scholarly journals An Intramolecular Association between Two Domains of the Protein Kinase Fused Is Necessary for Hedgehog Signaling

2004 ◽  
Vol 24 (23) ◽  
pp. 10397-10405 ◽  
Author(s):  
Manuel Ascano ◽  
David J. Robbins
Keyword(s):  
Signal Transduction ◽  
Protein Kinase ◽  
Hedgehog Signaling ◽  
Membrane Vesicles ◽  
Kinase Domain ◽  
Kinase Assay ◽  
Dependent Manner ◽  
Intramolecular Association

ABSTRACT The protein kinase Fused (Fu) is an integral member of the Hedgehog (Hh) signaling pathway. Although genetic studies demonstrate that Fu is required for the regulation of the Hh pathway, the mechanistic role that it plays remains largely unknown. Given our difficulty in developing an in vitro kinase assay for Fu, we reasoned that the catalytic activity of Fu might be highly regulated. Several mechanisms are known to regulate protein kinases, including self-association in either an intra- or an intermolecular fashion. Here, we provide evidence that Hh regulates Fu through intramolecular association between its kinase domain (ΔFu) and its carboxyl-terminal domain (Fu-tail). We show that ΔFu and Fu-tail can interact in trans, with or without the kinesin-related protein Costal 2 (Cos2). However, since the majority of Fu is found associated with Cos2 in vivo, we hypothesized that Fu-tail, which binds Cos2 directly, would be able to tether ΔFu to Cos2. We demonstrate that ΔFu colocalizes with Cos2 in the presence of Fu-tail and that this colocalization occurs on a subset of membrane vesicles previously characterized to be important for Hh signal transduction. Additionally, expression of Fu-tail in fu mutant flies that normally express only the kinase domain rescues the fu wing phenotype. Therefore, reestablishing the association between these two domains of Fu in trans is sufficient to restore Hh signal transduction in vivo. In such a manner we validate our hypothesis, demonstrating that Fu self-associates and is functional in an Hh-dependent manner. Our results here enhance our understanding of one of the least characterized, yet critical, components of Hh signal transduction.

scholarly journals Normal cellular and transformation-associated abl proteins share common sites for protein kinase C phosphorylation.

1987 ◽  
Vol 7 (12) ◽  
pp. 4280-4289 ◽  
Author(s):  
A M Pendergast ◽  
J A Traugh ◽  
O N Witte
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Growth Factor Receptor ◽  
Kinase Domain ◽  
Kinase Assay ◽  
Amino Terminal ◽  
Kinase C ◽  
Carboxy Terminal

Viral transduction and chromosomal translocations of the c-abl gene result in the synthesis of abl proteins with structurally altered amino termini. These altered forms of the abl protein, but not the c-abl proteins, are detectably phosphorylated on tyrosine in vivo. In contrast, all forms of the abl protein are phosphorylated on serine following in vivo labeling with Pi. Treatment of NIH-3T3 cells with protein kinase C activators resulted in a four- to eightfold increase in the phosphorylation of murine c-abl due to modification of two serines on the c-abl protein. Purified protein kinase C phosphorylated all abl proteins at the same two sites. Both sites are precisely conserved in murine and human abl proteins. The sites on the abl proteins were found near the carboxy terminus. In contrast, for the epidermal growth factor receptor (T. Hunter, N. Ling, and J. A. Cooper, Nature [London] 311:480-483, 1984) and pp60src (K. L. Gould, J. R. Woodgett, J. A. Cooper, J. E. Buss, D. Shalloway, and T. Hunter, Cell 42:849-857, 1985), the sites of protein kinase C phosphorylation are amino-terminal to the kinase domain. The abl carboxy-terminal region is not necessary for the tyrosine kinase activity or transformation potential of the viral abl protein and may represent a regulatory domain. Using an in vitro immune complex kinase assay, we were not able to correlate reproducible changes in c-abl activity with phosphorylation by protein kinase C. However, the high degree of conservation of the phosphorylation sites for protein kinase C between human and mouse abl proteins suggests an important functional role.

