Variant IL-1 Receptor-Associated Kinase-1 Mediates Increased NF-κB Activity
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IL-1R-associated kinase (IRAK)-1 is a critical mediator of TLR/IL-1R-induced activation of the transcription factor NF-kappaB. We previously described that a commonly occurring IRAK-1 variant haplotype, containing amino acid changes from serine to phenylalanine at position 196 and from leucine to serine at position 532, is associated with increased activation of NF-kappaB in LPS-stimulated neutrophils from patients with sepsis-induced acute lung injury and also higher mortality and more severe clinical outcomes in such patients. To investigate the underlying molecular mechanisms, we examined the ability of wild-type and variant IRAK-1 to modulate NF-kappaB activation. We found increased NF-kappaB transcriptional activity and expression of NF-kappaB-dependent proinflammatory cytokines in IL-1beta-stimulated IRAK-1-deficient cells transfected with variant IRAK-1 as compared with IRAK-1 wild type. IkappaB-alpha degradation was faster and p65 phosphorylation more prolonged after IL-1beta stimulation in cells expressing the IRAK-1 variant. However, IL-1-induced activation of MAPKs and nuclear translocation of NF-kappaB are comparable in both IRAK-1 variant- and IRAK-1 wild-type-expressing cells. Autophosphorylation of the IRAK-1 variant is greater than that found with wild-type IRAK-1. Additionally, variant IRAK-1 has greater interaction with TNFR-associated factor 6 than does wild-type IRAK-1. The enhanced activity of variant IRAK-1 appeared to be due to the alteration at aa 532, with only minimal effects being associated with change at aa 196. These results demonstrate that variant IRAK-1 is associated with alterations in multiple intracellular events that are likely to contribute to increased NF-kappaB activation and inflammatory responses in individuals with this IRAK-1 haplotype.Keywords:
Proinflammatory cytokine
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Flexible linkers in CaMKII control the balance between activating and inhibitory autophosphorylation
Abstract The activity of Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) depends on the balance between activating and inhibitory autophosphorylation (Thr 286 and Thr 305/306, respectively, in the human α isoform). Variation in the lengths of the flexible linkers that connect the kinase domains of CaMKII to a central oligomeric hub could alter transphosphorylation rates within a holoenzyme, thereby affecting the balance of autophosphorylation outcomes. Using a single-molecule assay for visualization of CaMKII phosphorylation on glass, we show that the balance of autophosphorylation is flipped between CaMKII-α and CaMKII-β, the two principal isoforms in the brain. CaMKII-α, with a ∼30 residue kinase-hub linker, readily acquires activating autophosphorylation, which we show is resistant to removal by phosphatases. CaMKII-β, with a ∼200 residue kinase-hub linker, is biased towards inhibitory autophosphorylation. Thus, the responsiveness of CaMKII to calcium signals can be tuned by varying the relative levels of the α and β isoforms.
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Receptor tyrosine kinases undergo ligand-induced dimerization that promotes kinase domain trans-autophosphorylation. However, the kinase domains of the insulin receptor are effectively dimerized because of the covalent α2β2 holomeric structure. This fact has made it difficult to determine the molecular mechanism of intraholomeric autophosphorylation, but there is evidence for both cis- and trans-autophosphorylation in the absence and presence of insulin. Here, using the cytoplasmic kinase domain (CKD) of the human insulin receptor, we demonstrate that autophosphorylation in the juxtamembrane (JM) subdomain follows a cis-reaction pathway. JM autophosphorylation was independent of CKD concentration over the range 6 nM−3 μM and was characterized kinetically: Half-saturation (KATP) was observed at 75 μM ATP [5 mM Mn(CH3CO2)2] with a maximal rate of 0.24 mol of PO4 (mol of CKD)-1 min-1. Pairwise substitutions of Phe for Tyr in the other two autophosphorylation subdomains, generated by site-directed mutagenesis, altered the kinetics of JM autophosphorylation but did not change the pathway from a cis-reaction. Tyr1328,1334 to Phe (in the carboxy-terminal subdomain) yielded <2-fold increase in the efficiency of JM autophosphorylation, whereas Tyr1162,1163 to Phe (in the activation loop subdomain) yielded ≈38-fold increased efficiency of JM autophosphorylation, due predominantly to a 23-fold decreased KATP. These findings demonstrate basal state binding of ATP to the CKD leading to cis-autophosphorylation and novel basal state regulatory interactions among the subdomains of the insulin receptor kinase. On the basis of these results and the crystal structure of the conserved catalytic core of this kinase [Hubbard, S. R., et al. (1994) Nature 372, 746], a model is proposed which reconciles the JM cis-reaction and the activation loop cis-inhibition/trans-reaction with the complex kinetics of insulin receptor autophosphorylation [Kohanski, R. A. (1993) Biochemistry 32, 5766].
