scholarly journals Inhibition of ULK2 Autophagic Activity by Its Association with YAP

2016 ◽  
Vol 8 (1) ◽  
pp. 12 ◽  
Author(s):  
Sung Hwa Shin ◽  
Eun Jeoung Lee ◽  
Sunghee Hyun ◽  
Dowonkyoung Park ◽  
Sang Sun Kang
Keyword(s):  
Mammalian Cells ◽  
Signal Transduction Pathway ◽  
Transduction Pathway ◽  
Wild Type ◽  
Therapeutic Approaches ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Py Motif

Uncoordinated 51-like kinase 2 (ULK2) is a member of the serine/threonine kinase family that functions an essential role regulating autophagy in mammalian cells. As autophagy is implicated in normal cellular homeostasis and multiple diseases, better mechanisticinsight will drive thedevelopment of novel therapeutic approaches. Here, we present evidence that ULK2 interacts withYAP for its degradation and subcellular localization. A potential PPxY motif (328PPnY331) was identified, which is similar with the consensus PPxY motif in ULK2 S/P domain. TheP329A (PA) mutation in the PY motif of ULK2 abolished the YAP-ULK2 association.At first, we observed that ULK2 physically interacted to YAP in vivo and in vitro, using a pull-down approach. Secondly, ULK2 and YAP co- localized at the apical (or tight junction) membrane as visualized by confocal microscopy. Furthermore, the PA mutant substantially increased during autophagy than that of wild-type ULK2 or the P242A mutant in transient transfection assays. Thus, the association between ULK2 and YAP through the WW domain links autophagy and the Hippo signal transduction pathway.

scholarly journals Inhibition of Bcr serine kinase by tyrosine phosphorylation.

1996 ◽  
Vol 16 (3) ◽  
pp. 998-1005 ◽  
Author(s):  
J Liu ◽  
Y Wu ◽  
G Z Ma ◽  
D Lu ◽  
L Haataja ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Tyrosine Phosphorylation ◽  
Kinase Activity ◽  
Philadelphia Chromosome ◽  
Wild Type ◽  
Serine Kinase ◽  
Serine Threonine Kinase ◽  
Threonine Kinase

The first exon of the BCR gene encodes a new serine/threonine protein kinase. Abnormal fusion of the BCR and ABL genes, resulting from the formation of the Philadelphia chromosome (Ph), is the hallmark of Ph-positive leukemia. We have previously demonstrated that the Bcr protein is tyrosine phosphorylated within first-exon sequences by the Bcr-Abl oncoprotein. Here we report that in addition to tyrose 177 (Y-177), Y-360 and Y283 are phosphorylated in Bcr-Abl proteins in vitro. Moreover, Bcr tyrosine 360 is phosphorylated in vivo within both Bcr-Abl and Bcr. Bcr mutant Y177F had a greatly reduced ability to transphosphorylate casein and histone H1, whereas Bcr mutants Y177F and Y283F had wild-type activities. In contrast, the Y360F mutation had little effect on Bcr's autophosphorylation activity. Tyrosine-phosphorylated Bcr, phosphorylated in vitro by Bcr-Abl, was greatly inhibited in its serine/threonine kinase activity, impairing both auto- and transkinase activities of Bcr. Similarly, the isolation of Bcr from cells expressing Bcr-Abl under conditions that preserve phosphotyrosine residues also reduced Bcr's kinase activity. These results indicate that tyrosine 360 of Bcr is critical for the transphosphorylation activity of Bcr and that in Ph-positive leukemia, Bcr serine/threonine kinase activity is seriously impaired.

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.

Novel pathway for thyroid hormone receptor action through interaction with jun and fos oncogene activities

1991 ◽  
Vol 11 (12) ◽  
pp. 6016-6025
Author(s):  
X K Zhang ◽  
K N Wills ◽  
M Husmann ◽  
T Hermann ◽  
M Pfahl
Keyword(s):  
Signal Transduction ◽  
Dna Binding ◽  
Thyroid Hormone Receptor ◽  
Gene Activation ◽  
Signal Transduction Pathway ◽  
Large Family ◽  
Transduction Pathway ◽  
Regulatory Pathway

Many essential biological pathways, including cell growth, development, and metabolism, are regulated by thyroid hormones (THs). TH action is mediated by intracellular receptors that belong to a large family of ligand-dependent transcription factors, including the steroid hormone and retinoic acid receptors. So far it has been assumed that TH receptors (TRs) regulate gene transcription only through the classical protein-DNA interaction mechanism. Here we provide evidence for a regulatory pathway that allows cross-talk between TRs and the signal transduction pathway used by many growth factors, oncogenes, and tumor promoters. In transient transfection studies, we observed that the oncogenes c-jun and c-fos inhibit TR activities, while TRs inhibit induction of the c-fos promoter and repress AP-1 site-dependent gene activation. A truncated TR that lacks only 17 amino acids from the carboxy terminus can no longer antagonize AP-1 activity. The cross-regulation between TRs and the signal transduction pathway appears to be based on the ability of TRs to inhibit DNA binding of the transcription factor AP-1 in the presence of THs. The constituents of AP-1, c-Jun, and c-Fos, vice versa, can inhibit TR-induced gene activation in vivo, and c-Jun inhibits TR DNA binding in vitro. This novel regulatory pathway is likely to play a major role in growth control and differentiation by THs.