Scutellarin Suppresses RPMI7951 Melanoma Cell Proliferation by Targeting TOPK

2020 ◽  
Vol 20 ◽  
Author(s):  
Xin Mu ◽  
Lijuan Wang ◽  
Zixi Zhang ◽  
Rui Ge ◽  
Jian Zhang ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinases ◽  
Histone H3 ◽  
Soft Agar ◽  
Kinase Assay ◽  
Dependent Manner ◽  
Dose Dependent ◽  
Extracellular Regulated Protein Kinases

Background: T-LAK cell-originated protein kinase (TOPK) belongs to the serine/threonine protein kinase family. It is highly expressed in RPMI7951 melanoma cells. Scutellarin (SCU) is an active ingredient extracted from Erigeron breviscapus (Vant.) Hand.–Mazz. Its main physiological functions are related to its anti-inflammatory and antitumour activities. Methods: The relationship between SCU and TOPK was assessed by molecular docking, an in vitro binding assay and an in vitro kinase assay. The effect of SCU on RPMI7951 cells was detected by MTS and soft agar assays. TOPK knockdown was induced by lentiviral infection. The TOPK downstream signalling pathway was detected by western blot and immunohistochemical analyses in vitro and in vivo. Results: SCU was found to directly bind with TOPK and inhibit TOPK activity in vitro. SCU inhibited the proliferation and colony formation of RPMI7951 cells in a dose-dependent manner. Silencing TOPK decreased the sensitivity of colon cancer cells to SCU. SCU inhibited the phosphorylation levels of extracellular regulated protein kinases 1/2 (ERK1/2) and histone H3 in a time- and dose-dependent manner in RPMI7951 cells. In addition, SCU inhibited the growth of xenograft tumours of RPMI7951 cells and decreased the phosphorylation levels of extracellular regulated protein kinases 1/2 and histone H3 in vivo. Conclusion: The results showed that SCU exerts promising antitumour effects on human RPMI7951 cells by inhibiting the activity of TOPK.

Normal cellular and transformation-associated abl proteins share common sites for protein kinase C phosphorylation

1987 ◽  
Vol 7 (12) ◽  
pp. 4280-4289
Author(s):  
A M Pendergast ◽  
J A Traugh ◽  
O N Witte
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Growth Factor Receptor ◽  
Kinase Domain ◽  
Kinase Assay ◽  
Amino Terminal ◽  
Kinase C ◽  
Carboxy Terminal

Viral transduction and chromosomal translocations of the c-abl gene result in the synthesis of abl proteins with structurally altered amino termini. These altered forms of the abl protein, but not the c-abl proteins, are detectably phosphorylated on tyrosine in vivo. In contrast, all forms of the abl protein are phosphorylated on serine following in vivo labeling with Pi. Treatment of NIH-3T3 cells with protein kinase C activators resulted in a four- to eightfold increase in the phosphorylation of murine c-abl due to modification of two serines on the c-abl protein. Purified protein kinase C phosphorylated all abl proteins at the same two sites. Both sites are precisely conserved in murine and human abl proteins. The sites on the abl proteins were found near the carboxy terminus. In contrast, for the epidermal growth factor receptor (T. Hunter, N. Ling, and J. A. Cooper, Nature [London] 311:480-483, 1984) and pp60src (K. L. Gould, J. R. Woodgett, J. A. Cooper, J. E. Buss, D. Shalloway, and T. Hunter, Cell 42:849-857, 1985), the sites of protein kinase C phosphorylation are amino-terminal to the kinase domain. The abl carboxy-terminal region is not necessary for the tyrosine kinase activity or transformation potential of the viral abl protein and may represent a regulatory domain. Using an in vitro immune complex kinase assay, we were not able to correlate reproducible changes in c-abl activity with phosphorylation by protein kinase C. However, the high degree of conservation of the phosphorylation sites for protein kinase C between human and mouse abl proteins suggests an important functional role.

