scholarly journals XIAP controls RIPK2 signaling by preventing its deposition in speck-like structures

2019 ◽  
Author(s):  
Kornelia Ellwanger ◽  
Christine Arnold ◽  
Selina Briese ◽  
Ioannis Kienes ◽  
Jens Pfannstiel ◽  
...  
Keyword(s):  
Pattern Recognition ◽  
Complex Formation ◽  
Posttranslational Modifications ◽  
Molecular Structures ◽  
Host Cells ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Enteropathogenic Bacteria ◽  
Binding Partners

ABSTRACTThe receptor interacting serine/threonine kinase 2 (RIPK2) is essential for linking activation of the pattern recognition receptors NOD1 and NOD2 to cellular signaling events.Recently, it was shown that RIPK2 forms higher order molecular structuresin vitro, which were proposed to activate signaling. Here, we demonstrate that RIPK2 forms detergent insoluble complexes in the cytosol of host cells upon infection with invasive enteropathogenic bacteria. Formation of these structures occurred after NF-κB activation and depends on the CARD of NOD1 or NOD2. Complex formation upon activation was dependent on RIPK2 autophosphorylation at Y474 and influenced by phosphorylation at S176. Inhibition of activity of the cIAP protein XIAP induced spontaneous complex formation of RIPK2 but blocked NOD1-dependet NF-κB activation. Using immunoprecipitation, we identified 14-3-3 proteins as novel binding partners of non-activated RIPK2, whereas complexed RIPK2 was bound by the prohibitin proteins Erlin-1 and Erlin-2.Taken together, our work reveals novel roles of XIAP, 14-3-3 and Erlin proteins in the regulation of RIPK2 and expands our knowledge on the function of RIPK2 posttranslational modifications in NOD1/2 signaling.

scholarly journals XIAP controls RIPK2 signaling by preventing its deposition in speck-like structures

2019 ◽  
Vol 2 (4) ◽  
pp. e201900346 ◽  
Author(s):  
Kornelia Ellwanger ◽  
Selina Briese ◽  
Christine Arnold ◽  
Ioannis Kienes ◽  
Valentin Heim ◽  
...  
Keyword(s):  
Complex Formation ◽  
Posttranslational Modifications ◽  
Molecular Structures ◽  
Host Cells ◽  
Inhibitor Of Apoptosis ◽  
Caspase Activation ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Enteropathogenic Bacteria

The receptor interacting serine/threonine kinase 2 (RIPK2) is essential for linking activation of the pattern recognition receptors NOD1 and NOD2 to cellular signaling events. Recently, it was shown that RIPK2 can form higher order molecular structures in vitro. Here, we demonstrate that RIPK2 forms detergent insoluble complexes in the cytosol of host cells upon infection with invasive enteropathogenic bacteria. Formation of these structures occurred after NF-κB activation and depended on the caspase activation and recruitment domain of NOD1 or NOD2. Complex formation upon activation required RIPK2 autophosphorylation at Y474 and was influenced by phosphorylation at S176. We found that the E3 ligase X-linked inhibitor of apoptosis (XIAP) counteracts complex formation of RIPK2, accordingly mutation of the XIAP ubiquitylation sites in RIPK2 enhanced complex formation. Taken together, our work reveals novel roles of XIAP in the regulation of RIPK2 and expands our knowledge on the function of RIPK2 posttranslational modifications in NOD1/2 signaling.

scholarly journals Sit4p/PP6 regulates ER-to-Golgi traffic by controlling the dephosphorylation of COPII coat subunits

2013 ◽  
Vol 24 (17) ◽  
pp. 2727-2738 ◽  
Author(s):  
Deepali Bhandari ◽  
Jinzhong Zhang ◽  
Shekar Menon ◽  
Christopher Lord ◽  
Shuliang Chen ◽  
...  
Keyword(s):  
Golgi Complex ◽  
Loss Of Function ◽  
Serine Threonine Kinase ◽  
Vesicle Budding ◽  
Threonine Kinase ◽  
Binding Partners ◽  
Copii Vesicle ◽  
Membrane Bound

