Salt-inducible kinase 2 regulates fibrosis during bleomycin-induced lung injury.

Idiopathic pulmonary fibrosis is a progressive and normally fatal disease with limited treatment options. The tyrosine kinase inhibitor nintedanib has recently been approved for the treatment of idiopathic pulmonary fibrosis, and its effectiveness has been linked to its ability to inhibit a number of receptor tyrosine kinases including the platelet-derived growth factor, vascular endothelial growth factor, and fibroblast growth factor receptors. We show here that nintedanib also inhibits salt-inducible kinase 2 (SIK2), with a similar IC50 to its reported tyrosine kinase targets. Nintedanib also inhibited the related kinases SIK1 and SIK3, although with 12-fold and 72-fold higher IC50s, respectively. To investigate if the inhibition of SIK2 may contribute to the effectiveness of nintedanib in treating lung fibrosis, mice with kinase-inactive knockin mutations were tested using a model of bleomycin-induced lung fibrosis. We found that loss of SIK2 activity protects against bleomycin-induced fibrosis, as judged by collagen deposition and histological scoring. Loss of both SIK1 and SIK2 activity had a similar effect to loss of SIK2 activity. Total SIK3 knockout mice have a developmental phenotype making them unsuitable for analysis in this model; however, we determined that conditional knockout of SIK3 in the immune system did not affect bleomycin-induced lung fibrosis. Together, these results suggest that SIK2 is a potential drug target for the treatment of lung fibrosis.

Bronchial gene expression signature associated with rate of subsequent FEV1 decline in individuals with and at risk of COPD.

BACKGROUND: COPD is characterised by progressive lung function decline. Leveraging prior work demonstrating bronchial airway COPD-associated gene expression alterations, we sought to determine if there are alterations associated with differences in the rate of FEV1 decline. METHODS: We examined gene expression among ever smokers with and without COPD who at baseline had bronchial brushings profiled by Affymetrix microarrays and had longitudinal lung function measurements (n=134; mean follow-up=6.38±2.48 years). Gene expression profiles associated with the rate of FEV1 decline were identified by linear modelling. RESULTS: Expression differences in 171 genes were associated with rate of FEV1 decline (false discovery rate <0.05). The FEV1 decline signature was replicated in an independent dataset of bronchial biopsies from patients with COPD (n=46; p=0.018; mean follow-up=6.76±1.32 years). Genes elevated in individuals with more rapid FEV1 decline are significantly enriched among the genes altered by modulation of XBP1 in two independent datasets (Gene Set Enrichment Analysis (GSEA) p<0.05) and are enriched in mucin-related genes (GSEA p<0.05). CONCLUSION: We have identified and replicated an airway gene expression signature associated with the rate of FEV1 decline. Aspects of this signature are related to increased expression of XBP1-regulated genes, a transcription factor involved in the unfolded protein response, and genes related to mucin production. Collectively, these data suggest that molecular processes related to the rate of FEV1 decline can be detected in airway epithelium, identify a possible indicator of FEV1 decline and make it possible to detect, in an early phase, ever smokers with and without COPD most at risk of rapid FEV1 decline.

Rapid Cycle Evaluation and Adaptation of an Inpatient Tobacco Treatment Service at a U.S. Safety-Net Hospital.

