Browsing by Subject "Protein Kinases"

Now showing 1 - 8 of 8
  • Thumbnail Image
    Item
  • Thumbnail Image
    Item
    Haemophilus ducreyi RpoE and CpxRA Appear To Play Distinct yet Complementary Roles in Regulation of Envelope-Related Functions
    (Journal of Bacteriology, 2014-12) Gangaiah, Dharanesh; Zhang, Xinjun; Baker, Beth; Fortney, Kate R.; Liu, Yunlong; Munson, Robert S. Jr.; Spinola, Stanley M.; Department of Microbiology & Immunology, IU School of Medicine
    Haemophilus ducreyi causes the sexually transmitted disease chancroid and a chronic limb ulceration syndrome in children. In humans, H. ducreyi is found in an abscess and overcomes a hostile environment to establish infection. To sense and respond to membrane stress, bacteria utilize two-component systems (TCSs) and extracytoplasmic function (ECF) sigma factors. We previously showed that activation of CpxRA, the only intact TCS in H. ducreyi, does not regulate homologues of envelope protein folding factors but does downregulate genes encoding envelope-localized proteins, including many virulence determinants. H. ducreyi also harbors a homologue of RpoE, which is the only ECF sigma factor in the organism. To potentially understand how H. ducreyi responds to membrane stress, here we defined RpoE-dependent genes using transcriptome sequencing (RNA-Seq). We identified 180 RpoE-dependent genes, of which 98% were upregulated; a major set of these genes encodes homologues of envelope maintenance and repair factors. We also identified and validated a putative RpoE promoter consensus sequence, which was enriched in the majority of RpoE-dependent targets. Comparison of RpoE-dependent genes to those controlled by CpxR showed that each transcription factor regulated a distinct set of genes. Given that RpoE activated a large number of genes encoding envelope maintenance and repair factors and that CpxRA represses genes encoding envelope-localized proteins, these data suggest that RpoE and CpxRA appear to play distinct yet complementary roles in regulating envelope homeostasis in H. ducreyi.
  • Thumbnail Image
    Item
    JNK2 modulates the CD1d-dependent and -independent activation of iNKT cells
    (Wiley, 2019-02) Liu, Jianyun; Gallo, Richard M.; Khan, Masood A.; Iyer, Abhirami K.; Kratzke, Ian M.; Brutkiewicz, Randy R.; Microbiology and Immunology, School of Medicine
    Invariant Natural Killer T (iNKT) cells play critical roles in autoimmune, anti-tumor and anti-microbial immune responses, and are activated by glycolipids presented by the MHC class I-like molecule, CD1d. How the activation of signaling pathways impacts antigen (Ag)-dependent iNKT cell activation is not well-known. In the current study, we found that the MAPK JNK2 not only negatively regulates CD1d-mediated Ag presentation in APCs, but also contributes to CD1d-independent iNKT cell activation. A deficiency in the JNK2 (but not JNK1) isoform enhanced Ag presentation by CD1d. Using a vaccinia virus (VV) infection model known to cause a loss in iNKT cells in a CD1d-independent, but IL-12-dependent manner, we found the virus-induced loss of iNKT cells in JNK2 KO mice was substantially lower than that observed in JNK1 KO or wildtype (WT) mice. Importantly, compared to WT mice, JNK2 KO mouse iNKT cells were found to express less surface IL-12 receptors. As with a VV infection, an IL-12 injection also resulted in a smaller decrease in JNK2 KO iNKT cells as compared to WT mice. Overall, our work strongly suggests JNK2 is a negative regulator of CD1d-mediated Ag presentation and contributes to IL-12-induced iNKT cell activation and loss during viral infections.
