scholarly journals Caspase processing activates atypical protein kinase C ζ by relieving autoinhibition and destabilizes the protein

2003 ◽  
Vol 375 (3) ◽  
pp. 663-671 ◽  
Author(s):  
Lucinda SMITH ◽  
Zhi WANG ◽  
Jeffrey B. SMITH
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Hela Cells ◽  
Half Life ◽  
Kinase Activity ◽  
Ubiquitin Proteasome System ◽  
Kinase Domain ◽  
Kinase C ◽  
Pkc Ζ

Treatment of HeLa cells with tumour necrosis factor α (TNFα) induced caspase processing of ectopic PKC (protein kinase C) ζ, which converted most of the holoenzyme into the freed kinase domain and increased immune-complex kinase activity. The goal of the present study was to determine the basis for the increased kinase activity that is associated with caspase processing of PKC ζ. Atypical PKC ι is largely identical with PKC ζ, except for a 60-amino-acid segment that lacks the caspase-processing sites of the ζ isoform. Replacement of this segment of PKC ζ with the corresponding segment of PKC ι prevented caspase processing and activation of the kinase function. Processing of purified recombinant PKC ζ by caspase 3 in vitro markedly increased its kinase activity. Caspase processing activated PKC ζ in vitro or intracellularly without increasing the phosphorylation of Thr410 of PKC ζ, which is required for catalytic competency. The freed kinase domain of PKC ζ had a much shorter half-life than the holoenzyme in transfected HeLa cells and in non-transfected kidney epithelial cells. Treatment with TNF-α shortened the half-life of the kinase domain protein, and proteasome blockade stabilized the protein. Studies of kinase-domain mutants indicate that a lack of negative charge at Thr410 can shorten the half-life of the freed kinase domain. The present findings indicate that the freed kinase domain has substantially higher kinase activity and a much shorter half-life than the holoenzyme because of accelerated degradation by the ubiquitin–proteasome system.

scholarly journals 6,6′-Dihydroxythiobinupharidine (DTBN) Purified from Nuphar lutea Leaves Is an Inhibitor of Protein Kinase C Catalytic Activity

Molecules ◽  
2021 ◽  
Vol 26 (9) ◽  
pp. 2785
Author(s):  
Kamran Waidha ◽  
Nikhil Ponnoor Anto ◽  
Divya Ram Jayaram ◽  
Avi Golan-Goldhirsh ◽  
Saravanakumar Rajendran ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Kinase Activity ◽  
Kinase Domain ◽  
Water Lily ◽  
Activity Data ◽  
Nuphar Lutea ◽  
Kinase C ◽  
Disease Biology

Water lily (Nuphar) bioactive extracts have been widely used in traditional medicine owing to their multiple applications against human ailments. Phyto-active Nuphar extracts and their purified and synthetic derivatives have attracted the attention of ethnobotanists and biochemists. Here, we report that 6,6′-dihydroxythiobinupharidine (DTBN), purified from extracts of Nuphar lutea (L.) Sm. leaves, is an effective inhibitor of the kinase activity of members of the protein kinase C (PKC) family using in vitro and in silico approaches. We demonstrate that members of the conventional subfamily of PKCs, PKCα and PKCγ, were more sensitive to DTBN inhibition as compared to novel or atypical PKCs. Molecular docking analysis demonstrated the interaction of DTBN, with the kinase domain of PKCs depicting the best affinity towards conventional PKCs, in accordance with our in vitro kinase activity data. The current study reveals novel targets for DTBN activity, functioning as an inhibitor for PKCs kinase activity. Thus, this and other data indicate that DTBN modulates key cellular signal transduction pathways relevant to disease biology, including cancer.

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.