Background: To address disparities in smoking rates, our safety-net hospital implemented an inpatient tobacco treatment intervention: an "opt-out" electronic health record (EHR)-based Best Practice Alert + order-set, which triggers consultation to a Tobacco Treatment Consult (TTC) service for all hospitalized patients who smoke cigarettes. We report on development, implementation, and adaptation of the intervention, informed by a pre-implementation needs assessment and two rapid-cycle evaluations guided by the Consolidated Framework for Implementation Research (CFIR) and Expert Recommendations for Implementing Change (ERIC) compilation. Methods: We identified stakeholders affected by implementation and conducted a local needs assessment starting 6 months-pre-launch. We then conducted two rapid-cycle evaluations during the first 6 months post-implementation. The CFIR informed survey and interview guide development, data collection, assessment of barriers and facilitators, and selection of ERIC strategies to implement and adapt the intervention. Results: Key themes were: (1) Understanding the hospital's priority to improving tobacco performance metrics was critical in gaining leadership buy-in (CFIR Domain: Outer setting; Construct: External Policy and Incentives). (2) CFIR-based rapid-cycle evaluations allowed us to recognize implementation challenges early and select ERIC strategies clustering into 3 broad categories (conducting needs assessment; developing stakeholder relationships; training and educating stakeholders) to make real-time adaptations, creating an acceptable clinical workflow. (3) Minimizing clinician burden allowed the successful implementation of the TTC service. (4) Demonstrating improved 6-month quit rates and tobacco performance metrics were key to sustaining the program. Conclusions: Rapid-cycle evaluations to gather pre-implementation and early-implementation data, focusing on modifiable barriers and facilitators, allowed us to develop and refine the intervention to improve acceptability, adoption, and sustainability, enabling us to improve tobacco performance metrics in a short timeline. Future directions include spreading rapid-cycle evaluations to promote implementation of inpatient tobacco treatment programs to other settings and assessing long-term sustainability and return on investment of these programs.

Feasibility, Acceptability, and Adoption of an Inpatient Tobacco Treatment Service at a Safety-Net Hospital: A Mixed-Methods Study.

Rationale: Hospitalization is an opportunity to engage smokers who may not seek tobacco treatment. Our safety-net hospital developed and implemented an inpatient intervention consisting of an "opt-out" electronic health record-based Best Practice Alert (BPA)+order-set, designed to trigger referral to the Tobacco Treatment Consult (TTC) service (a team staffed by tobacco treatment specialists) for all hospitalized smokers, regardless of motivation to quit.Objectives: We performed a sequential explanatory mixed-methods study to evaluate the feasibility, acceptability, and adoption of the TTC service.Methods: Among all admissions of adult "current smokers" between July 2016 and June 2017, we calculated the percentage of patients whose clinicians accepted the order-set (through a simple "order" click), thus generating the TTC referral. We then determined the extent of clinician follow-through of TTC recommendations for prescribing nicotine replacement therapy among 1,651 consecutive smokers seen by the TTC service. Finally, we conducted qualitative interviews with inpatient clinicians (n = 25) to understand their rationale for adoption or nonuse of the TTC intervention, including perceived usefulness, barriers to adoption, and strategies to improve the utility of the service.Results: Clinicians accepted the TTC order-set for 4,100 out of 6,598 "current smokers" (62.1%) for whom the BPA fired, typically after initially deferring the BPA. Rates of acceptance significantly differed across clinical services (range: 8% [obstetrics-gynecology] to 82.2% [cardiology]; P < 0.00001). A chart review showed that 43.5% (719/1,651) of the patients seen by the TTC service desired outpatient nicotine replacement therapy, but only half of these patients (48.8%; 351/719) received a discharge prescription from the inpatient team. Clinicians expressed that they valued the TTC service, but that BPA fatigue, time constraints, competing priorities, and poor communication with the TTC service were barriers to using the service and following recommendations. Clinicians suggested strategies to address barriers to the use of tobacco treatment interventions during hospitalization and after discharge.Conclusions: Implementing a large-scale "opt-out" tobacco treatment service for hospitalized smokers at a safety-net hospital is feasible and acceptable, but suffers from inconsistent adoption due to a variety of clinician barriers. System-level changes are needed to increase uptake and sustain inpatient tobacco treatment interventions to promote smoking cessation.

The NEDD8 E3 ligase DCNL5 is phosphorylated by IKK alpha during Toll-like receptor activation.

