Inhibition of the c-Jun N-terminal Kinase/AP-1 and NF-κB Pathways by PICOT, a Novel Protein Kinase C-interacting Protein with a Thioredoxin Homology Domain

Abstract

Protein kinase C-(PKC ) is a Ca 2؉ -independent PKC isoform that is selectively expressed in T lymphocytes (and muscle), and is thought to play an important role in T cell receptor-induced activation. To gain a better understanding of the function and regulation of PKC , we have employed the yeast two-hybrid system to identify PKC -interacting proteins. We report the isolation and characterization of a cDNA encoding a novel 335-amino acid (37.5-kDa) PKC -interacting protein termed PICOT (for PKC-interacting cousin of thioredoxin). PICOT is expressed in various tissues, including in T cells, where it colocalizes with PKC . PICOT displays an N-terminal thioredoxin homology domain, which is required for the interaction with PKC. Comparison of the unique C-terminal region of PICOT with expressed sequence tag data bases revealed two tandem repeats of a novel domain that is highly conserved from plants to mammals. Transient overexpression of full-length PICOT (but not its N-or C-terminal fragments) in T cells inhibited the activation of c-Jun N-terminal kinase (but not extracellular signal-regulated kinase), and the transcription factors AP-1 or NF-B. These findings suggest that PICOT and its evolutionary conserved homologues may interact with PKC-related kinases in multiple organisms and, second, that it plays a role in regulating the function of the thioredoxin system.Members of the protein kinase C (PKC) 1 family of intracellular serine/threonine kinases play critical roles in the regulation of cellular differentiation and proliferation in many cell types and in response to diverse stimuli, including hormones, neurotransmitters, and growth factors (1-3). The PKC family consists of 11 known mammalian members that are expressed in a wide variety of tissues and cell types. Based on sequence similarities, domain structures, and cofactor requirements, PKC isoenzymes can be grouped into three subfamilies. 1) The Ca 2ϩ -dependent conventional enzymes, consisting of PKC-␣, -␤I, -␤II, and -␥, contain three conserved domains, namely the diacylglycerol/phorbol ester binding C1 domain, which contains two repeats of a cysteine-rich zinc finger, the phospholipid-and calcium-binding C2 domain, and the catalytic C3 and C4 domains.2) The Ca 2ϩ -independent enzymes (PKC-␦, -⑀, -, -and -) are termed novel PKCs. The C2-like N-terminal domain of these enzymes can bind acidic phospholipids but not Ca 2ϩ . 3) A third PKC subfamily, termed atypical PKCs, includes PKCand -/ that possess a single cysteine-rich domain, lacking the ability to bind phospholipids or phorbol esters. PKC activity is regulated by defined cofactors that interact with specific regions of the regulatory domain as well as transphosphorylation by serine/threonine kinases and autophosphorylation. The activation is accompanied by a conformational change that releases the basic pseudosubstrate region from the catalytic cleft of the kinase domain. In addition, interaction with specific proteins, termed receptors for activated PKC, that function as selective scaffolds for a...

Chemogenomic identification of Ref-1/AP-1 as a therapeutic target for asthma

“…Recently, it has become evident that some of the members of this superfamily of oxidoreductases are involved in regulating inflammatory processes. Other members of the oxidoreductase superfamily that may play important roles in inflammatory processes include nucleoredoxin (41), which significantly enhances tumor necrosis factor-␣-induced NF-B reporter activity (42); PICOT, a 37.5-kDa PKC -interacting protein that regulates AP-1 and NF-B transcription (43); and TRP32, a 32-kDa protein that interacts with the catalytic domain of the protein kinase MST (44 …”

Protein expression changes in a cell system of beta-cell maturation reflect an acquired sensitivity to IL-1?

“…A protein spot containing PKC-interacting cousin of thioredoxin (PICOT), a Jun N-terminal kinase (JNK) inhibitor [45], was down-regulated during beta-cell maturation. JNK is activated in response to different stress factors such as cytokines, heat and oxi-dative compounds.…”

Menin represses JunD transcriptional activity in protein kinase Cθ-mediated Nur77 expression

“…It has been known that PKC is a predominant PKC isoform for T cell development and/or activation . To determine whether PKC is involved in Nur77 induction, we transiently transfected DO11.10 cells with HA-tagged PICOT, an endogenous PKCspecific inhibitor along with Nur77 promoter-driven luciferase reporter plasmid (Witte et al, 2000). Similar to bis-IM, PICOT also inhibited the activation of the Nur77 reporter gene in a dose-dependent fashion, indicating that PKC serves as an activator for Nur77 induction in T cell ( Figure 1D).…”