Protein kinase C activation by acidic proteins including 14-3-3

2000 ◽  
Vol 347 (3) ◽  
pp. 781-785 ◽  
Author(s):  
Paulus C. J. VAN DER HOEVEN ◽  
José C. M. VAN DER WAL ◽  
Paula RUURS ◽  
Wim J. VAN BLITTERSWIJK
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Mitogen Activated Protein Kinase ◽  
Kinase Assay ◽  
Kinase C ◽  
Mitogen Activated Protein ◽  
Dose Dependent Fashion ◽  
Acidic Proteins ◽  
Pkc Ζ

14-3-3 proteins may function as adapter or scaffold proteins in signal transduction pathways. We reported previously that several 14-3-3 isotypes bind to protein kinase C (PKC)-ζ and facilitate coupling of PKC-ζ to Raf-1 [van der Hoeven, van der Wal, Ruurs, van Dijk and van Blitterswijk (2000) Biochem. J. 345, 297-306], an event that boosts the mitogen-activated protein kinase (ERK) pathway in Rat-1 fibroblasts. The present work investigated whether bound 14-3-3 would affect PKC-ζ activity. Using recombinant 14-3-3 proteins and purified PKC-ζ in a convenient, newly developed in vitro kinase assay, we found that 14-3-3 proteins stimulated PKC-ζ activity in a dose-dependent fashion up to approx. 2.5-fold. Activation of PKC-ζ by 14-3-3 isotypes was unrelated to their mutual affinity, estimated by co-immunoprecipitation from COS cell lysates. Accordingly, PKC-ζ with a defective (point-mutated) 14-3-3-binding site, showed the same 14-3-3-stimulated activity as wild-type PKC-ζ. As 14-13-3 proteins are acidic, we tested several other acidic proteins, which turned out to stimulate PKC-ζ activity in a similar fashion, whereas neutral or basic proteins did not. These effects were not restricted to the atypical PKC-ζ, but were also found for classical PKC. Together, the results suggest that the stimulation of PKC activity by 14-3-3 proteins is non-specific and solely due to the acidic nature of these proteins, quite similar to that known for acidic lipids.

scholarly journals Mechanism of A-kinase-anchoring protein 79 (AKAP79) and protein kinase C interaction

1999 ◽  
Vol 343 (2) ◽  
pp. 443-452 ◽  
Author(s):  
Maree C. FAUX ◽  
Emily N. ROLLINS ◽  
Amelia S. EDWARDS ◽  
Lorene K. LANGEBERG ◽  
Alexandra C. NEWTON ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Protein Interactions ◽  
Hippocampal Neurons ◽  
Kinase Activity ◽  
Catalytic Core ◽  
Kinase C ◽  
Anchoring Protein ◽  
Arginine Residues

The A-kinase-anchoring protein AKAP79 co-ordinates the location of cAMP-dependent protein kinase, phosphatase 2B (PP2B/calcineurin) and protein kinase C (PKC) at postsynaptic sites in neurons. In this report we focus on the mechanism of interaction between AKAP79 and PKC. We show that neither lipid activators nor kinase activation are required for association with AKAP79. The anchoring protein binds and inhibits the conserved catalytic core of PKCβII. AKAP79 also associates with conventional, novel and atypical isoforms of PKC in vitro andin vivo, and immunofluorescence staining of rat hippocampal neurons demonstrates that the murine anchoring-protein homologue AKAP150 is co-distributed with PKCα/β, PKCε or PKCℓ. Binding of the AKAP79(31-52) peptide, which inhibits kinase activity, exposes the pseudosubstrate domain of PKCβII, allowing endoproteinase Arg-C proteolysis in the absence of kinase activators. Reciprocal experiments have identified two arginine residues at positions 39 and 40 that are essential for AKAP79(31-52) peptide inhibition of PKCβII. Likewise, the same mutations in the full-length anchoring protein reduced inhibition of PKCβII. Thus AKAP79 associates with multiple PKC isoforms through a mechanism involving protein-protein interactions at the catalytic core where binding of the anchoring protein inhibits kinase activity through displacement of the pseudosubstrate.

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.

scholarly journals Three Distinct Mechanisms for Translocation and Activation of the δ Subspecies of Protein Kinase C

1998 ◽  
Vol 18 (9) ◽  
pp. 5263-5271 ◽  
Author(s):  
Shiho Ohmori ◽  
Yasuhito Shirai ◽  
Norio Sakai ◽  
Motoko Fujii ◽  
Hiroaki Konishi ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Fusion Protein ◽  
Kinase Activity ◽  
Fluorescent Protein ◽  
Simultaneous Treatment ◽  
Kinase C ◽  
Green Fluorescent ◽  
Number Of Cells

