scholarly journals Inhibition of protein kinase C catalytic activity by additional regions within the human protein kinase Calpha-regulatory domain lying outside of the pseudosubstrate sequence

2003 ◽  
Vol 373 (2) ◽  
pp. 571-581 ◽  
Author(s):  
Angie F. KIRWAN ◽  
Ashley C. BIBBY ◽  
Thierry MVILONGO ◽  
Heimo RIEDEL ◽  
Thomas BURKE ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Kinase ◽  
Catalytic Domain ◽  
Dependent Manner ◽  
Regulatory Domain ◽  
Direct Role ◽  
Independent Manner ◽  
Inhibitory Capacity ◽  
Pseudosubstrate Sequence

The N-terminal pseudosubstrate site within the protein kinase Cα (PKCα)-regulatory domain has long been regarded as the major determinant for autoinhibition of catalytic domain activity. Previously, we observed that the PKC-inhibitory capacity of the human PKCα-regulatory domain was only reduced partially on removal of the pseudosubstrate sequence [Parissenti, Kirwan, Kim, Colantonio and Schimmer (1998) J. Biol. Chem. 273, 8940–8945]. This finding suggested that one or more additional region(s) contributes to the inhibition of catalytic domain activity. To assess this hypothesis, we first examined the PKC-inhibitory capacity of a smaller fragment of the PKCα-regulatory domain consisting of the C1a, C1b and V2 regions [GST-Rα39–177: this protein contained the full regulatory domain of human PKCα fused to glutathione S-transferase (GST), but lacked amino acids 1–38 (including the pseudosubstrate sequence) and amino acids 178–270 (including the C2 region)]. GST-Rα39–177 significantly inhibited PKC in a phorbol-independent manner and could not bind the peptide substrate used in our assays. These results suggested that a region within C1/V2 directly inhibits catalytic domain activity. Providing further in vivo support for this hypothesis, we found that expression of N-terminally truncated pseudosubstrate-less bovine PKCα holoenzymes in yeast was capable of inhibiting cell growth in a phorbol-dependent manner. This suggested that additional autoinhibitory force(s) remained within the truncated holoenzymes that could be relieved by phorbol ester. Using tandem PCR-mediated mutagenesis, we observed that mutation of amino acids 33–86 within GST-Rα39–177 dramatically reduced its PKC-inhibitory capacity when protamine was used as substrate. Mutagenesis of a broad range of sequences within C2 (amino acids 159–242) also significantly reduced PKC-inhibitory capacity. Taken together, these observations support strongly the existence of multiple regions within the PKCα-regulatory domain that play a direct role in the inhibition of catalytic domain activity.

A protein kinase C cDNA without the regulatory domain is active after transfection in vivo in the absence of phorbol ester

1989 ◽  
Vol 9 (2) ◽  
pp. 831-836
Author(s):  
M Muramatsu ◽  
K Kaibuchi ◽  
K Arai
Keyword(s):  
Amino Acids ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Phorbol Ester ◽  
Interleukin 2 ◽  
Regulatory Domain ◽  
Coding Region ◽  
Kinase C ◽  
Pkc Alpha

We constructed mutant protein kinase C (PKC) cDNAs which expressed PKC activity in vivo in the absence of phorbol ester activation. A hybrid PKC gene, PKAC, was constructed by substituting the coding region for the N-terminal 253 amino acids of PKC alpha with the N-terminal 17 amino acids of the cyclic AMP-dependent protein kinase catalytic subunit (PKA). A truncated PKC gene, delta PKC beta, lacking the coding region for amino acid positions 6 to 159 of PKC beta was also constructed. These mutant kinase genes expressed under the control of the SR alpha promoter activated the c-fos gene enhancer in Jurkat cells and initiated maturation of Xenopus laevis oocytes. Phorbol ester binding activity was absent in both constructs but was preserved in another hybrid gene, PKCA, which was composed of the coding region for 1 to 253 amino acids of PKC alpha at the N-terminal side and the coding region for 18 to 350 amino acids of PKA at the C-terminal side. These results indicate that elimination of the regulatory domain of PKC produces constitutively active PKC that can bypass activation by the phorbol ester. delta PKC beta, in synergy with a calcium ionophore, was capable of activating the interleukin 2 promoter, indicating that cooperation of PKC-dependent and calcium-dependent pathways is necessary for activation of the interleukin 2 gene.

scholarly journals A protein kinase C cDNA without the regulatory domain is active after transfection in vivo in the absence of phorbol ester.