scholarly journals Current translational potential and underlying molecular mechanisms of necroptosis

2019 ◽  
Vol 10 (11) ◽  
Author(s):  
Tamás Molnár ◽  
Anett Mázló ◽  
Vera Tslaf ◽  
Attila Gábor Szöllősi ◽  
Gabriella Emri ◽  
...  
Keyword(s):  
Cell Death ◽  
Protein Kinase ◽  
Posttranslational Modifications ◽  
Molecular Mechanisms ◽  
Inflammatory Diseases ◽  
Immune Surveillance ◽  
Kinase Domain ◽  
Tumor Formation

Abstract Cell death has a fundamental impact on the evolution of degenerative disorders, autoimmune processes, inflammatory diseases, tumor formation and immune surveillance. Over the past couple of decades extensive studies have uncovered novel cell death pathways, which are independent of apoptosis. Among these is necroptosis, a tightly regulated, inflammatory form of cell death. Necroptosis contribute to the pathogenesis of many diseases and in this review, we will focus exclusively on necroptosis in humans. Necroptosis is considered a backup mechanism of apoptosis, but the in vivo appearance of necroptosis indicates that both caspase-mediated and caspase-independent mechanisms control necroptosis. Necroptosis is regulated on multiple levels, from the transcription, to the stability and posttranslational modifications of the necrosome components, to the availability of molecular interaction partners and the localization of receptor-interacting serine/threonine-protein kinase 1 (RIPK1), receptor-interacting serine/threonine-protein kinase 3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL). Accordingly, we classified the role of more than seventy molecules in necroptotic signaling based on consistent in vitro or in vivo evidence to understand the molecular background of necroptosis and to find opportunities where regulating the intensity and the modality of cell death could be exploited in clinical interventions. Necroptosis specific inhibitors are under development, but >20 drugs, already used in the treatment of various diseases, have the potential to regulate necroptosis. By listing necroptosis-modulated human diseases and cataloging the currently available drug-repertoire to modify necroptosis intensity, we hope to kick-start approaches with immediate translational potential. We also indicate where necroptosis regulating capacity should be considered in the current applications of these drugs.

14-3-3 Isotypes facilitate coupling of protein kinase C-ζ to Raf-1: negative regulation by 14-3-3 phosphorylation

2000 ◽  
Vol 345 (2) ◽  
pp. 297-306 ◽  
Author(s):  
Paulus C. J. VAN DER HOEVEN ◽  
José C. M. VAN DER WAL ◽  
Paula RUURS ◽  
Marc C. M. VAN DIJK ◽  
Wim J. VAN BLITTERSWIJK
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Complex Formation ◽  
Dependent Manner ◽  
Cos Cells ◽  
Kinase C ◽  
Pkc Ζ ◽  
Co Precipitation

14-3-3 Proteins may function as adapters or scaffold in signal-transduction pathways. We found previously that protein kinase C-ζ (PKC-ζ) can phosphorylate and activate Raf-1 in a signalling complex [van Dijk, Hilkmann and van Blitterswijk (1997) Biochem. J. 325, 303-307]. We report now that PKC-ζ-Raf-1 interaction is mediated by 14-3-3 proteins in vitro and in vivo. Co-immunoprecipitation experiments in COS cells revealed that complex formation between PKC-ζ and Raf-1 is mediated strongly by the 14-3-3β and -θ isotypes, but not by 14-3-3ζ. Far-Western blotting revealed that 14-3-3 binds PKC-ζ directly at its regulatory domain, where a S186A mutation in a putative 14-3-3-binding domain strongly reduced the binding and the complex formation with 14-3-3β and Raf-1. Treatment of PKC-ζ with lambda protein phosphatase also reduced its binding to 14-3-3β in vitro. Preincubation of an immobilized Raf-1 construct with 14-3-3β facilitated PKC-ζ binding. Together, the results suggest that 14-3-3 binds both PKC-ζ (at phospho-Ser-186) and Raf-1 in a ternary complex. Complex formation was much stronger with a kinase-inactive PKC-ζ mutant than with wild-type PKC-ζ, supporting the idea that kinase activity leads to complex dissociation. 14-3-3β and -θ were substrates for PKC-ζ, whereas 14-3-3ζ was not. Phosphorylation of 14-3-3β by PKC-ζ negatively regulated their physical association. 14-3-3β with its putative PKC-ζ phosphorylation sites mutated enhanced co-precipitation between PKC-ζ and Raf-1, suggesting that phosphorylation of 14-3-3 by PKC-ζ weakens the complex in vivo. We conclude that 14-3-3 facilitates coupling of PKC-ζ to Raf-1 in an isotype-specific and phosphorylation-dependent manner. We suggest that 14-3-3 is a transient mediator of Raf-1 phosphorylation and activation by PKC-ζ.

