Details

Autor(en) / Beteiligte

• 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.

Titel

Mechanism of Inhibition of Protein Phosphatase 1 by DARPP-32: Studies with Recombinant DARPP-32 and Synthetic Peptides

Ist Teil von

• Biochemical and biophysical research communications, 1995-01, Vol.206 (2), p.652-658

Ort / Verlag

United States: Elsevier Inc

Erscheinungsjahr

1995

Quelle

Elsevier Journal Backfiles on ScienceDirect (DFG Nationallizenzen)

Beschreibungen/Notizen

• 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 ≍ 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 ≍ 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.