The leucine-rich repeat kinase2 (LRRK2) has been identified to be the gene causing autosomal dominant inherited Parkinson's disease(PD)8. The clinical features of this type of PD are similar to those of idiopathic PD, but the pathological changes are diverse. The mutation types and frequencies of the LRRK2 distribute unevenly in different populations. LRRK2 is a large complex protein with multiple functions and expresses widely in human body. Sequence alignment shows that LRRK2 might be a multiple function kinase for substrate phosphorylation and might also act as a scaffolding protein. Further study on the physiological function and pathogenic mechanism of LRRK2 will help to find out the possible pathogenesis and new treatment for PD.
Animals ; Continental Population Groups ; genetics ; Humans ; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 ; Mutation ; Parkinson Disease ; genetics ; pathology ; Protein-Serine-Threonine Kinases ; chemistry ; genetics ; metabolism ; Sequence Alignment

Arabidopsis ; chemistry ; genetics ; metabolism ; Arabidopsis Proteins ; chemistry ; genetics ; metabolism ; Crystallography, X-Ray ; Intercellular Signaling Peptides and Proteins ; chemistry ; genetics ; metabolism ; Protein Conformation ; Protein-Serine-Threonine Kinases ; chemistry ; genetics ; metabolism

Antirheumatic gold compounds have been shown to inhibit NF-kB activation by blocking IkB kinase (IKK) activity. To examine the possible inhibitory mechanism of gold compounds, we expressed wild type and mutant forms of IKk alpha and beta subunits in COS-7 cells and determined the effect of gold on the activity of these enzymes both in vivo and in vitro. Substitution of Cys-179 of IKK beta with alanine (C179A) rendered the enzyme to become resistant to inhibition by a gold compound auranofin, however, similar protective effect was not observed with an equivalent level of IKK alpha (C178A) mutant expressed in the cells. Auranofin inhibited constitutively active IKK alpha and beta and variants; IKK alpha (S176E, S180E) or IKK beta (S177E, S181E), suggesting that gold directly cause inhibition of activated enzyme. The different inhibitory effect of auranofin on IKK alpha (C178A) and IKK beta (C179A) mutants indicates that gold could inhibit the two subunits of IKK in a different mode, and the inhibition of NF- kB and IKK activation induced by inflammatory signals in gold-treated cells appears through its interaction with Cys-179 of IKK beta.
Amino Acid Substitution ; Animals ; Auranofin/*pharmacology ; COS Cells ; Cysteine/genetics/*metabolism ; Enzyme Activation/drug effects ; Gold Compounds/*pharmacology ; Protein Subunits/chemistry ; Protein-Serine-Threonine Kinases/*antagonists & inhibitors/chemistry/genetics/*metabolism ; Sulfhydryl Compounds/pharmacology

In recent years, human cancer genome projects provide unprecedented opportunities for the discovery of cancer genes and signaling pathways that contribute to tumor development. While numerous gene mutations can be identified from each cancer genome, what these mutations mean for cancer is a challenging question to address, especially for those from less understood putative new cancer genes. As a powerful approach, in silico bioinformatics analysis could efficiently sort out mutations that are predicted to damage gene function. Such an analysis of human large tumor suppressor genes, LATS1 and LATS2, has been carried out and the results support a role of hLATS1//2 as negative growth regulators and tumor suppressors.
Adaptor Proteins, Signal Transducing ; chemistry ; metabolism ; Animals ; Carrier Proteins ; chemistry ; metabolism ; Computational Biology ; Genes, Neoplasm ; Humans ; LIM Domain Proteins ; chemistry ; metabolism ; Mice ; Mutation ; Neoplasms ; genetics ; pathology ; Phosphoproteins ; chemistry ; metabolism ; Phosphorylation ; Protein Binding ; Protein Structure, Tertiary ; Protein-Serine-Threonine Kinases ; chemistry ; genetics ; metabolism ; Transferases (Other Substituted Phosphate Groups) ; chemistry ; metabolism ; Tumor Suppressor Proteins ; chemistry ; genetics ; metabolism

OBJECTIVETo investigate the role of interleukin-17 (IL-17) in alveolar fluid clearance in mice with acute lung injury (ALI) and explore the possible mechanism.

METHODSSixteen IL-17-knockout mice and 16 wild-type mice were both randomized for intratracheal instillation of PBS (control) on lipopolysaccharide (LPS) to induce ALI. Forty-eight hours after the treatments, the wet-dry ratio (W/D) of the lungs, IL-8 in the bronchoalveolar lavage fluid (BALF) and histopathological changes of the lung tissues were examined. The expressions of epithelial sodium channel α subunit (α-ENaC) was detected with Western blotting and liver kinase B1 (LKB1) was detected with immunohistochemistry.

RESULTSCompared with wild-type mice treated with LPS, IL-17 knockout mice showed significantly decreased W/D of the lungs (9.739∓3.3 vs 5.351∓0.56) and IL-8 level in the BALF (67.50∓7.33 vs 41.00∓3.16 pg/mL) following LPS challenge. Pathological examination revealed reduced alveolar edema fluid aggregations and lower lung injury score in IL-17 knockout mice with also higher expression levels of ENaC and LKB1 compared with the wild-type mice.