The activity of Cullin-RING ubiquitin E3 ligases (CRL) is regulated by NEDD8 modification. DCN-like proteins promote Cullin neddylation as scaffold-like E3s. One DCNL, DCNL5, is highly expressed in immune tissue. Here, we provide evidence that DCNL5 may be involved in innate immunity, as it is a direct substrate of the kinase IKKα during immune signalling. We find that upon activation of Toll-like receptors, DCNL5 gets rapidly and transiently phosphorylated on a specific N-terminal serine residue (S41). This phosphorylation event is specifically mediated by IKKα and not IKKß. Our data for the first time provides evidence that DCNL proteins are post-translationally modified in an inducible manner. Our findings also provide the first example of a DCNL member as a kinase substrate in a signalling pathway, indicating that the activity of at least some DCNLs may be regulated.

Inhibition of SIK2 and SIK3 during differentiation enhances the anti-inflammatory phenotype of macrophages.

The salt-inducible kinases (SIKs) control a novel molecular switch regulating macrophage polarization. Pharmacological inhibition of the SIKs induces a macrophage phenotype characterized by the secretion of high levels of anti-inflammatory cytokines, including interleukin (IL)-10, and the secretion of very low levels of pro-inflammatory cytokines, such as tumour necrosis factor α. The SIKs, therefore, represent attractive new drug targets for the treatment of macrophage-driven diseases, but which of the three isoforms, SIK1, SIK2 or SIK3, would be appropriate to target remains unknown. To address this question, we developed knock-in (KI) mice for SIK1, SIK2 and SIK3, in which we introduced a mutation that renders the enzymes catalytically inactive. Characterization of primary macrophages from the single and double KI mice established that all three SIK isoforms, and in particular SIK2 and SIK3, contribute to macrophage polarization. Moreover, we discovered that inhibition of SIK2 and SIK3 during macrophage differentiation greatly enhanced the production of IL-10 compared with their inhibition in mature macrophages. Interestingly, macrophages differentiated in the presence of SIK inhibitors, MRT199665 and HG-9-91-01, still produced very large amounts of IL-10, but very low levels of pro-inflammatory cytokines, even after the SIKs had been reactivated by removal of the drugs. Our data highlight an integral role for SIK2 and SIK3 in innate immunity by preventing the differentiation of macrophages into a potent and stable anti-inflammatory phenotype.

The TRPM7 interactome defines a cytoskeletal complex linked to neuroblastoma progression.

Neuroblastoma is the second-most common solid tumor in children and originates from poorly differentiated neural crest-derived progenitors. Although most advanced stage metastatic neuroblastoma patients initially respond to treatment, a therapy resistant pool of poorly differentiated cells frequently arises, leading to refractory disease. A lack of insight into the molecular mechanisms that underlie neuroblastoma progression hampers the development of effective new therapies for these patients. Normal neural crest development and maturation is guided by physical interactions between the cell and its surroundings, in addition to soluble factors such as growth factors. This mechanical crosstalk is mediated by actin-based adhesion structures and cell protrusions that probe the cellular environment to modulate migration, proliferation, survival and differentiation. Whereas such signals preserve cellular quiescence in non-malignant cells, perturbed adhesion signaling promotes de-differentiation, uncontrolled cell proliferation, tissue invasion and therapy resistance. We previously reported that high expression levels of the channel-kinase TRPM7, a protein that maintains the progenitor state of embryonic neural crest cells, are closely associated with progenitor-like features of tumor cells, accompanied by extensive cytoskeletal reorganization and adhesion remodeling. To define mechanisms by which TRPM7 may contribute to neuroblastoma progression, we applied a proteomics approach to identify TRPM7 interacting proteins. We show that TRPM7 is part of a large complex of proteins, many of which function in cytoskeletal organization, cell protrusion formation and adhesion dynamics. Expression of a subset of these TRPM7 interacting proteins strongly correlates with neuroblastoma progression in independent neuroblastoma patient datasets. Thus, TRPM7 is part of a large cytoskeletal complex that may affect the malignant potential of tumor cells by regulating actomyosin dynamics and cell-matrix interactions.