1989 ◽  
Vol 9 (2) ◽  
pp. 831-836 ◽  
Author(s):  
M Muramatsu ◽  
K Kaibuchi ◽  
K Arai
Keyword(s):  
Amino Acids ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Phorbol Ester ◽  
Interleukin 2 ◽  
Regulatory Domain ◽  
Coding Region ◽  
Kinase C ◽  
Pkc Alpha

We constructed mutant protein kinase C (PKC) cDNAs which expressed PKC activity in vivo in the absence of phorbol ester activation. A hybrid PKC gene, PKAC, was constructed by substituting the coding region for the N-terminal 253 amino acids of PKC alpha with the N-terminal 17 amino acids of the cyclic AMP-dependent protein kinase catalytic subunit (PKA). A truncated PKC gene, delta PKC beta, lacking the coding region for amino acid positions 6 to 159 of PKC beta was also constructed. These mutant kinase genes expressed under the control of the SR alpha promoter activated the c-fos gene enhancer in Jurkat cells and initiated maturation of Xenopus laevis oocytes. Phorbol ester binding activity was absent in both constructs but was preserved in another hybrid gene, PKCA, which was composed of the coding region for 1 to 253 amino acids of PKC alpha at the N-terminal side and the coding region for 18 to 350 amino acids of PKA at the C-terminal side. These results indicate that elimination of the regulatory domain of PKC produces constitutively active PKC that can bypass activation by the phorbol ester. delta PKC beta, in synergy with a calcium ionophore, was capable of activating the interleukin 2 promoter, indicating that cooperation of PKC-dependent and calcium-dependent pathways is necessary for activation of the interleukin 2 gene.

scholarly journals Characterization of a calmodulin-regulated Ca2+-dependent-protein-kinase-related protein kinase, AtCRK1, from Arabidopsis

2004 ◽  
Vol 383 (1) ◽  
pp. 73-81 ◽  
Author(s):  
Ying WANG ◽  
Shuping LIANG ◽  
Qi-Guang XIE ◽  
Ying-Tang LU
Keyword(s):  
Protein Kinase ◽  
Catalytic Domain ◽  
Dependent Manner ◽  
Amino Acid Residues ◽  
Related Protein ◽  
Dependent Protein Kinase ◽  
Independent Manner ◽  
Calmodulin Binding ◽  
Kinase Catalytic Domain ◽  
Dependent Protein

An AtCRK1 [Arabidopsis thaliana CDPK (Ca2+-dependent protein kinase)-related protein kinase 1] has been characterized molecularly and biochemically. AtCRK1 contains the kinase catalytic domain and a CaM (calmodulin)-binding site. Our results demonstrated that AtCRK1 could bind CaM in a Ca2+-dependent manner. This kinase phosphorylated itself and substrates such as histone IIIS and syntide-2 in a Ca2+-independent manner and the activity was stimulated by several CaM isoforms through its CaM-binding domain. This domain was localized within a stretch of 39 amino acid residues at positions from 403 to 441 with Kd=67 nM for CaM binding. However, the stimulation amplification of the kinase activity of AtCRK1 by different CaM isoforms was similar.

scholarly journals The Transcriptional Activator Mirk/Dyrk1B Is Sequestered by p38α/β MAP Kinase

2002 ◽  
Vol 277 (51) ◽  
pp. 49438-49445 ◽  
Author(s):  
Seunghwan Lim ◽  
Yonglong Zou ◽  
Eileen Friedman
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Nuclear Export ◽  
Binding Protein ◽  
Transcriptional Activator ◽  
Nuclear Body ◽  
Scaffolding Protein ◽  
Dependent Manner ◽  
Independent Manner

