Structure of the Catalytic Domain of Human Protein Kinase C β II Complexed with a Bisindolylmaleimide Inhibitor

Abstract

The conventional protein kinase C isoform, PKCII, is a signaling kinase activated during the hyperglycemic state and has been associated with the development of microvascular abnormalities associated with diabetes. PKCII, therefore, has been identified as a therapeutic target where inhibitors of its kinase activity are being pursued for treatment of microvascular-related diabetic complications. In this report, we describe the crystal structure of the catalytic domain of PKCbetaII complexed with an inhibitor at 2.6 A resolution. The kinase domain of PKCbetaII was cleaved and purified from full-length PKCbetaII expressed in baculovirus-infected insect cells. The overall kinase domain structure follows the classical bilobal fold and is in its fully activated conformation with three well-defined phosphorylated residues: Thr-500, Thr-641, and Ser-660. Different from the crystal structures of nonconventional PKC isoforms, the C-terminus of the PKCbetaII catalytic domain is almost fully ordered and features a novel alpha helix in the turn motif. An ATP-competitive inhibitor, 2-methyl-1H-indol-3-yl-BIM-1, was crystallized with the PKCbetaII catalytic domain as a dimer of two enzyme-inhibitor complexes. The bound inhibitor adopts a nonplanar conformation in the ATP-binding site, with the kinase domain taking on an intermediate, open conformation. This PKCbetaII-inhibitor complex represents the first structural description of any conventional PKC kinase domain. Given the pathogenic role of PKCbetaII in the development of diabetic complications, this structure can serve as a template for the rational design of inhibitors as potential therapeutic agents.

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

“…3A, graph). This protection from dephosphorylation was exaggerated in cells overexpressing a PKC ␤II construct containing a destabilizing mutation within the highly conserved NFD motif of the AGC protein kinase family, corresponding to F629A in PKC ␤II (33)(34)(35). This construct was more readily down-regulated by PDBu when compared with wild-type enzyme (Fig.…”

Regulation of Protein Kinase C function by phosphorylation on conserved and non-conserved sites

“…7 molecular basis of A-loop phosphorylation in controlling catalytic activity has been revealed with the publication of the crystal structures of the kinase domains of PKC , PKC and PKC II : phosphorylation at the A-loop makes important contributions in stabilising the active conformation of these kinases by forming ionic contacts with positively-charged residues in the vicinity of the kinase domain [27][28][29].…”

“…10 of the enzyme [28,29,50]. While phosphorylation at the TM influences enzyme stability, differences have been reported as to the importance of this site for catalytic activity (Table 1).…”

“…Molecular modelling studies have shown that the negatively charged phosphate at the HM site on PKC II and PKC forms hydrogen bonds with an invariant glycine residue and thus serves to stabilise the enzyme [27,29]. While this phosphorylation site is not conserved in aPKCs, the negatively charged glutamic acid residue at the HM site on PKC performs a similar function [28].…”

Kinase-activity-independent functions of atypical protein kinase C inDrosophila

“…Amino acids 532-555 were taken from the homologous structure 2I0E (Grodsky et al, 2006) (sequence identity 49%) with the respective amino acids mutated/inserted with WHATIF (Vriend, 1990). The resulting structure was then solvated, energy minimized, and slowly heated up from 20 K to 300 K while position restraining all non-modeled atoms.…”