Catalytic Domain Crystal Structure of Protein Kinase C-θ (PKCθ)

Abstract

A member of the novel protein kinase C (PKC) subfamily, PKC, is an essential component of the T cell synapse and is required for optimal T cell activation and interleukin-2 production. Selective involvement of PKC in TCR signaling makes this enzyme an attractive therapeutic target in T cell-mediated disease processes. In this report we describe the crystal structure of the catalytic domain of PKC at 2.0-Å resolution. Human recombinant PKC kinase domain was expressed in bacteria as catalytically active phosphorylated enzyme and co-crystallized with its subnanomolar, ATP site inhibitor staurosporine. The structure follows the classic bilobal kinase fold and shows the enzyme in its active conformation and phosphorylated state. Inhibitory interactions between conserved features of staurosporine and the ATP-binding cleft are accompanied by closing of the glycine-rich loop, which also maintains an inhibitory arrangement by blocking the phosphate recognition subsite. The two major phosphorylation sites, Thr-538 in the activation loop and Ser-695 in the hydrophobic motif, are both occupied in the structure, playing key roles in stabilizing active conformation of the enzyme and indicative of PKC autocatalytic phosphorylation and activation during bacterial expression. The PKC-staurosporine complex represents the first kinase domain crystal structure of any PKC isotypes to be determined and as such should provide valuable insight into PKC specificity and into rational drug design strategies for PKC selective leads. Inhibitors of PKC1 are currently being used in clinical trials for various types of cancer, and a PKC␤ inhibitor is being used in clinical trials for diabetes-related retinopathy (1).PKC and PKB/AKT kinase domains are related by sequence homology; however, there are key structural differences in the regulatory domains and second messenger cofactor requirements. PKB/AKT contains an N-terminal pleckstrin homology domain regulated by phosphoinositide second messengers, a central catalytic kinase domain, and a C-terminal regulatory region facilitating key protein-protein interactions with signal-

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].…”

“…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].…”

Conformational Transitions of Adenylate Kinase: Switching by Cracking

“…Interaction energies of adjacent beads are harmonic in bond length and angle, with the geometry of the native state included through a dihedral term and non-local bead-bead interaction terms. Non-local contacts are included via (4) which has a minimum of depth ε n at r = σ ij , with σ ij being the native distance between the C α (i) and C α (j) atoms in the crystal structure. In homogenous structure-based models, there is a single value for ε n .…”

“…The activity of many signal transducing proteins is associated with large conformational changes. For example, C-terminal Src Kinase protein [2], the Cyclin Dependent Kinase family [3], the Protein Kinase C family [4] and Adenylate Kinase (AKE) [5] have stable inactive conformations, in addition to active forms. Since the balance between conformations regulates protein activity, conformational transitions play important roles in the machinery of the cell [6].…”

Anticorpos conformacionais para PKCs clássicas e suas aplicações

“…A estrutura cristalográfica do domínio catalítico das PKCs só foi resolvida em 2004 (Xu et al, 2004). O domínio catalítico é bastante conservado nas quinases em geral, possuindo cerca de 250 aminoácidos que formam os lóbulos N e C. O lóbulo N é composto principalmente por folhas  e também possui uma alça rica em glicinas responsável por ligar o ATP e posicionar o fosfato  para a catálise.…”