CrossRefGoogle Scholar
  33. Stewart AA, Ingebristen TS,. Manalan AS, Draetta GF, Newton DL (1985) The protein phosphatases involved in cellular regulation. Eur J Biochem 132:289–295CrossRefGoogle Scholar
  34. Yan L, Wei Q (1999) High activity of the CaN A subunit with a V314 deletion. Biol Chem 380(11):1281–1285CrossRefGoogle Scholar
  35. Yang SA, Klee CB (2000) Low affinity Ca2+ binding sites of CaN B mediate conformational changes in CaN A. Biochemistry 51:16147–16154CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  • Sarojini R. Bulbule
    • 1
  • P. Aravind
    • 1
  • N. Hemalatha
    • 2
  • K. S. Devaraju
    • 1
    Email author
  1. 1.Department of BiochemistryKarnatak UniversityDharwadIndia
  2. 2.Department of Biochemistry and NutritionCFTRIMysoreIndia

Personalised recommendations