Mirk/Dyrk1B protein kinase was shown in an earlier study to function as a transcriptional activator of HNF1α, which Mirk phosphorylates at Ser249within its CREB (cAMP-response element-binding protein)-binding protein (CBP) binding domain (1). The MAPK kinase MKK3 was also shown to activate Mirk as a protein kinase, implicating Mirk in the biological response to certain stress agents. Another MKK3 substrate, p38MAPK, is now shown to inhibit the function of Mirk as a transcriptional activator in a kinase-independent manner. Co-immunoprecipitation experiments demonstrated that kinase-inactive p38AF, as well as wild-type p38, sequestered Mirk and prevented its association with MKK3. Only the p38α and p38β isoforms, but not the γ or δ isoforms, complexed with Mirk. p38αMAPK blocked Mirk activation of HNF1α in a dose-dependent manner, with high levels of kinase-inactive p38αAF completely suppressing the activity of Mirk. Size fractionation by fast protein liquid chromatography on Superdex 200 demonstrated that Mirk is not found as a monomerin vivo, but is found within 150–700 kDa subnuclear complexes, which co-migrate with the nuclear body scaffolding protein PML. Endogenous Mirk, p38, and MKK3 co-migrate within 500–700-kDa protein complexes, which accumulate when nuclear export is blocked by leptomycin B. Stable overexpression of Mirk increases the fraction of Mirk protein and p38 protein within these 500–700 kDa complexes, suggesting that the complexes act as nuclear depots for Mirk and p38. Sequestration of Mirk by p38 may occur within these subnuclear complexes. Synchronization experiments demonstrated that Mirk levels fluctuate about 10-fold within the cell cycle, while p38 levels do not, leading to the speculation that endogenous p38 could only block Mirk function when Mirk levels were low in S phase and not when Mirk levels were elevated in G0/G1. These data suggest a novel cell cycle-dependent function for p38, suppression of the function of Mirk as a transcriptional activator only when cells are proliferating, and thus limiting Mirk function to growth-arrested cells.

scholarly journals The Amino Terminus of Varicella-Zoster Virus (VZV) Glycoprotein E Is Required for Binding to Insulin-Degrading Enzyme, a VZV Receptor

2007 ◽  
Vol 81 (16) ◽  
pp. 8525-8532 ◽  
Author(s):  
Qingxue Li ◽  
Tammy Krogmann ◽  
Mir A. Ali ◽  
Wei-Jen Tang ◽  
Jeffrey I. Cohen
Keyword(s):  
Amino Acids ◽  
Varicella Zoster Virus ◽  
Catalytic Domain ◽  
Amino Terminus ◽  
Dependent Manner ◽  
Insulin Degrading Enzyme ◽  
Concentration Dependent Manner ◽  
Glycoprotein E ◽  
Degrading Enzyme ◽  
Varicella Zoster

ABSTRACT Varicella-zoster virus (VZV) glycoprotein E (gE) is required for VZV infection. Although gE is well conserved among alphaherpesviruses, the amino terminus of VZV gE is unique. Previously, we showed that gE interacts with insulin-degrading enzyme (IDE) and facilitates VZV infection and cell-to-cell spread of the virus. Here we define the region of VZV gE required to bind IDE. Deletion of amino acids 32 to 71 of gE, located immediately after the predicted signal peptide, resulted in loss of the ability of gE to bind IDE. A synthetic peptide corresponding to amino acids 24 to 50 of gE blocked its interaction with IDE in a concentration-dependent manner. However, a chimeric gE in which amino acids 1 to 71 of VZV gE were fused to amino acids 30 to 545 of herpes simplex virus type 2 gE did not show an increased level of binding to IDE compared with that of full-length HSV gE. Thus, amino acids 24 to 71 of gE are required for IDE binding, and the secondary structure of gE is critical for the interaction. VZV gE also forms a heterodimer with glycoprotein gI. Deletion of amino acids 163 to 208 of gE severely reduced its ability to form a complex with gI. The amino portion of IDE, as well an IDE mutant in the catalytic domain of the protein, bound to gE. Therefore, distinct motifs of VZV gE are important for binding to IDE or to gI.

scholarly journals Nerve Growth Factor Stimulates Multisite Tyrosine Phosphorylation and Activation of the Atypical Protein Kinase C's via a src Kinase Pathway

2001 ◽  
Vol 21 (24) ◽  
pp. 8414-8427 ◽  
Author(s):  
Marie W. Wooten ◽  
Michel L. Vandenplas ◽  
M. Lamar Seibenhener ◽  
Thangiah Geetha ◽  
Maria T. Diaz-Meco
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Protein Kinase ◽  
Tyrosine Phosphorylation ◽  
Pc12 Cells ◽  
Catalytic Domain ◽  
Src Kinase ◽  
Dependent Manner ◽  
Nerve Growth ◽  
Atypical Protein Kinase