PSM Peptides of Staphylococcus aureus Activate the p38-CREB Pathway in Dendritic Cells, Thereby Modulating Cytokine Production and T Cell Priming.

The challenging human pathogen Staphylococcus aureus has highly efficient immune evasion strategies for causing a wide range of diseases, from skin and soft tissue to life-threatening infections. Phenol-soluble modulin (PSM) peptides are major pathogenicity factors of community-associated methicillin-resistant S. aureus strains. In previous work, we demonstrated that PSMs in combination with TLR2 ligand from S. aureus induce tolerogenic dendritic cells (DCs) characterized by the production of high amounts of IL-10, but no proinflammatory cytokines. This in turn promotes the activation of regulatory T cells while impairing Th1 response; however, the signaling pathways modulated by PSMs remain elusive. In this study, we analyzed the effects of PSMs on signaling pathway modulation downstream of TLR2. TLR2 stimulation in combination with PSMα3 led to increased and prolonged phosphorylation of NF-κB, ERK, p38, and CREB in mouse bone marrow-derived DCs compared with single TLR2 activation. Furthermore, inhibition of p38 and downstream MSK1 prevented IL-10 production, which in turn reduced the capacity of DCs to activate regulatory T cells. Interestingly, the modulation of the signaling pathways by PSMs was independent of the known receptor for PSMs, as shown by experiments with DCs lacking the formyl peptide receptor 2. Instead, PSMs penetrate the cell membrane most likely by transient pore formation. Moreover, colocalization of PSMs and p38 was observed near the plasma membrane in the cytosol, indicating a direct interaction. Thus, PSMs from S. aureus directly modulate the signaling pathway p38-CREB in DCs, thereby impairing cytokine production and in consequence T cell priming to increase the tolerance toward the pathogen.

The clinically approved drugs dasatinib and bosutinib induce anti-inflammatory macrophages by inhibiting the salt-inducible kinases.

Macrophages switch to an anti-inflammatory, 'regulatory'-like phenotype characterized by the production of high levels of interleukin (IL)-10 and low levels of pro-inflammatory cytokines to promote the resolution of inflammation. A potential therapeutic strategy for the treatment of chronic inflammatory diseases would be to administer drugs that could induce the formation of 'regulatory'-like macrophages at sites of inflammation. In the present study, we demonstrate that the clinically approved cancer drugs bosutinib and dasatinib induce several hallmark features of 'regulatory'-like macrophages. Treatment of macrophages with bosutinib or dasatinib elevates the production of IL-10 while suppressing the production of IL-6, IL-12p40 and tumour necrosis factor α (TNFα) in response to Toll-like receptor (TLR) stimulation. Moreover, macrophages treated with bosutinib or dasatinib express higher levels of markers of 'regulatory'-like macrophages including LIGHT, SPHK1 and arginase 1. Bosutinib and dasatinib were originally developed as inhibitors of the protein tyrosine kinases Bcr-Abl and Src but we show that, surprisingly, the effects of bosutinib and dasatinib on macrophage polarization are the result of the inhibition of the salt-inducible kinases. Consistent with the present finding, bosutinib and dasatinib induce the dephosphorylation of CREB-regulated transcription co-activator 3 (CRTC3) and its nuclear translocation where it induces a cAMP-response-element-binding protein (CREB)-dependent gene transcription programme including that of IL-10. Importantly, these effects of bosutinib and dasatinib on IL-10 gene expression are lost in macrophages expressing a drug-resistant mutant of salt-inducible kinase 2 (SIK2). In conclusion, our study identifies the salt-inducible kinases as major targets of bosutinib and dasatinib that mediate the effects of these drugs on the innate immune system and provides novel mechanistic insights into the anti-inflammatory properties of these drugs.

