Abstract
BackgroundThe NLRP3 inflammasome is a critical component of sterile inflammation, which is involved in many diseases. However, there is currently no known proximal biomarker for measuring NLRP3 activation in pathological conditions. Protein kinase D (PKD) has emerged as an important NLRP3 kinase that catalyzes the release of a phosphorylated NLRP3 species that is competent for inflammasome complex assembly.MethodsTo explore the potential for PKD activation to serve as a selective biomarker of the NLRP3 pathway, we tested various stimulatory conditions in THP-1 and U937 cell lines, probing the inflammasome space beyond NLRP3. We analyzed the correlation between PKD activation (monitored by its auto-phosphorylation) and functional inflammasome readouts.ResultsPKD activation/auto-phosphorylation always preceded cleavage of caspase-1 and gasdermin D, and treatment with the PKD inhibitor CRT0066101 could block NLRP3 inflammasome assembly and interleukin-1β production. Conversely, blocking NLRP3 either genetically or using the MCC950 inhibitor prevented PKD auto-phosphorylation, indicating a bidirectional functional crosstalk between NLRP3 and PKD. Further assessments of the pyrin and NLRC4 pathways, however, revealed that PKD auto-phosphorylation can be triggered by a broad range of stimuli unrelated to NLRP3 inflammasome assembly.ConclusionAlthough PKD and NLRP3 become functionally interconnected during NLRP3 activation, the promiscuous reactivity of PKD challenges its potential use for tracing the NLRP3 inflammasome pathway.
Highlights
The (nucleotide-binding oligomerization domain) (NOD)-like receptor (NLR) family, pyrin domain-containing protein 3 (NLRP3) inflammasome is involved in several pathophysiological conditions featuring sterile inflammation [1]
Further assessments of the pyrin and NLRC4 pathways, revealed that Protein kinase D (PKD) auto-phosphorylation can be triggered by a broad range of stimuli unrelated to (NOD-like receptor family (NLRP3) inflammasome assembly
NLRP3 and PKD are functionally interconnected as vehicle for compound dilutions (Sigma #D2650), MCC950, CGP084892 and AFN700, CRT0066101 (Tocris #4975)
Summary
The NOD-like receptor (NLR) family, pyrin domain-containing protein 3 (NLRP3) inflammasome is involved in several pathophysiological conditions featuring sterile inflammation [1]. I.e., NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), Caspase-1, and NIMA-related kinase 7 (NEK7) can undergo post-translational modifications, such as phosphorylation, ubiquitination, acetylation or neddylation, but how these events are coordinated for effective inflammasome licensing and activation remains largely unknown. Phosphorylation of S5 in the pyrin domain appears to be an early event Removal of this phosphate group by protein phosphatase 2A [8] allows for subsequent activation steps. Phosphorylation of S198 between the pyrin and the NACHT domains, likely by c-Jun kinase (JNK)-1, is a critical licensing step [9]. Dephosphorylation is required for activation at the membrane and subsequent re-phosphorylation by protein kinase D (PKD) allows for cytoplasmic release and association with ASC [10, 11]. Protein kinase D (PKD) has emerged as an important NLRP3 kinase that catalyzes the release of a phosphorylated NLRP3 species that is competent for inflammasome complex assembly
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