CONCLUSIONKnocking out IL-17 in mice not only alleviates inflammation of the lung tissue following ALI but also reduces the loss of ENaC protein and promotes alveolar fluid clearance, mechanism of which is probably associated with LKB1.

Acute Lung Injury ; metabolism ; Animals ; Bronchoalveolar Lavage Fluid ; chemistry ; Epithelial Sodium Channels ; metabolism ; Gene Knockout Techniques ; Interleukin-17 ; genetics ; metabolism ; Interleukin-8 ; metabolism ; Lipopolysaccharides ; Lung ; pathology ; Mice ; Protein-Serine-Threonine Kinases ; metabolism

Checkpoint kinase 1 (Chk1) and Chk2 are effector kinases in the cellular DNA damage response and impairment of their function is closely related to tumorigenesis. Previous studies revealed several substrate proteins of Chk1 and Chk2, but identification of additional targets is still important in order to understand their tumor suppressor functions. In this study, we screened novel substrates for Chk1 and Chk2 using substrate target motifs determined previously by an oriented peptide library approach. The potential candidates were selected by genome-wide peptide database searches and were examined by in vitro kinase assays. ST5, HDAC5, PGC-1alpha, PP2A PR130, FANCG, GATA3, cyclin G, Rad51D and MAD1alpha were newly identified as in vitro substrates for Chk1 and/or Chk2. Among these, HDAC5 and PGC-1alpha were further analyzed to substantiate the screening results. Immunoprecipitation kinase assay of full-length proteins and site-directed mutagenesis analysis of the target motifs demonstrated that HDAC5 and PGC-1alpha were specific targets for Chk1 and/or Chk2 at least in vitro.
Amino Acid Motifs ; Amino Acid Sequence ; *Consensus Sequence ; Genome, Human/*genetics ; Heat-Shock Proteins/chemistry/metabolism ; Histone Deacetylases/chemistry/metabolism ; Humans ; Molecular Sequence Data ; Peptide Fragments/chemistry/metabolism ; Phosphorylation ; Phosphoserine/metabolism ; Protein Kinases/*metabolism ; Protein-Serine-Threonine Kinases/*metabolism ; Substrate Specificity ; Transcription Factors/chemistry/metabolism

The research of p53 is being conducted to find the mechanisms of tumorigenesis and to treat various cancers. Homeodomain-interacting protein kinase2 (HIPK2) is an important factor to regulate p53 and to increase the stability of p53. Activation of HIPK2 leads to the selective phosphorylation of p53, resulting in growth arrest and the enhancement of apoptosis. In this study, the canine HIPK2 cDNA fragments were obtained, and their overlapping regions were aligned to give a total sequence of 3489 bp. The canine HIPK2 cDNA (GenBank accession number; AY800385) shares 93% and 90% sequence identity with those of human and mouse HIPK2, respectively. The canine HIPK2 cDNA contains an open reading frame encoding 1163 amino acid residues and the predicted amino acid sequence has 98% and 96% identity with those of human and mouse, respectively. The deduced amino acid sequence of canine HIPK2 has also all domains' sites compared with human and mouse HIPK2. Therefore, these structural similarities suggested that the canine HIPK2 shares the basic biological functions that HIPK2 exhibit in other species.
Amino Acid Sequence ; Animals ; Base Sequence ; Cloning, Molecular ; DNA, Complementary/chemistry/genetics ; Dogs/metabolism/*physiology ; Male ; Molecular Sequence Data ; Polymerase Chain Reaction/veterinary ; Protein-Serine-Threonine Kinases/*genetics ; Sequence Alignment ; Sequence Analysis, DNA

We previously reported that the p53 tumor suppressor protein plays an essential role in the induction of tetraploid G1 arrest in response to perturbation of the actin cytoskeleton, termed actin damage. In this study, we investigated the role of p53, ataxia telangiectasia mutated protein (ATM), and catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) in tetraploid G1 arrest induced by actin damage. Treatment with actin-damaging agents including pectenotoxin-2 (PTX-2) increases phosphorylation of Ser-15 and Ser-37 residues of p53, but not Ser-20 residue. Knockdown of ATM and DNA-PKcs do not affect p53 phosphorylation induced by actin damage. However, while ATM knockdown does not affect tetraploid G1 arrest, knockdown of DNA-PKcs not only perturbs tetraploid G1 arrest, but also results in formation of polyploidy and induction of apoptosis. These results indicate that DNA-PKcs is essential for the maintenance of actin damage induced-tetraploid G1 arrest in a p53-independent manner. Furthermore, actin damage-induced p53 expression is not observed in cells synchronized at G1/S of the cell cycle, implying that p53 induction is due to actin damage-induced tetraploidy rather than perturbation of actin cytoskeleton. Therefore, these results suggest that p53 and DNA-PKcs independently function for tetraploid G1 arrest and preventing polyploidy formation.
Actins/*metabolism ; Apoptosis ; Catalytic Domain ; Cell Cycle Proteins/genetics/*metabolism ; Cell Line ; Cell Line, Tumor ; DNA-Activated Protein Kinase/chemistry/genetics/*metabolism ; DNA-Binding Proteins/genetics/*metabolism ; Furans/pharmacology ; *G1 Phase ; Gene Knockdown Techniques ; Humans ; Phosphorylation/drug effects ; Protein-Serine-Threonine Kinases/genetics/*metabolism ; Pyrans/pharmacology ; Tumor Suppressor Protein p53/*metabolism ; Tumor Suppressor Proteins/genetics/*metabolism