An unexpected twist to the activation of IKKß: TAK1 primes IKKß for activation by autophosphorylation.

IKKß {IκB [inhibitor of NF-κB (nuclear factor κB)] kinase ß} is required to activate the transcription factor NF-κB, but how IKKß itself is activated in vivo is still unclear. It was found to require phosphorylation by one or more 'upstream' protein kinases in some reports, but by autophosphorylation in others. In the present study, we resolve this contro-versy by demonstrating that the activation of IKKß induced by IL-1 (interleukin-1) or TNF (tumour necrosis factor) in embryonic fibroblasts, or by ligands that activate Toll-like receptors in macrophages, requires two distinct phosphorylation events: first, the TAK1 [TGFß (transforming growth factor ß)-activated kinase-1]-catalysed phosphorylation of Ser¹77 and, secondly, the IKKß-catalysed autophosphorylation of Ser¹8¹. The phosphorylation of Ser¹77 by TAK1 is a priming event required for the subsequent autophosphorylation of Ser¹8¹, which enables IKKß to phosphorylate exogenous substrates. We also provide genetic evidence which indicates that the IL-1-stimulated, LUBAC (linear ubiquitin chain assembly complex)-catalysed formation of linear ubiquitin chains and their interaction with the NEMO (NF-κB essential modulator) component of the canonical IKK complex permits the TAK1-catalysed priming phosphorylation of IKKß at Ser¹77 and IKKα at Ser¹76. These findings may be of general significance for the activation of other protein kinases.

Molecular control of the NEMO family of ubiquitin-binding proteins.

Research over the past decade has revealed how NF-κB essential modulator (NEMO; also known as IKKγ) regulates the IKKα-IKKß signalling axis in the innate immune system. The discovery that NEMO is a polyubiquitin-binding protein and that the IKK complex is modulated by other protein kinases that are themselves controlled by polyubiquitin chains has provided a deeper molecular understanding of the non-degradative roles of ubiquitylation. New mechanistic insights of NEMO and related polyubiquitin-binding proteins have become a paradigm for how the interplay between phosphorylation and ubiquitylation controls cell signalling networks in health and disease.

PGE(2) induces macrophage IL-10 production and a regulatory-like phenotype via a protein kinase A-SIK-CRTC3 pathway.

The polarization of macrophages into a regulatory-like phenotype and the production of IL-10 plays an important role in the resolution of inflammation. We show in this study that PGE(2), in combination with LPS, is able to promote an anti-inflammatory phenotype in macrophages characterized by high expression of IL-10 and the regulatory markers SPHK1 and LIGHT via a protein kinase A-dependent pathway. Both TLR agonists and PGE(2) promote the phosphorylation of the transcription factor CREB on Ser(133). However, although CREB regulates IL-10 transcription, the mutation of Ser(133) to Ala in the endogenous CREB gene did not prevent the ability of PGE(2) to promote IL-10 transcription. Instead, we demonstrate that protein kinase A regulates the phosphorylation of salt-inducible kinase 2 on Ser(343), inhibiting its ability to phosphorylate CREB-regulated transcription coactivator 3 in cells. This in turn allows CREB-regulated transcription coactivator 3 to translocate to the nucleus where it serves as a coactivator with the transcription factor CREB to induce IL-10 transcription. In line with this, we find that either genetic or pharmacological inhibition of salt-inducible kinases mimics the effect of PGE(2) on IL-10 production.

Phosphorylation of CRTC3 by the salt-inducible kinases controls the interconversion of classically activated and regulatory macrophages.