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Protein kinases regulate diverse physiological processes. Because many kinases preserve inherent autophosphorylation capability, autophosphorylation appears to be one of the most important mechanisms for cellular signaling. However, physiological functions of autophosphorylation are still largely unknown, other than the self-activation by phosphorylation of activation loop in the catalytic domain. REPRESSION OF SHOOT GROWTH (RSG) is the transcription factor involved in gibberellin (GA) feedback regulation. The tobacco (Nicotiana tabacum) Ca2+-dependent protein kinase, NtCDPK1, phosphorylates RSG, resulting in the negative regulation of RSG. NtCDPK1 was previously shown to be autophosphorylated in a Ca2+-dependent manner. Here, we investigated the functional importance of autophosphorylation in NtCDPK1. Ser-6 and Thr-21 were identified as autophosphorylation sites of NtCDPK1. Autophosphorylation not only reduced the binding affinity of NtCDPK1 for RSG, but also inhibited the homodimerization of NtCDPK1. Furthermore, autophosphorylation decreased the phosphorylation efficiency of RSG yet increased that of myelin basic protein. Ser-6 and Thr-21 of NtCDPK1 were phosphorylated in response to GAs in plants. The substitution of these autophosphorylation sites with Ala enhanced the NtCDPK1 overexpression-induced sensitization of seeds to a GA biosynthetic inhibitor during germination. These results suggest new functions of autophosphorylation in CDPKs, namely, autophosphorylation can prevent the excessive phosphorylation of substrates and alter the substrate preference of CDPKs.
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Objective To study the effect of NF-κB antisense oligodeoxynucleotides (AODN) on expression of NF-κB and proinflammatory factors. Methods The synthesized AODN modified by phosphorothioate at different concentrations were added to the medium of cultured RAW 264.7 cells (preincubation with lipopolysaccharide). Protein expressions of NF-κB p 65 、ICAM、iNOS were detected by SABC immuno-histochemical technique. Results The expressions of NF-κB p 65 、ICAM and iNOS of AODN groups were significantly lower than blank control and MODN groups. From the concentration of 1.0 μM to 5.0 μM , the inhibitory effects increased with the increasing concentration. Conclusion AODN can effectively inhibit the expression of NF-κB protein, thus can depress the expression of proinflammatory factors.
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Flexible linkers in CaMKII control the balance between activating and inhibitory autophosphorylation
The many variants of human Ca2+/calmodulin-dependent protein kinase II (CaMKII) differ in the lengths and sequences of disordered linkers connecting the kinase domains to the oligomeric hubs of the holoenzyme. CaMKII activity depends on the balance between activating and inhibitory autophosphorylation (on Thr 286 and Thr 305/306, respectively, in the human α isoform). Variation in the linkers could alter transphosphorylation rates within a holoenzyme and the balance of autophosphorylation outcomes. We show, using mammalian cell expression and a single-molecule assay, that the balance of autophosphorylation is flipped between CaMKII variants with longer and shorter linkers. For the principal isoforms in the brain, CaMKII-α, with a ~30 residue linker, readily acquires activating autophosphorylation, while CaMKII-β, with a ~200 residue linker, is biased towards inhibitory autophosphorylation. Our results show how the responsiveness of CaMKII holoenzymes to calcium signals can be tuned by varying the relative levels of isoforms with long and short linkers.
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Calmodulin-dependent protein kinase IV from rat cerebral cortex undergoes autophosphorylation in response to Ca2+ and calmodulin, resulting in its marked enzymatic activation. Autophosphorylation occurred at several sites on CaM-kinase IV, depending upon the enzyme concentration. Among them, Ser437 was almost exclusively phosphorylated at enzyme concentrations lower than 10 μg/ml, and autophosphorylation at Ser437 was responsible for marked activation of the enzyme through decreases in the Km values for its substrates and an increase in the Vmax value. The Ca2+/calmodulin-independent activity of CaM-kinase IV was also markedly stimulated by autophosphorylation, but even after autophosphorylation it amounted only about 17% of the total enzyme activity detected in the presence of Ca2+/calmodulin.