ABSTRACT We expressed δ subspecies of protein kinase C (δ-PKC) fused with green fluorescent protein (GFP) in CHO-K1 cells and observed the movement of this fusion protein in living cells after three different stimulations. The δ-PKC–GFP fusion protein had enzymological characteristics very similar to those of the native δ-PKC and was present throughout the cytoplasm in CHO-K1 cells. ATP at 1 mM caused a transient translocation of δ-PKC–GFP to the plasma membrane approximately 30 s after the stimulation and a sequent retranslocation to the cytoplasm within 3 min. A tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA; 1 μM), induced a slower translocation of δ-PKC–GFP, and the translocation was unidirectional. Concomitantly, the kinase activity of δ-PKC–GFP was increased by these two stimulations, when the kinase activity of the immunoprecipitated δ-PKC–GFP was measured in vitro in the absence of PKC activators such as phosphatidylserine and diacylglycerol. Hydrogen peroxide (H2O2; 5 mM) failed to translocate δ-PKC–GFP but increased its kinase activity more than threefold. δ-PKC–GFP was strongly tyrosine phosphorylated when treated with H2O2 but was tyrosine phosphorylated not at all by ATP stimulation and only slightly by TPA treatment. Both TPA and ATP induced the translocation of δ-PKC–GFP even after treatment with H2O2. Simultaneous treatment with TPA and H2O2 further activated δ-PKC–GFP up to more than fivefold. TPA treatment of cells overexpressing δ-PKC–GFP led to an increase in the number of cells in G2/M phase and of dikaryons, while stimulation with H2O2 increased the number of cells in S phase and induced no significant change in cell morphology. These results indicate that at least three different mechanisms are involved in the translocation and activation of δ-PKC.

scholarly journals Cell Volume Regulation in Response to Hypotonicity Is Impaired in HeLa Cells Expressing a Protein Kinase C α Mutant Lacking Kinase Activity

2004 ◽  
Vol 279 (17) ◽  
pp. 17681-17689 ◽  
Author(s):  
Marcela Hermoso ◽  
Pablo Olivero ◽  
Rubén Torres ◽  
Ana Riveros ◽  
Andrew F. G. Quest ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cell Volume ◽  
Hela Cells ◽  
Volume Regulation ◽  
Kinase Activity ◽  
Cell Volume Regulation ◽  
Kinase C

Coordinate regulation of pp90rsk and a distinct protein-serine/threonine kinase activity that phosphorylates recombinant pp90rsk in vitro

1991 ◽  
Vol 11 (4) ◽  
pp. 1868-1874
Author(s):  
J Chung ◽  
R H Chen ◽  
J Blenis
Keyword(s):  
Protein Kinase C ◽  
Growth Factor ◽  
Protein Kinase ◽  
Protein Kinases ◽  
Kinase Activity ◽  
Cultured Cells ◽  
Protein Kinase Activity ◽  
Kinase C ◽  
Dependent Protein

Protein kinase assays that use recombinant pp90rsk as a substrate were developed in an attempt to identify growth-regulated enzymes responsible for the phosphorylation and activation of pp90rsk S6 phosphotransferase activity. With this assay we have ientified a pp60v-src-, growth factor-, phorbol ester-, and vanadate-regulated serine/threonine protein kinase activity that is not related to two other cofactor-independent, growth-regulated protein kinases, pp70-S6 protein kinase and pp90rsk. The pp90rsk-protein kinase activity (referred to as rsk-kinase) is also not related to cofactor-dependent signal transducing protein kinases such as the cyclic AMP-dependent protein kinases, members of the protein kinase C family, or other Ca2(+)-dependent protein kinases. In vitro, partially purified rsk-kinase phosphorylates several of the sites (serine and threonine) that are phosphorylated in growth-stimulated cultured cells. A detailed examination of the mitogen-regulated activation kinetics of rsk-kinase and pp90rsk activities demonstrated that they are coordinately regulated. In addition, protein kinase C is not absolutely required for epidermal and fibroblast growth factor-stimulated activation of rsk-kinase, whereas, like pp90rsk, platelet-derived growth factor- and vanadate-stimulated rsk-kinase activity exhibits a greater dependence on protein kinase C-mediated signal transduction. The characterization and future purification of the rsk-kinase(s) will improve our understanding of the early signaling events regulating cell growth.

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