Macrophages acquire strikingly different properties that enable them to play key roles during the initiation, propagation, and resolution of inflammation. Classically activated (M1) macrophages produce proinflammatory mediators to combat invading pathogens and respond to tissue damage in the host, whereas regulatory macrophages (M2b) produce high levels of anti-inflammatory molecules, such as IL-10, and low levels of proinflammatory cytokines, like IL-12, and are important for the resolution of inflammatory responses. A central problem in this area is to understand how the formation of regulatory macrophages can be promoted at sites of inflammation to prevent and/or alleviate chronic inflammatory and autoimmune diseases. Here, we demonstrate that the salt-inducible kinases (SIKs) restrict the formation of regulatory macrophages and that their inhibition induces striking increases in many of the characteristic markers of regulatory macrophages, greatly stimulating the production of IL-10 and other anti-inflammatory molecules. We show that SIK inhibitors elevate IL-10 production by inducing the dephosphorylation of cAMP response element-binding protein (CREB)-regulated transcriptional coactivator (CRTC) 3, its dissociation from 14-3-3 proteins and its translocation to the nucleus where it enhances a gene transcription program controlled by CREB. Importantly, the effects of SIK inhibitors on IL-10 production are lost in macrophages that express a drug-resistant mutant of SIK2. These findings identify SIKs as a key molecular switch whose inhibition reprograms macrophages to an anti-inflammatory phenotype. The remarkable effects of SIK inhibitors on macrophage function suggest that drugs that target these protein kinases may have therapeutic potential for the treatment of inflammatory and autoimmune diseases.

The IkappaB kinase family phosphorylates the Parkinson's disease kinase LRRK2 at Ser935 and Ser910 during Toll-like receptor signaling.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are strongly associated with late-onset autosomal dominant Parkinson's disease. LRRK2 is highly expressed in immune cells and recent work points towards a link between LRRK2 and innate immunity. Here we demonstrate that stimulation of the Toll-Like Receptor (TLR) pathway by MyD88-dependent agonists in bone marrow-derived macrophages (BMDMs) or RAW264.7 macrophages induces marked phosphorylation of LRRK2 at Ser910 and Ser935, the phosphorylation sites that regulate the binding of 14-3-3 to LRRK2. Phosphorylation of these residues is prevented by knock-out of MyD88 in BMDMs, but not the alternative TLR adaptor protein TRIF. Utilising both pharmacological inhibitors, including a new TAK1 inhibitor, NG25, and genetic models, we provide evidence that both the canonical (IKKα and IKKß) and IKK-related (IKKε and TBK1) kinases mediate TLR agonist induced phosphorylation of LRRK2 in vivo. Moreover, all four IKK members directly phosphorylate LRRK2 at Ser910 and Ser935 in vitro. Consistent with previous work describing Ser910 and Ser935 as pharmacodynamic biomarkers of LRRK2 activity, we find that the TLR independent basal phosphorylation of LRRK2 at Ser910 and Ser935 is abolished following treatment of macrophages with LRRK2 kinase inhibitors. However, the increased phosphorylation of Ser910 and Ser935 induced by activation of the MyD88 pathway is insensitive to LRRK2 kinase inhibitors. Finally, employing LRRK2-deficient BMDMs, we present data indicating that LRRK2 does not play a major role in regulating the secretion of inflammatory cytokines induced by activation of the MyD88 pathway. Our findings provide the first direct link between LRRK2 and the IKKs that mediate many immune responses. Further work is required to uncover the physiological roles that phosphorylation of LRRK2 by IKKs play in controlling macrophage biology and to determine how phosphorylation of LRRK2 by IKKs impacts upon the use of Ser910 and Ser935 as pharmacodynamic biomarkers.

The TRAF-associated protein TANK facilitates cross-talk within the IkappaB kinase family during Toll-like receptor signaling.