Traffic from the endoplasmic reticulum (ER) to the Golgi complex is initiated when the activated form of the GTPase Sar1p recruits the Sec23p-Sec24p complex to ER membranes. The Sec23p-Sec24p complex, which forms the inner shell of the COPII coat, sorts cargo into ER-derived vesicles. The coat inner shell recruits the Sec13p-Sec31p complex, leading to coat polymerization and vesicle budding. Recent studies revealed that the Sec23p subunit sequentially interacts with three different binding partners to direct a COPII vesicle to the Golgi. One of these binding partners is the serine/threonine kinase Hrr25p. Hrr25p phosphorylates the COPII coat, driving the membrane-bound pool into the cytosol. The phosphorylated coat cannot rebind to the ER to initiate a new round of vesicle budding unless it is dephosphorylated. Here we screen all known protein phosphatases in yeast to identify one whose loss of function alters the cellular distribution of COPII coat subunits. This screen identifies the PP2A-like phosphatase Sit4p as a regulator of COPII coat dephosphorylation. Hyperphosphorylated coat subunits accumulate in the sit4Δ mutant in vivo. In vitro, Sit4p dephosphorylates COPII coat subunits. Consistent with a role in coat recycling, Sit4p and its mammalian orthologue, PP6, regulate traffic from the ER to the Golgi complex.

Ability of PknA, a mycobacterial eukaryotic-type serine/threonine kinase, to transphosphorylate MurD, a ligase involved in the process of peptidoglycan biosynthesis

2008 ◽  
Vol 415 (1) ◽  
pp. 27-33 ◽  
Author(s):  
Meghna Thakur ◽  
Pradip K. Chakraborti
Keyword(s):  
Protein Kinases ◽  
Solid Phase ◽  
Morphological Changes ◽  
Binding Assays ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Peptidoglycan Biosynthesis ◽  
Vitro Binding ◽  
Solid Phase Binding

Eukaryotic-type serine/threonine protein kinases in bacteria have been implicated in controlling a host of cellular activities. PknA is one of eleven such protein kinases from Mycobacterium tuberculosis which regulates morphological changes associated with cell division. In the present study we provide the evidence for the ability of PknA to transphosphorylate mMurD (mycobacterial UDP-N-acetylmuramoyl-L-alanine:D-glutamate-ligase), the enzyme involved in peptidoglycan biosynthesis. Its co-expression in Escherichia coli along with PknA resulted in phosphorylation of mMurD. Consistent with these observations, results of the solid-phase binding assays revealed a high-affinity in vitro binding between the two proteins. Furthermore, overexpression of m-murD in Mycobacterium smegmatis yielded a phosphorylated protein. The results of the present study therefore point towards the possibility of mMurD being a substrate of PknA.

scholarly journals Regulation of the tumour suppressor Fbw7α by PKC-dependent phosphorylation and cancer-associated mutations

2010 ◽  
Vol 432 (1) ◽  
pp. 77-87 ◽  
Author(s):  
Joanne Durgan ◽  
Peter J. Parker
Keyword(s):  
Mammalian Cells ◽  
Tumour Suppressor ◽  
Dependent Manner ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Pkc Protein Kinase C ◽  
Specific Regulation ◽  
The Relationship ◽  
Substrate Binding Domain

Fbw7 (F-box WD40 protein 7) is a major tumour suppressor, which mediates the degradation of several potent oncogenes. PKC (protein kinase C) comprises a serine/threonine kinase family that can promote transformation when dysregulated. In the present study, we investigated the relationship between Fbw7 and PKC. Multiple members of the PKC superfamily interact with the substrate-binding domain of Fbw7. However, we find no evidence for Fbw7-mediated degradation of PKC. Instead, we demonstrate that Fbw7 is a novel substrate for PKC. Two residues within the isoform-specific N-terminus of Fbw7α are phosphorylated in a PKC-dependent manner, both in vitro and in mammalian cells (Ser10 and Ser18). Mutational analyses reveal that phosphorylation of Fbw7α at Ser10 can regulate its nuclear localization. Cancer-associated mutations in nearby residues (K11R and the addition of a proline residue at position 16) influence Fbw7α localization in a comparable manner, suggesting that mislocalization of this protein may be of pathological significance. Together these results provide evidence for both physical and functional interactions between the PKC and Fbw7 families, and yield insights into the isoform-specific regulation of Fbw7α.