  • Thumbnail Image
    Item
  • Thumbnail Image
    Item
    Phosphorylation of a Myosin Motor by TgCDPK3 Facilitates Rapid Initiation of Motility during Toxoplasma gondii egress
    (Public Library of Science (PLoS), 2015) Gaji, Rajshekhar Y.; Johnson, Derrick E.; Treeck, Moritz; Wang, Mu; Hudmon, Andy; Arrizabalaga, Gustavo; Department of Pharmacology and Toxicology, IU School of Medicine
    Members of the family of calcium dependent protein kinases (CDPK's) are abundant in certain pathogenic parasites and absent in mammalian cells making them strong drug target candidates. In the obligate intracellular parasite Toxoplasma gondii TgCDPK3 is important for calcium dependent egress from the host cell. Nonetheless, the specific substrate through which TgCDPK3 exerts its function during egress remains unknown. To close this knowledge gap we applied the proximity-based protein interaction trap BioID and identified 13 proteins that are either near neighbors or direct interactors of TgCDPK3. Among these was Myosin A (TgMyoA), the unconventional motor protein greatly responsible for driving the gliding motility of this parasite, and whose phosphorylation at serine 21 by an unknown kinase was previously shown to be important for motility and egress. Through a non-biased peptide array approach we determined that TgCDPK3 can specifically phosphorylate serines 21 and 743 of TgMyoA in vitro. Complementation of the TgmyoA null mutant, which exhibits a delay in egress, with TgMyoA in which either S21 or S743 is mutated to alanine failed to rescue the egress defect. Similarly, phosphomimetic mutations in the motor protein overcome the need for TgCDPK3. Moreover, extracellular Tgcdpk3 mutant parasites have motility defects that are complemented by expression of S21+S743 phosphomimetic of TgMyoA. Thus, our studies establish that phosphorylation of TgMyoA by TgCDPK3 is responsible for initiation of motility and parasite egress from the host-cell and provides mechanistic insight into how this unique kinase regulates the lytic cycle of Toxoplasma gondii.
  • Thumbnail Image
    Item
    Pyruvate Dehydrogenase Kinase 4 Promotes Vascular Calcification via SMAD1/5/8 Phosphorylation
    (Nature Publishing Group, 2015-11-12) Lee, Sun Joo; Jeong, Ji Yun; Oh, Chang Joo; Park, Sungmi; Kim, Joon-Young; Kim, Han-Jong; Doo Kim, Nam; Choi, Young-Keun; Do, Ji-Yeon; Go, Younghoon; Ha, Chae-Myung; Choi, Je-Yong; Huh, Seung; Ho Jeoung, Nam; Lee, Ki-Up; Choi, Hueng-Sik; Wang, Yu; Park, Keun-Gyu; Harris, Robert A.; Lee, In-Kyu; Department of Biochemistry & Molecular Biology, IU School of Medicine
    Vascular calcification, a pathologic response to defective calcium and phosphate homeostasis, is strongly associated with cardiovascular mortality and morbidity. In this study, we have observed that pyruvate dehydrogenase kinase 4 (PDK4) is upregulated and pyruvate dehydrogenase complex phosphorylation is increased in calcifying vascular smooth muscle cells (VSMCs) and in calcified vessels of patients with atherosclerosis, suggesting that PDK4 plays an important role in vascular calcification. Both genetic and pharmacological inhibition of PDK4 ameliorated the calcification in phosphate-treated VSMCs and aortic rings and in vitamin D3-treated mice. PDK4 augmented the osteogenic differentiation of VSMCs by phosphorylating SMAD1/5/8 via direct interaction, which enhances BMP2 signaling. Furthermore, increased expression of PDK4 in phosphate-treated VSMCs induced mitochondrial dysfunction followed by apoptosis. Taken together, our results show that upregulation of PDK4 promotes vascular calcification by increasing osteogenic markers with no adverse effect on bone formation, demonstrating that PDK4 is a therapeutic target for vascular calcification.
  • Thumbnail Image
    Item
    Recognition of DNA Termini by the C-Terminal Region of the Ku80 and the DNA-Dependent Protein Kinase Catalytic Subunit
    (Public Library of Science, 2015) Woods, Derek S.; Sears, Catherine R.; Turchi, John J.; Department of Medicine, IU School of Medicine
    DNA double strand breaks (DSBs) can be generated by endogenous cellular processes or exogenous agents in mammalian cells. These breaks are highly variable with respect to DNA sequence and structure and all are recognized in some context by the DNA-dependent protein kinase (DNA-PK). DNA-PK is a critical component necessary for the recognition and repair of DSBs via non-homologous end joining (NHEJ). Previously studies have shown that DNA-PK responds differentially to variations in DSB structure, but how DNA-PK senses differences in DNA substrate sequence and structure is unknown. Here we explore the enzymatic mechanisms by which DNA-PK is activated by various DNA substrates and provide evidence that the DNA-PK is differentially activated by DNA structural variations as a function of the C-terminal region of Ku80. Discrimination based on terminal DNA sequence variations, on the other hand, is independent of the Ku80 C-terminal interactions and likely results exclusively from DNA-dependent protein kinase catalytic subunit interactions with the DNA. We also show that sequence differences in DNA termini can drastically influence DNA repair through altered DNA-PK activation. These results indicate that even subtle differences in DNA substrates influence DNA-PK activation and ultimately the efficiency of DSB repair.
  • Thumbnail Image
    Item