Toll-like receptor (TLR) ligands that signal via TIR-domain-containing adapter-inducing IFNß (TRIF) activate the IκB kinase (IKK)-related kinases, TRAF associated NFκB activator (TANK)-binding kinase-1 (TBK1) and IKKε, which then phosphorylate IRF3 and induce the production of IFNß. Here we show that TBK1 and IKKε are also activated by TLR ligands that signal via MyD88. Notably, the activation of IKKε is rapid, transient, and it precedes a more prolonged activation of TBK1. The MyD88- and TRIF-dependent signaling pathways activate the IKK-related kinases by two signaling pathways. One is mediated by the canonical IKKs, whereas the other culminates in the autoactivation of the IKK-related kinases. Once activated, TBK1/IKKε then phosphorylate and inhibit the canonical IKKs. The negative regulation of the canonical IKKs by the IKK-related kinases occurs in both the TRIF- and MyD88-dependent TLR pathways, whereas IRF3 phosphorylation is restricted to the TRIF-dependent signaling pathway. We have discovered that the activation of IKKε is abolished, the activation of TBK1 is reduced, and the interaction between the IKK-related kinases and the canonical IKKs is suppressed in TANK(-/-) macrophages, preventing the IKK-related kinases from negatively regulating the canonical IKKs. In contrast, IRF3 phosphorylation and IFNß production was normal in TANK(-/-) macrophages. Our results demonstrate a key role for TANK in enabling the canonical IKKs and the IKK-related kinases to regulate each other, which is required to limit the strength of TLR signaling and ultimately, prevent autoimmunity.

Novel cross-talk within the IKK family controls innate immunity.

Members of the IKK {IκB [inhibitor of NF-κB (nuclear factor κB)] kinase} family play a central role in innate immunity by inducing NF-κB- and IRF [IFN (interferon) regulatory factor]-dependent gene transcription programmes required for the production of pro-inflammatory cytokines and IFNs. However, the molecular mechanisms that activate these protein kinases and their complement of physiological substrates remain poorly defined. Using MRT67307, a novel inhibitor of IKKϵ/TBK1 (TANK {TRAF [TNF (tumour-necrosis-factor)-receptor-associated factor]-associated NF-κB activator}-binding kinase 1) and BI605906, a novel inhibitor of IKKß, we demonstrate that two different signalling pathways participate in the activation of the IKK-related protein kinases by ligands that activate the IL-1 (interleukin-1), TLR (Toll-like receptor) 3 and TLR4 receptors. One signalling pathway is mediated by the canonical IKKs, which directly phosphorylate and activate IKKϵ and TBK1, whereas the second pathway appears to culminate in the autocatalytic activation of the IKK-related kinases. In contrast, the TNFα-induced activation of the IKK-related kinases is mediated solely by the canonical IKKs. In turn, the IKK-related kinases phosphorylate the catalytic subunits of the canonical IKKs and their regulatory subunit NEMO (NF-κB essential modulator), which is associated with reduced IKKα/ß activity and NF-κB-dependent gene transcription. We also show that the canonical IKKs and the IKK-related kinases not only have unique physiological substrates, such as IκBα, p105, RelA (IKKα and IKKß) and IRF3 (IKKϵ and TBK1), but also have several substrates in common, including the catalytic and regulatory (NEMO and TANK) subunits of the IKKs themselves. Taken together, our studies reveal that the canonical IKKs and the IKK-related kinases regulate each other by an intricate network involving phosphorylation of their catalytic and regulatory (NEMO and TANK) subunits to balance their activities during innate immunity.

The alpha-kinase family: an exceptional branch on the protein kinase tree.

The alpha-kinase family represents a class of atypical protein kinases that display little sequence similarity to conventional protein kinases. Early studies on myosin heavy chain kinases in Dictyostelium discoideum revealed their unusual propensity to phosphorylate serine and threonine residues in the context of an alpha-helix. Although recent studies show that some members of this family can also phosphorylate residues in non-helical regions, the name alpha-kinase has remained. During evolution, the alpha-kinase domains combined with many different functional subdomains such as von Willebrand factor-like motifs (vWKa) and even cation channels (TRPM6 and TRPM7). As a result, these kinases are implicated in a large variety of cellular processes such as protein translation, Mg(2+) homeostasis, intracellular transport, cell migration, adhesion, and proliferation. Here, we review the current state of knowledge on different members of this kinase family and discuss the potential use of alpha-kinases as drug targets in diseases such as cancer.