scholarly journals WNK4 Kinase: from structure to physiology

2021 ◽  
Author(s):  
Adrian Rafael Murillo-de-Ozores ◽  
Alejandro Rodriguez-Gama ◽  
Hector Carbajal-Contreras ◽  
Gerardo Gamba ◽  
Maria Castaneda-Bueno
Keyword(s):  
Oxidative Stress ◽  
Mouse Models ◽  
Serine Threonine Kinase ◽  
Gene Encoding ◽  
Threonine Kinase ◽  
Physiological Conditions ◽  
Different Levels ◽  
Familial Hyperkalemic Hypertension

With No Lysine (K) kinase 4 (WNK4) belongs to a serine-threonine kinase family characterized by the atypical positioning of its catalytic lysine. Despite the fact that WNK4 has been found in many tissues, the majority of its study has revolved around its function in the kidney, specifically as a positive regulator of the thiazide-sensitive NaCl cotransporter (NCC) in the distal convoluted tubule (DCT) of the nephron. This is explained by the description of gain-of-function mutations in the gene encoding WNK4 that cause Familial Hyperkalemic Hypertension (FHHt). This disease is mainly driven by increased downstream activation of the Ste20-related Proline Alanine Rich Kinase (SPAK)/Oxidative Stress Responsive Kinase 1 (OSR1)-NCC pathway, which increases salt reabsorption in the DCT and indirectly impairs renal K+ secretion. Here, we review the large volume of information that has accumulated about different aspects of WNK4 function. We first review the knowledge on WNK4 structure and enumerate the functional domains and motifs that have been characterized. Then, we discuss WNK4 physiological functions based on the information obtained from in vitro studies and from a diverse set of genetically modified mouse models with altered WNK4 function. We then review in vitro and in vivo evidence on the different levels of regulation of WNK4. Finally, we go through the evidence that has suggested how different physiological conditions act through WNK4 to modulate NCC activity.

Arabidopsis MLK3, a Plant-specific Casein Kinase 1, Negatively Regulated Flowering and Phosphorylated Histone H3 in vivo

2019 ◽  
Author(s):  
Zhen Wang ◽  
Junmei Kang ◽  
Shangang Jia ◽  
Tiejun Zhang ◽  
Zhihai Wu ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Histone H3 ◽  
Casein Kinase ◽  
Kinase Assay ◽  
Wild Type ◽  
Serine Threonine Kinase ◽  
Altered Expression ◽  
Casein Kinase 1 ◽  
Threonine Kinase

Abstract Background: Casein kinase 1 (CK1) family members are highly conserved serine/threonine kinase present in most eukaryotes with multiple biological functions. Arabidopsis MUT9-like kinases ( MLKs ) belong to a clade CK1 specific to the plant kingdom and have been implicated collectively in modulating flowering related processes. Three of the four MLKs ( MLK1/2/4 ) have been characterized, however, little is known about MLK3 , the most divergent MLKs. Results: We demonstrated that compared with wild type, mlk3 , a truncated MLK3 , flowered slightly early under long day conditions and ectopic expression of MLK3 rescued the morphological defects of mlk3 , indicating that MLK3 negatively regulates flowering. GA 3 application accelerated flowering of both wild type and mlk3 , suggesting that mlk3 had normal GA response. The recombinant MLK3-GFP was localized in the nucleus exclusively. In vitro kinase assay revealed that the nuclear protein MLK3 phosphorylated histone 3 at threonine 3 (H3T3ph). Mutation of a conserved catalytic residue (Lysine 175) abolished the kinase activity and resulted in failure to complement the early flowering phenotype of mlk3 . Interestingly, the global level of H3T3 phosphorylation in mlk3 did not differ significantly from wild type, suggesting the redundant roles of MLKs in flowering regulation. The transcriptomic analysis demonstrated that 425 genes significantly altered expression level in mlk3 relative to wild type. The mlk3 mlk4 double mutant generated by crossing mlk3 with mlk4 , a loss-of-function mutant of MLK4 showing late flowering, flowered between the two parental lines, suggesting that MLK3 played an antagonistic role to MLK4 in plant transition to flowering. Conclusions: A serine/threonine kinase encoding gene MLK3 is a casein kinase 1 specific to the plant species and represses flowering slightly. MLK3 located in nucleus catalyzes the phosphorylation of histone H3 at threonine 3 in vitro and an intact lysine residue (K175) is indispensible for the kinase activity. This study sheds new light on the delicate control of flowering by the plant-specific CK1 in Arabidopsis.

