Mechanism of inhibition of protein phosphatase 1 by DARPP-32: Studies with recombinant DARPP-32 and synthetic peptides

F. Desdouits, J. J. Cheetham, H. B. Huang, Y. G. Kwon, E. F.D.E. Silva, P. Denefle, M. E. Ehrlich, A. C. Nairn, P. Greengard, J. A. Girault

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Abstract

The mechanism of inhibition of protein phosphatase-1 catalytic subunit (PP-1c) by recombinant DARPP-32 and synthetic peptides was studied. DARPP-32 was expressed in Escherichia coli as a non-fusion protein using a pEt-3a plasmid, purified to homogeneity and shown to have physicochemical properties similar to those of the protein purified from bovine brain. Recombinant DARPP-32 phosphorylated on threonine-34 by cAMP-dependent protein kinase inhibited PP-1c with an IC50 (equivalent to) 0.5 nM, comparable to that obtained with bovine DARPP-32. Non-phosphorylated DARPP-32, and mutated forms in which threonine-34 was replaced by an alanine or a glutamic acid, inhibited PP-1c with an IC50 (equivalent to) 1 μM. Surface plasmon resonance analysis showed binding of PP-1c to nonphospho- and phospho-DARPP-32-(8-38) synthetic peptides with apparent Kd values of 1.2 and 0.3 μM, respectively, supporting the existence of an interaction between non-phosphorylated DARPP-32 and PP-1c that is increased by phosphorylation of DARPP-32 at threonine-34. These results suggest a model in which DARPP-32 interacts with PP-1c by at least two low affinity sites, the combination of which is responsible for the high affinity (nM) inhibition.

Original languageEnglish
Pages (from-to)652-658
Number of pages7
JournalBiochemical and Biophysical Research Communications
Volume206
Issue number2
DOIs
Publication statusPublished - 1995 Jan 17

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Desdouits, F., Cheetham, J. J., Huang, H. B., Kwon, Y. G., Silva, E. F. D. E., Denefle, P., Ehrlich, M. E., Nairn, A. C., Greengard, P., & Girault, J. A. (1995). Mechanism of inhibition of protein phosphatase 1 by DARPP-32: Studies with recombinant DARPP-32 and synthetic peptides. Biochemical and Biophysical Research Communications, 206(2), 652-658. https://doi.org/10.1006/bbrc.1995.1092