Use of the pharmacological inhibitor BX795 to study the regulation and physiological roles of TBK1 and IkappaB kinase epsilon: a distinct upstream kinase mediates Ser-172 phosphorylation and activation.

TANK-binding kinase 1 (TBK1) and IkappaB kinase epsilon (IKKepsilon) regulate the production of Type 1 interferons during bacterial and viral infection, but the lack of useful pharmacological inhibitors has hampered progress in identifying additional physiological roles of these protein kinases and how they are regulated. Here we demonstrate that BX795, a potent and relatively specific inhibitor of TBK1 and IKKepsilon, blocked the phosphorylation, nuclear translocation, and transcriptional activity of interferon regulatory factor 3 and, hence, the production of interferon-beta in macrophages stimulated with poly(I:C) or lipopolysaccharide (LPS). In contrast, BX795 had no effect on the canonical NFkappaB signaling pathway. Although BX795 blocked the autophosphorylation of overexpressed TBK1 and IKKepsilon at Ser-172 and, hence, the autoactivation of these protein kinases, it did not inhibit the phosphorylation of endogenous TBK1 and IKKepsilon at Ser-172 in response to LPS, poly(I:C), interleukin-1alpha (IL-1alpha), or tumor necrosis factor alpha and actually enhanced the LPS, poly(I:C), and IL-1alpha-stimulated phosphorylation of this residue. These results demonstrate that the phosphorylation of Ser-172 and the activation of TBK1 and IKKepsilon are catalyzed by a distinct protein kinase(s) in vivo and that TBK1 and IKKepsilon control a feedback loop that limits their activation by LPS, poly(I:C) and IL-1alpha (but not tumor necrosis factor alpha) to prevent the hyperactivation of these enzymes.

TRPM7 regulates myosin IIA filament stability and protein localization by heavy chain phosphorylation.

Deregulation of myosin II-based contractility contributes to the pathogenesis of human diseases, such as cancer, which underscores the necessity for tight spatial and temporal control of myosin II activity. Recently, we demonstrated that activation of the mammalian alpha-kinase TRPM7 inhibits myosin II-based contractility in a Ca(2+)- and kinase-dependent manner. However, the molecular mechanism is poorly defined. Here, we demonstrate that TRPM7 phosphorylates the COOH-termini of both mouse and human myosin IIA heavy chains--the COOH-terminus being a region that is critical for filament stability. Phosphorylated residues were mapped to Thr1800, Ser1803 and Ser1808. Mutation of these residues to alanine and that to aspartic acid lead to an increase and a decrease, respectively, in myosin IIA incorporation into the actomyosin cytoskeleton and accordingly affect subcellular localization. In conclusion, our data demonstrate that TRPM7 regulates myosin IIA filament stability and localization by phosphorylating a short stretch of amino acids within the alpha-helical tail of the myosin IIA heavy chain.

Myosin II and mechanotransduction: a balancing act.

Adherent cells respond to mechanical properties of the surrounding extracellular matrix. Mechanical forces, sensed at specialized cell-matrix adhesion sites, promote actomyosin-based contraction within the cell. By manipulating matrix rigidity and adhesion strength, new roles for actomyosin contractility in the regulation of basic cellular functions, including cell proliferation, migration and stem cell differentiation, have recently been discovered. These investigations demonstrate that a balance of forces between cell adhesion on the outside and myosin II-based contractility on the inside of the cell controls many aspects of cell behavior. Disturbing this balance contributes to the pathogenesis of various human diseases. Therefore, elaborate signaling networks have evolved that modulate myosin II activity to maintain tensional homeostasis. These include signaling pathways that regulate myosin light chain phosphorylation as well as myosin II heavy chain interactions.