Active Site Inhibitors Protect Protein Kinase C from Dephosphorylation and Stabilize Its Mature Form

Christine M Gould,Corina E Antal,Gloria Reyes,Maya T Kunkel,Ryan A Adams,Ahdad Ziyar,Tania Riveros,Alexandra C Newton

doi:10.1074/jbc.m111.272526

Abstract

Conformational changes acutely control protein kinase C (PKC). We have previously shown that the autoinhibitory pseudosubstrate must be removed from the active site in order for 1) PKC to be phosphorylated by its upstream kinase phosphoinositide-dependent kinase 1 (PDK-1), 2) the mature enzyme to bind and phosphorylate substrates, and 3) the mature enzyme to be dephosphorylated by phosphatases. Here we show an additional level of conformational control; binding of active site inhibitors locks PKC in a conformation in which the priming phosphorylation sites are resistant to dephosphorylation. Using homogeneously pure PKC, we show that the active site inhibitor Gö 6983 prevents the dephosphorylation by pure protein phosphatase 1 (PP1) or the hydrophobic motif phosphatase, pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP). Consistent with results using pure proteins, treatment of cells with the competitive inhibitors Gö 6983 or bisindolylmaleimide I, but not the uncompetitive inhibitor bisindolylmaleimide IV, prevents the dephosphorylation and down-regulation of PKC induced by phorbol esters. Pulse-chase analyses reveal that active site inhibitors do not affect the net rate of priming phosphorylations of PKC; rather, they inhibit the dephosphorylation triggered by phorbol esters. These data provide a molecular explanation for the recent studies showing that active site inhibitors stabilize the phosphorylation state of protein kinases B/Akt and C.

Highlights

The PKC family is composed of nine genes [9, 10], of which the conventional (␣, the alternatively spliced ␤I and ␤II, and brain-enriched ␥) and novel (␦, ⑀, ␩, and hematopoietic-enriched ␪) isozymes are matured by phosphorylation on three conserved sites originally identified in PKC ␤II: the activation loop at the entrance to the active site and two C-terminal sites, the turn motif and hydrophobic motif [3]
This construct, in which the Lys that coordinates the ␣-␤ phosphates of ATP is mutated to Met or Arg, accumulates as the non-phosphorylated species under normal conditions, but accumulates as the phosphorylated species when cells are preincubated with ATP analogues such as bisindolylmaleimide I (Bis I)
We show that active site inhibitors prevent the dephosphorylation of pure protein in vitro and prevent the phorbol ester-mediated dephosphorylation of endogenous and overexpressed PKC in cells

Summary

Introduction

The PKC family is composed of nine genes [9, 10], of which the conventional (␣, the alternatively spliced ␤I and ␤II, and brain-enriched ␥) and novel (␦, ⑀, ␩, and hematopoietic-enriched ␪) isozymes are matured by phosphorylation on three conserved sites originally identified in PKC ␤II: the activation loop at the entrance to the active site and two C-terminal sites, the turn motif and hydrophobic motif [3].

Results

Conclusion