343 PHOSPHATIDYLINOSITOL 3-KINASE IS INVOLVED IN THE MAINTENANCE OF METAPHASE II ARREST IN PORCINE OOCYTES MATURED IN VITRO

2010 ◽  
Vol 22 (1) ◽  
pp. 328
Author(s):  
N. Kashiwazaki ◽  
M. Shimada ◽  
J. Ito
Keyword(s):  
Protein Kinase ◽  
Time Dependent ◽  
Kinase Assay ◽  
Dependent Manner ◽  
Phosphatidylinositol 3 Kinase ◽  
Mapk Activity ◽  
Pronuclear Formation ◽  
Pkb Phosphorylation ◽  
Phosphatidylinositol 3

It has been reported that phosphatidylinositol 3-kinase (PI3K)-protein kinase B (PKB) pathway plays a crucial role in the meiotic resumption and progression to the metaphase II (MII) stage of oocytes. However, the role of this pathway in meiotic arrest at the MII stage (cytostatic activity) is not well understood. In this study, the effect of a PI3K inhibitor, LY294002, on the mitogen-activated protein kinase (MAPK) and p34cdc2 kinase activities of matured porcine oocytes was examined. Immature oocytes were collected from ovaries and cultured in modified NCSU37 up to 48 hr. After culture, cumulus cells were removed and oocytes were cultured up to 24 h in medium supplemented with 25 or 50 μM LY294002. Groups of 10 or 20 oocytes were collected at each culture period for in vitro kinase assay of p34cdc2 kinase and MAPK, respectively. Groups of 40 oocytes were also used for detection of PKB phosphorylation by Western blotting. After maturation culture, both the p34cdc2 kinase and MAPK activities in the oocytes were gradually decreased in a time-dependent manner. Although 25 μM LY294002 did not affect either the p34cdc2 kinase or MAPK activities, 50 μM LY294002 suppressed the PKB phosphorylation and slightly decreased MAPK activity, but not the p34cdc2 kinase activity. Next, the effect of 10 μM Ca2+ ionophore which was reported as inducing a transient decrease of p342+ kinase but not MAPK activities, was examined in LY294002-treated oocytes. Pronuclear formation of the oocytes was also evaluated by the aceto-orcein staining. By additional treatment with LY294002 after Ca2+ ionophore, both the MAPK and p34cdc2 kinase activities were decreased in a time-dependent manner, concomitantly with improvement of pronuclear formation. Therefore, we concluded that PI3K is possibly involved in the maintenance of MAPK activity in matured porcine oocytes. The work was supported in part by Grant-in-Aid for Scientific Research from JSPS (KAKENHI) (21789253) to J.I. This work was also supported in part by the Promotion and Mutual Aid Corporation for Private Schools of Japan through a Grant-in-Aid for Matching Fund Subsidy for Private Universities to J.I. and N.K.