The Serine/Threonine Kinase Pim-1 Stabilizes 130 Kilodalton FLT3 and Promotes Aberrant Signaling through STAT5 in Acute Myeloid Leukemia Cells with FLT3 Internal Tandem Duplication: Synergistic Apoptosis Induction by Pim-1 and FLT3 Inhibitors.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 942-942 ◽  
Author(s):  
Yingqiu Xie ◽  
Mehmet Burcu ◽  
Maria R. Baer
Keyword(s):  
Half Life ◽  
Myeloid Leukemia ◽  
Tandem Duplication ◽  
Kinase Activity ◽  
Serine Threonine Kinase ◽  
Internal Tandem Duplication ◽  
Threonine Kinase ◽  
Flt3 Inhibitors ◽  
Flt3 Internal Tandem Duplication

Abstract Abstract 942 Fms-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) results in FLT3 constitutive activation and aberrant signaling in acute myeloid leukemia (AML) cells. FLT3-ITD is associated with adverse treatment outcome in AML, but FLT3 inhibitors have had limited therapeutic efficacy. The oncogenic serine/threonine kinase Pim-1 is upregulated in AML cells with FLT3-ITD. Pim-1 inhibitors are entering clinical trials, and we sought to characterize the role of Pim-1 and the effects of Pim-1 inhibition in FLT3-ITD cells. Wild-type (WT) FLT3 exists predominantly in a 150 kDa complex glycosylated form. In contrast, FLT3-ITD is partially retained in the endoplasmic reticulum (ER) as a misfolded 130 kDa underglycosylated, or high-mannose, species in association with the ER transmembrane chaperone calnexin. In addition, FLT3-ITD also associates with and is stabilized by the cytosolic chaperone heat shock protein (HSP) 90. FLT3-ITD activates signal transducer and activation of transcription (STAT) 5 and upregulates the STAT5 downstream target Pim-1. FLT3 contains a putative Pim-1 substrate consensus serine phosphorylation site, and we hypothesized that FLT3 might be a Pim-1 substrate. FLT3-ITD cell lines studied included MV4-11, MOLM-14 and transfected Ba/F3-ITD, and FLT3 WT cells included BV173, EOL-1 and transfected Ba/F3-WT. Pim-1 activity was measured by an in vitro kinase assay of BAD phosphorylation at serine 112, and Pim-1 expression, FLT3 expression, phosphorylation and co-immunoprecipitation, and STAT5 phosphorylation and expression by Western blot analysis. Pim-1 knockdown was accomplished by infection with lentivirus containing Pim-1 small hairpin RNA (shRNA) or non-target control, and Pim-1 kinase inhibition by incubation with the Pim-1-selective inhibitor quercetagetin. Pim-1 was found to directly interact with and serine-phosphorylate FLT3 from FLT3-ITD, but not FLT3-WT, cells in vitro. Inhibition of Pim-1 kinase disrupted binding of FLT3 to its chaperones calnexin and HSP90, and resulted in decreased expression and half-life of 130 kDa FLT3 and increased expression and half-life of 150 kDa FLT3. The decrease in expression and half-life of 130 kDa FLT3 was partially abrogated by co-incubation with the proteasome inhibitor MG132. Moreover, the increase in 150 Kda FLT3 was abrogated by co-incubation with the glycosylation inhibitor 2-deoxy-D-glucose. Thus Pim-1 maintains FLT3 as a 130 kDa species by enhancing its binding to its chaperones calnexin and HSP90, protecting it from proteasomal degradation and inhibiting its glycosylation to form 150 kDa FLT3. Inhibition of Pim-1 kinase activity also decreased phosphorylation of FLT3 at tyrosine 591, a docking site for binding of FLT3-ITD, but not FLT3-WT, to STAT5, and decreased both STAT5 phosphorylation and expression of Pim-1 itself. In contrast, Pim-1 inhibition had no effect on FLT3 tyrosine kinase activity nor on expression of Pim-2, another Pim kinase family member implicated in promoting survival of FLT3-ITD cells. Finally, the Pim-1 kinase inhibitor quercetagetin and the FLT3 inhibitor PKC412 had a synergistic effect in inducing apoptosis of Ba/F3-ITD cells: We conclude that Pim-1, which is transcriptionally upregulated through STAT5 in FLT3-ITD cells, serine-phosphorylates FLT3-ITD, thereby maintaining it in an underglycosylated form, and promotes STAT5 signaling, and that inhibition of Pim-1 and of FLT3 is synergistic in inducing apoptosis of FLT3-ITD cells. Thus Pim-1 inhibitors should inhibit aberrant signaling upstream as well as downstream of Pim-1 in FLT3-ITD cells, and have the potential to enhance the therapeutic efficacy of FLT3 inhibitors in patients with AML with FLT3-ITD Disclosures: No relevant conflicts of interest to declare.