Functional interaction between p21rap1A and components of the budding pathway in Saccharomyces cerevisiae

1992 ◽  
Vol 12 (9) ◽  
pp. 4084-4092
Author(s):  
P C McCabe ◽  
H Haubruck ◽  
P Polakis ◽  
F McCormick ◽  
M A Innis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mammalian Cells ◽  
Bound States ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Amino Terminal ◽  
Loop Region

The rap1A gene encodes a 21-kDa, ras-related GTP-binding protein (p21rap1A) of unknown function. A close structural homolog of p21rap1A (65% identity in the amino-terminal two-thirds) is the RSR1 gene product (Rsr1p) of Saccharomyces cerevisiae. Although Rsr1p is not essential for growth, its presence is required for nonrandom selection of bud sites. To assess the similarity of these proteins at the functional level, wild-type and mutant forms of p21rap1A were tested for complementation of activities known to be fulfilled by Rsr1p. Expression of p21rap1A, like multicopy expression of RSR1, suppressed the conditional lethality of a temperature-sensitive cdc24 mutation. Point mutations predicted to affect the localization of p21rap1A or its ability to cycle between GDP and GTP-bound states disrupted suppression of cdc24ts, while other mutations in the 61-65 loop region improved suppression. Expression of p21rap1A could not, however, suppress the random budding phenotype of rsr1 cells. p21rap1A also apparently interfered with the normal activity of Rsrlp, causing random budding in diploid wild-type cells, suggesting an inability of p21rap1A to interact appropriately with Rsr1p regulatory proteins. Consistent with this hypothesis, we found an Rsr1p-specific GTPase-activating protein (GAP) activity in yeast membranes which was not active toward p21rap1A, indicating that p21rap1A may be predominantly GTP bound in yeast cells. Coexpression of human Rap1-specific GAP suppressed the random budding due to expression of p21rap1A or its derivatives, including Rap1AVal-12. Although Rap1-specific GAP stimulated the GTPase of Rsr1p in vitro, it did not dominantly interfere with Rsr1p function in vivo. A chimera consisting of Rap1A1-165::Rsr1p166-272 did not exhibit normal Rsr1p function in the budding pathway. These results indicated that p21rap1A and Rsr1p share at least partial functional homology, which may have implications for p21rap1A function in mammalian cells.

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.

scholarly journals Erythropoietin-Induced Activation of STAT5 Is Impaired in the Myelodysplastic Syndrome

Blood ◽  
1997 ◽  
Vol 89 (5) ◽  
pp. 1690-1700 ◽  
Author(s):  
Lies H. Hoefsloot ◽  
Martine P. van Amelsvoort ◽  
Lianne C.A.M. Broeders ◽  
Dorien C. van der Plas ◽  
Kirsten van Lom ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Myelodysplastic Syndrome ◽  
Signal Transduction Pathway ◽  
Binding Activity ◽  
Signal Transducer ◽  
Transduction Pathway ◽  
Normal Bone ◽  
Marrow Cells

Abstract Patients with myelodysplastic syndrome (MDS) have ineffective in vivo and in vitro erythropoiesis, characterized by an impaired response to erythropoietin (Epo). We examined proliferation and maturation of MDS marrow cells in response to Epo in more detail. Epo-dependent DNA synthesis as well as induction of GATA-1 binding activity in marrow cells from 15 MDS cases were severely reduced as compared with normal bone marrow (NBM). Additionally, the appearance of morphologically identifiable erythroid cells was decreased in MDS cell cultures. These data indicate that both the Epo-dependent proliferation as well as the differentiation induction by Epo is suppressed. To study more upstream events of the Epo signal transduction route we investigated activation of the signal transducer and activator of transcription (STAT) 5. In all 15 MDS samples tested, STAT5 activation was absent or greatly suppressed in response to Epo. In contrast, interleukin-3 induced a normal STAT5 response in MDS cells. Further, in MDS the subset of CD71+ BM cells that is phenotypically similar to Epo-responsive cells in normal marrow, was present. We conclude that the Epo response in MDS is disturbed at an early point in the Epo receptor (EpoR) signal transduction pathway.

scholarly journals CP-31398 Restores DNA-binding Activity to Mutant p53in Vitrobut Does Not Affect p53 Homologs p63 and p73