Taurine Activates BMP-2/Wnt3a-Mediated Osteoblast Differentiation and Mineralization via Akt and MAPK Signaling

2020 ◽  
Author(s):  
Minsu PARK ◽  
Hyeon Kyeong CHOI ◽  
Jeung Hee AN
Keyword(s):  
Protein Kinase ◽  
Osteoblast Differentiation ◽  
Integration Site ◽  
Mapk Signaling ◽  
Mitogen Activated Protein Kinase ◽  
Bone Morphogenetic Protein 2 ◽  
Dependent Manner ◽  
Ovx Rats

Background: We aimed to elucidate the preventive effects of taurine against osteopenia in ovariectomized (OVX) rats and the mechanisms by which taurine regulates osteoblastogenesis in vitro and in vivo. Methods: The effects of the taurine on human osteoblast MG-63 cell differentiation and osteoblastogenesis effect in OVX rat were examined Konkuk University in 2018 by evaluating osteoblast differentiation, and expression of osteoblast-specific factors by western blotting analysis. Results: Taurine supplementation significantly improved alkaline phosphatase (ALP) activity and mineralization in a concentration-dependent manner. Further, taurine induced the expression of osteogenic growth factors such as bone morphogenetic protein-2 (BMP-2), runt-related transcription factor 2 (RUNX2), small mothers against decapentaplegic 1/5/8 (SMAD1/5/8), wingless-type MMTV integration site family member 3A (Wnt3a), and collagen type 1 (COL-1) via mitogen-activated protein kinase (MAPK) and serine/threonine protein kinase (Akt). Moreover, the RUNX2 activity of the taurine-treated group was enhanced by proteinprotein interactions such as Wnt3a-induced p-AKT/RUNX2 and BMP-mediated SMADs/MAPK/RUNX2 interactions. Conclusion: Our in vitro and in vivo results suggested that taurine can be considered as a potential therapeutic candidate agent for preventing bone loss in postmenopausal osteoporosis.

scholarly journals FUS3 phosphorylates multiple components of the mating signal transduction cascade: evidence for STE12 and FAR1.

1993 ◽  
Vol 4 (5) ◽  
pp. 495-510 ◽  
Author(s):  
E A Elion ◽  
B Satterberg ◽  
J E Kranz
Keyword(s):  
Signal Transduction ◽  
Map Kinase ◽  
Maximal Activity ◽  
Kinase Assay ◽  
G1 Arrest ◽  
Multicopy Suppressor ◽  
Signal Transduction Cascade ◽  
Kinase Homologue

The mitogen-activated protein (MAP) kinase homologue FUS3 mediates both transcription and G1 arrest in a pheromone-induced signal transduction cascade in Saccharomyces cerevisiae. We report an in vitro kinase assay for FUS3 and its use in identifying candidate substrates. The assay requires catalytically active FUS3 and pheromone induction. STE7, a MAP kinase kinase homologue, is needed for maximal activity. At least seven proteins that specifically associate with FUS3 are phosphorylated in the assay. Many of these substrates are physiologically relevant and are affected by in vivo levels of numerous signal transduction components. One substrate is likely to be the transcription factor STE12. A second is likely to be FAR1, a protein required for G1 arrest. FAR1 was isolated as a multicopy suppressor of a nonarresting fus3 mutant and interacts with FUS3 in a two hybrid system. Consistent with this FAR1 is a good substrate in vitro and generates a FUS3-associated substrate of expected size. These data support a model in which FUS3 mediates transcription and G1 arrest by direct activation of STE12 and FAR1 and phosphorylates many other proteins involved in the response to pheromone.

scholarly journals SMG-1 Is a Phosphatidylinositol Kinase-Related Protein Kinase Required for Nonsense-Mediated mRNA Decay in Caenorhabditis elegans

2004 ◽  
Vol 24 (17) ◽  
pp. 7483-7490 ◽  
Author(s):  
Andrew Grimson ◽  
Sean O'Connor ◽  
Carrie Loushin Newman ◽  
Philip Anderson
Keyword(s):  
Protein Kinase ◽  
Mammalian Cells ◽  
Mrna Decay ◽  
Null Alleles ◽  
Dependent Manner ◽  
Messenger Rnas ◽  
Phosphatidylinositol Kinase ◽  
Nonsense Mediated Mrna Decay