To observe the effects of Danshen-containing serum on SuFu and DYRK2 expression in the HSCs stimulated by leptin. SD rats (n = 60) were used to make danshen-containing serum by gastric perfusion for ten days with Danshen water decoction, normal saline and colchicine. The HSCs that were cultured in vitro would be stimulated for 24 hours by leptin (100 μg x L(-1)) except blank control group, after being intervened, the drug serum in each group would be cultured at 37 degrees C in 5% incubator. The cells would be collected after 24 hours, then the effects of danshen-containing serum on the proliferation of HSCs were detected by MTT, the expression of SuFu mRNA and DYRK2 mRNA were detected by RT-PCR, the expression of SuFu and DYRK2 proteins were tested by Western blot. Compared with blank control group, the expression of DYRK2 mRNA and DYRK2 proteins were enhanced obviously after stimulated the HSCs of rats by leptin (P < 0.01), but the expression of SuFu mRNA and SuFu proteins were decreased significantly (P < 0.01). Compared with the model group, after cyclopamine group (Hh pathway inhibitor), Danshen-containing serum and colchicine were interfered, the expression of DYRK2 mRNA and DYRK2 proteins were decreased clearly (P < 0.01), but the expression of SuFu mRNA and SuFu proteins were increased significantly (P < 0.01 or P < 0.05). Compared with model group, adding purmorphamine (Hh pathway agonist) to model group and making it activate could increase the expression of DYRK2 mRNA and DYRK2 proteins, but the expression of SuFu mRNA and SuFu proteins were decreased significantly (P < 0.01). Compared with the model group, using the Danshen-containing serum to interfere the purmorphamine group could make the expression of DYRK2 mRNA and DYRK2 proteins decrease and the expression of SuFu mRNA and SuFu proteins increase significantly (P < 0.01). Danshen-containing serum would inhibition the activation and increment of HSCs by interfering the expression of SuFu and DYRK2 which were induced by leptin.
Animals ; Drugs, Chinese Herbal ; administration & dosage ; Female ; Hepatic Stellate Cells ; drug effects ; metabolism ; Humans ; Liver Cirrhosis ; drug therapy ; genetics ; metabolism ; Male ; Protein-Serine-Threonine Kinases ; genetics ; metabolism ; Protein-Tyrosine Kinases ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Repressor Proteins ; genetics ; metabolism ; Salvia miltiorrhiza ; chemistry

SARS coronavirus (SARS-CoV) develops an antagonistic mechanism by which to evade the antiviral activities of interferon (IFN). Previous studies suggested that SARS-CoV papain-like protease (PLpro) inhibits activation of the IRF3 pathway, which would normally elicit a robust IFN response, but the mechanism(s) used by SARS PLpro to inhibit activation of the IRF3 pathway is not fully known. In this study, we uncovered a novel mechanism that may explain how SARS PLpro efficiently inhibits activation of the IRF3 pathway. We found that expression of the membrane-anchored PLpro domain (PLpro-TM) from SARS-CoV inhibits STING/TBK1/IKKε-mediated activation of type I IFNs and disrupts the phosphorylation and dimerization of IRF3, which are activated by STING and TBK1. Meanwhile, we showed that PLpro-TM physically interacts with TRAF3, TBK1, IKKε, STING, and IRF3, the key components that assemble the STING-TRAF3-TBK1 complex for activation of IFN expression. However, the interaction between the components in STING-TRAF3-TBK1 complex is disrupted by PLpro-TM. Furthermore, SARS PLpro-TM reduces the levels of ubiquitinated forms of RIG-I, STING, TRAF3, TBK1, and IRF3 in the STING-TRAF3-TBK1 complex. These results collectively point to a new mechanism used by SARS-CoV through which PLpro negatively regulates IRF3 activation by interaction with STING-TRAF3-TBK1 complex, yielding a SARS-CoV countermeasure against host innate immunity.
Dimerization ; HEK293 Cells ; Humans ; I-kappa B Kinase ; metabolism ; Interferon Regulatory Factor-3 ; metabolism ; Interferon Type I ; antagonists & inhibitors ; metabolism ; Membrane Proteins ; chemistry ; genetics ; metabolism ; Papain ; metabolism ; Peptide Hydrolases ; chemistry ; metabolism ; Phosphorylation ; Protein Binding ; Protein Structure, Tertiary ; Protein-Serine-Threonine Kinases ; metabolism ; SARS Virus ; enzymology ; Signal Transduction ; TNF Receptor-Associated Factor 3 ; metabolism ; Ubiquitination