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The Phosphorylation is the key activity of nm23-H1. The aim of this study is to explore the effect of different amino acid mutation on the phosphorylation status of nm23-H1.The wild type nm23-H1 was as the control of this study. Autoradiography was used for detecting the serine and histidine autophosphorylation of wild type (WT) and mutant nm23-H1 (P96S, H118F, S120G and S44A); RP-HPLC was used for detecting the NDPK activity of above proteins.The autophosphorylation activities of serine and histidine from high to low were P96S, WT, S44A, S120G and H118F, respectively, while the NDPK activities from high to low were WT, S120G, P96S, S44A, H118F. A highly positive correlation was found between serine and histidine autophosphorylation activity of above proteins (r =0.985, P <0.01), but no significant correlation was found between NDPK and serine or histidine autophosphorylation activity (r=0.458, P >0.05, and r =0.482, P >0.05, respectively).Site mutation of nm23-H1 can affect the phosphorylation activity. H118 site was the key amino acid of kinase activity, P96 site maybe related to phosphotransferring, S120 was the site of histidine autophosphorylation and serine autophosphorylation, while the S44 site may be another amino acid which possessed NDPK activity.
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Tobacco (Nicotiana tabacum) Ca2+-dependent protein kinase 1 (NtCDPK1) is involved in feedback regulation of the plant hormone gibberellin through the phosphorylation of the transcription factor, REPRESSION OF SHOOT GROWTH (RSG). Previously, Ser-6 and Thr-21 were identified as autophosphorylation sites in NtCDPK1. Autophosphorylation of Ser-6 and Thr-21 not only decreases the binding affinity of NtCDPK1 for RSG, but also inhibits the homodimerization of NtCDPK1. Furthermore, autophosphorylation decreases the phosphorylation efficiency of RSG. We demonstrated that Ser-6 and Thr-21 of NtCDPK1 are phosphorylated in response to GAs in plants. The substitution of these autophosphorylation sites with Ala enhances the NtCDPK1 overexpression-induced sensitization of seeds to a GA biosynthetic inhibitor during germination. These findings suggested that autophosphorylation of Ser-6 and Thr-21 prevents excessive phosphorylation of RSG. In this study, we attempted to determine which autophosphorylation site is responsible for the functional regulation of NtCDPK1. Ser-6 was autophosphorylated within 1 min, whereas Thr-21 required over 5 min to be completely autophosphorylated. Furthermore, we found that Ser-6 and Thr-21 were autophosphorylated by inter- and intramolecular mechanisms, respectively, which may be reflected in the faster autophosphorylation of Ser-6. Although both autophosphorylation sites were involved in the reduction of the binding affinity of NtCDPK1 for RSG and the inhibition of NtCDPK1 homodimerization, autophosphorylation of Ser-6 alone was sufficient to decrease the kinase activity of NtCDPK1 for RSG. These results suggest that autophosphorylation of Ser-6 is important for the rapid reduction of NtCDPK1 kinase activity for RSG, whereas that of Thr-21 may play an auxiliary role.
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By combining biochemical experiments with computer modelling of biochemical reactions we elucidated some of the currently unresolved aspects of calcium-calmodulin-dependent protein kinase II (CaMKII) activation and autophosphorylation that might be relevant for its physiological function and provided a model that incorporates in detail the mechanism of CaMKII activation and autophosphorylation at T286 that is based on experimentally determined binding constants and phosphorylation rates. To this end, we developed a detailed state model of CaMKII activation and autophosphorylation based on the currently available literature, and constrained it with data from CaMKII autophosphorylation essays. Our model takes exact phosphorylation patterns of CaMKII holoenzymes into account, and is valid at physiologically relevant conditions where the concentrations of calcium and calmodulin are not saturating. Our results strongly suggest that even when bound to less than fully calcium-bound calmodulin, CaMKII is in the active state, and indicate that the autophosphorylation of T286 by an active non-phosphorylated CaMKII subunit is significantly faster than by an autophosphorylated CaMKII subunit. These results imply that CaMKII can be efficiently activated at significantly lower calcium concentrations than previously thought, which may explain how CaMKII gets activated at calcium concentrations existing at synapses in vivo. We also investigated the significance of CaMKII holoenzyme structure on CaMKII autophosphorylation and obtained estimates of previously unknown binding constants.
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