ABSTRACT Eukaryotic messenger RNAs containing premature stop codons are selectively and rapidly degraded, a phenomenon termed nonsense-mediated mRNA decay (NMD). Previous studies with both Caenohabditis elegans and mammalian cells indicate that SMG-2/human UPF1, a central regulator of NMD, is phosphorylated in an SMG-1-dependent manner. We report here that smg-1, which is required for NMD in C. elegans, encodes a protein kinase of the phosphatidylinositol kinase superfamily of protein kinases. We identify null alleles of smg-1 and demonstrate that SMG-1 kinase activity is required in vivo for NMD and in vitro for SMG-2 phosphorylation. SMG-1 and SMG-2 coimmunoprecipitate from crude extracts, and this interaction is maintained in smg-3 and smg-4 mutants, both of which are required for SMG-2 phosphorylation in vivo and in vitro. SMG-2 is located diffusely through the cytoplasm, and its location is unaltered in mutants that disrupt the cycle of SMG-2 phosphorylation. We discuss the role of SMG-2 phosphorylation in NMD.

scholarly journals Adrenocorticotrophic hormone stimulates phosphotyrosine phosphatase SHP2 in bovine adrenocortical cells: phosphorylation and activation by cAMP-dependent protein kinase

2000 ◽  
Vol 352 (2) ◽  
pp. 483-490 ◽  
Author(s):  
Stéphane ROCCHI ◽  
Isabelle GAILLARD ◽  
Emmanuel VAN OBBERGHEN ◽  
Edmond M. CHAMBAZ ◽  
Isabelle VILGRAIN
Keyword(s):  
Protein Kinase ◽  
Kinase Assay ◽  
Dependent Manner ◽  
Adrenocorticotrophic Hormone ◽  
Adrenocortical Cells ◽  
Dependent Protein Kinase ◽  
Phosphotyrosine Phosphatase ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein

During activation of adrenocortical cells by adrenocorticotrophic hormone (ACTH), tyrosine dephosphorylation of paxillin is stimulated and this correlates with protrusion of filopodial structures and a decreased number of focal adhesions. These effects are inhibited by Na3VO4, a phosphotyrosine phosphatase inhibitor [Vilgrain, Chinn, Gaillard, Chambaz and Feige (1998) Biochem. J. 332, 533–540]. However, the tyrosine phosphatases involved in these processes remain to be identified. In this study, we provide evidence that the Src homology domain (SH)2-containing phosphotyrosine phosphatase (SHP)2, but not SHP1, is expressed in adrenocortical cells and is phosphorylated upon ACTH challenge. ACTH (10-8M) treatment of 32P-labelled adrenocortical cells resulted in an increase in phosphorylated SHP2. By probing SHP2-containing immunoprecipitates with an antibody to phosphoserine we found that SHP2 was phosphorylated on serine in ACTH-treated cells in a dose- and time-dependent manner. Furthermore, using an in vitro kinase assay, we showed that SHP2 was a target for cAMP-dependent protein kinase (PKA). Serine was identified as the only target amino acid phosphorylated in SHP2. Phosphorylation of SHP2 by PKA resulted in a dramatic stimulation of phosphatase activity measured either with insulin receptor substrate-1 or with the synthetic peptide [32P]poly(Glu/Tyr) as substrate. In an in-gel assay of SHP2-containing immunoprecipitates, phosphorylated in vitro by PKA or isolated from adrenocortical cells treated with 10nM ACTH, a pronounced activation of SHP2 activity was shown. These observations clearly support the idea that a PKA-mediated signal transduction pathway contributes to SHP2 regulation in adrenocortical cells and point to SHP2 as a possible mediator of the effects of ACTH.

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