scholarly journals Protein Serine/Threonine Kinase StkP Positively Controls Virulence and Competence in Streptococcus pneumoniae

2004 ◽  
Vol 72 (4) ◽  
pp. 2434-2437 ◽  
Author(s):  
Jose Echenique ◽  
Aras Kadioglu ◽  
Susana Romao ◽  
Peter W. Andrew ◽  
Marie-Claude Trombe
Keyword(s):  
Streptococcus Pneumoniae ◽  
Lung Infection ◽  
Signaling Network ◽  
Serine Threonine Kinase ◽  
Positive Regulation ◽  
Threonine Kinase

ABSTRACT In the Streptococcus pneumoniae genome, stkP, encoding a membrane-associated serine/threonine kinase, is not redundant (L. Novakova, S. Romao, J. Echenique, P. Branny, and M.-C. Trombe, unpublished results). The data presented here demonstrate that StkP belongs to the signaling network involved in competence triggering in vitro and lung infection and bloodstream invasion in vivo. In competence, functional StkP is required for activation of comCDE upstream of the autoregulated ring orchestrated by the competence-stimulating peptide. This is the first description of positive regulation of comCDE transcription in balance with its repression by CiaRH.

scholarly journals LKB1 kinase-dependent and -independent defects disrupt polarity and adhesion signaling to drive collagen remodeling during invasion

2016 ◽  
Vol 27 (7) ◽  
pp. 1069-1084 ◽  
Author(s):  
Jessica Konen ◽  
Scott Wilkinson ◽  
Byoungkoo Lee ◽  
Haian Fu ◽  
Wei Zhou ◽  
...  
Keyword(s):  
Cell Polarity ◽  
Cancer Metastasis ◽  
Kinase Activity ◽  
Rho Gtpase ◽  
Three Dimensional ◽  
Serine Threonine Kinase ◽  
Collagen Remodeling ◽  
Threonine Kinase ◽  
Three Dimensional Models

LKB1 is a serine/threonine kinase and a commonly mutated gene in lung adenocarcinoma. The majority of LKB1 mutations are truncations that disrupt its kinase activity and remove its C-terminal domain (CTD). Because LKB1 inactivation drives cancer metastasis in mice and leads to aberrant cell invasion in vitro, we sought to determine how compromised LKB1 function affects lung cancer cell polarity and invasion. Using three-dimensional models, we show that LKB1 kinase activity is essential for focal adhesion kinase–mediated cell adhesion and subsequent collagen remodeling but not cell polarity. Instead, cell polarity is overseen by the kinase-independent function of its CTD and more specifically its farnesylation. This occurs through a mesenchymal-amoeboid morphological switch that signals through the Rho-GTPase RhoA. These data suggest that a combination of kinase-dependent and -independent defects by LKB1 inactivation creates a uniquely invasive cell with aberrant polarity and adhesion signaling that drives invasion into the microenvironment.

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