2004 ◽  
Vol 279 (44) ◽  
pp. 45887-45896 ◽  
Author(s):  
Mark J. Demma ◽  
Serena Wong ◽  
Eugene Maxwell ◽  
Bimalendu Dasmahapatra
Keyword(s):  
Dna Binding ◽  
Mammalian Cells ◽  
P53 Protein ◽  
Binding Activity ◽  
Mutant P53 ◽  
Dependent Manner ◽  
Wild Type ◽  
Dna Binding Activity

The p53 protein plays a major role in the maintenance of genome stability in mammalian cells. Mutations of p53 occur in over 50% of all cancers and are indicative of highly aggressive cancers that are hard to treat. Recently, there has been a high degree of interest in therapeutic approaches to restore growth suppression functions to mutant p53. Several compounds have been reported to restore wild type function to mutant p53. One such compound, CP-31398, has been shown effectivein vivo, but questions have arisen to whether it actually affects p53. Here we show that mutant p53, isolated from cells treated with CP-31398, is capable of binding to p53 response elementsin vitro. We also show the compound restores DNA-binding activity to mutant p53 in cells as determined by a chromatin immunoprecipitation assay. In addition, using purified p53 core domain from two different hotspot mutants (R273H and R249S), we show that CP-31398 can restore DNA-binding activity in a dose-dependent manner. Using a quantitative DNA binding assay, we also show that CP-31398 increases significantly the amount of mutant p53 that binds to cognate DNA (Bmax) and its affinity (Kd) for DNA. The compound, however, does not affect the affinity (Kdvalue) of wild type p53 for DNA and only increasesBmaxslightly. In a similar assay PRIMA1 does not have any effect on p53 core DNA-binding activity. We also show that CP-31398 had no effect on the DNA-binding activity of p53 homologs p63 and p73.

Rhogtpase RAC2 Is Required for In Vivo Transformation Activity by P210 Bcr-Abl Fusion Oncogene.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 717-717
Author(s):  
Nithya Krishnan ◽  
Jeff R. Bailey ◽  
Victoria Summey-Harner ◽  
Claudio Brunstein ◽  
Catherine M. Verfaillie ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Philadelphia Chromosome ◽  
Protein Domain ◽  
Myelogenous Leukemia ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Cell Functions ◽  
Empty Vector

Abstract Bcr-Abl, the translocation product of the Philadelphia chromosome implicated in human chronic myelogenous leukemia (CML), is a kinase affecting hematopoietic stem cell (HSC) behavior with respect to proliferation, apoptosis, adhesion and migration. Rho GTPases, particularly the Rac subfamily, have been shown to regulate these same cell functions in normal HSC and also regulate gene expression in many mammalian cells. BCR contains a “GTPase-activating protein” domain and a guanine nucleotide exchange domain, the latter or which is preserved in p210 Bcr-Abl. Since HSC functions regulated by Bcr-Abl and Rac are similar, we studied the potential involvement of Rac activation in Bcr-Abl signaling cascade. Human CML samples demonstrate baseline activation of Rac proteins that is reversed by in vitro treatment with STI571. To study the specific involvement of Rac2, we used a gene targeted mouse model with Rac2 null bone marrow. Using retovirus-mediated gene transfer, we introduced p210 Bcr-Abl in the MSCV vector into wild-type or Rac2−/− HSC/P and studied the behavior of these cells in vitro and in vivo. Irradiated recipient mice injected with LDBM cells transduced with p210 developed a uniformly fatal myeloproliferative syndrome (Median survival: 45 days, N=12), while mice injected with p210 transduced Rac2−/− LDBM cells (N=12, 2 independent exp.) had 100% survival and no development of leukocytosis, splenomegaly or organ infiltration of hematopoietic cells. These data suggest that Rac GTPases are critical for the transformation of HSC by Bcr-Abl and provide an additional therapeutic target for intervention in CML. WILD TYPE Rac 2 −/− Empty Vector MSCV-p210 Empty vector MSCV-p210 *p < 0.01 vs WT-MIEG3, **p< 0.01 vs WT-p210 bcr-abl. Proliferation (CPM) Medium 562 ± 278 16,207± 1605* 819.7 ± 363 3,135.5 ± 498** SCF (100ng/ml) 856 ± 187 23,226 ± 2203* 853.7 ± 524 3,756.8 ± 207** Cytokines (SCF, GCSF, MGDF) 8011± 1412 42,711± 13393* 4833 ±1019 3,614.5 ± 1982** Migration (%) Fibronectin 7 ± 0.4 38 ± 1.9* 0.4 ± 0.0 0.8 ± 0.1** SDF-1α 30 ±2.8 13 ±1.1* 0.5 ± 0.0 0.6 ± 0.0** Adhesion (% ) Fibronectin 76± 2.9 40 ±3* 4 ±0.4 10 ±0.1 **

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