ABSTRACT Atypical protein kinase C (PKC) isoforms are required for nerve growth factor (NGF)-initiated differentiation of PC12 cells. In the present study, we report that PKC-ι becomes tyrosine phosphorylated in the membrane coincident with activation posttreatment with nerve growth factor. Tyrosine phosphorylation and activation of PKC-ι were inhibited in a dose-dependent manner by both PP2 and K252a, src and TrkA kinase inhibitors. Purified src was observed to phosphorylate and activate PKC-ι in vitro. In PC12 cells deficient in src kinase activity, both NGF-induced tyrosine phosphorylation and activation of PKC-ι were also diminished. Furthermore, we demonstrate activation of src by NGF along with formation of a signal complex including the TrkA receptor, src, and PKC-ι. Recruitment of PKC-ι into the complex was dependent on the tyrosine phosphorylation state of PKC-ι. The association of src and PKC-ι was constitutive but was enhanced by NGF treatment, with the src homology 3 domain interacting with a PXXP sequence within the regulatory domain of PKC-ι (amino acids 98 to 114). Altogether, these findings support a role for src in regulation of PKC-ι. Tyrosine 256, 271, and 325 were identified as major sites phosphorylated by src in the catalytic domain. Y256F and Y271F mutations did not alter src-induced activation of PKC-ι, whereas the Y325F mutation significantly reduced src-induced activation of PKC-ι. The functional relevance of these mutations was tested by determining the ability of each mutant to support TRAF6 activation of NF-κB, with significant impairment by the Y325F PKC-ι mutant. Moreover, when the Y352F mutant was expressed in PC12 cells, NGF's ability to promote survival in serum-free media was reduced. In summary, we have identified a novel mechanism for NGF-induced activation of atypical PKC involving tyrosine phosphorylation by c-Src.

scholarly journals A Possible Role of Allatostatin C in Inhibiting Ecdysone Biosynthesis Revealed in the Mud Crab Scylla paramamosain

2021 ◽  
Vol 8 ◽  
Author(s):  
An Liu ◽  
Wenyuan Shi ◽  
Dongdong Lin ◽  
Haihui Ye
Keyword(s):  
Scylla Paramamosain ◽  
Dependent Manner ◽  
Mud Crab ◽  
Methyl Farnesoate ◽  
Independent Manner ◽  
Wide Range ◽  
Negative Effect

C-type allatostatins (C-type ASTs) are a family of structurally related neuropeptides found in a wide range of insects and crustaceans. To date, the C-type allatostatin receptor in crustaceans has not been deorphaned, and little is known about its physiological functions. In this study, we aimed to functionally define a C-type ASTs receptor in the mud crab, Scylla paramamosian. We showed that C-type ASTs receptor can be activated by ScypaAST-C peptide in a dose-independent manner and by ScypaAST-CCC peptide in a dose-dependent manner with an IC50 value of 6.683 nM. Subsequently, in vivo and in vitro experiments were performed to investigate the potential roles of ScypaAST-C and ScypaAST-CCC peptides in the regulation of ecdysone (20E) and methyl farnesoate (MF) biosynthesis. The results indicated that ScypaAST-C inhibited biosynthesis of 20E in the Y-organ, whereas ScypaAST-CCC had no effect on the production of 20E. In addition, qRT-PCR showed that both ScypaAST-C and ScypaAST-CCC significantly decreased the level of expression of the MF biosynthetic enzyme gene in the mandibular organ, suggesting that the two neuropeptides have a negative effect on the MF biosynthesis in mandibular organs. In conclusion, this study provided new insight into the physiological roles of AST-C in inhibiting ecdysone biosynthesis. Furthermore, it was revealed that AST-C family peptides might inhibit MF biosynthesis in crustaceans.

Effects of inhibitors of protein kinase C and calpain in experimental delayed cerebral vasospasm

1992 ◽  
Vol 76 (1) ◽  
pp. 111-118 ◽  
Author(s):  
Nobutaka Minami ◽  
Eiichi Tani ◽  
Yukio Maeda ◽  
Ikuya Yamaura ◽  
Masahiro Fukami
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Catalytic Domain ◽  
Limited Proteolysis ◽  
Maximum Effect ◽  
Regulatory Domain ◽  
Calphostin C ◽  
Delayed Cerebral Vasospasm ◽  
Kinase C ◽  
Basilar Arteries

✓ Vasospasm was produced in adult mongrel dogs by a two-hemorrhage method, and the spastic basilar arteries were exposed via the transclival route on Day 7. Tonic contraction was produced in the normal canine basilar arteries by a local application of KCl or serotonin after transclival exposure. The exposed spastic and tonic basilar arteries then received a topical application of the following: 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine (H-7), a potent inhibitor of protein kinase C acting at the catalytic domain; calphostin C, a specific inhibitor of protein kinase C acting at the regulatory domain; or calpeptin, a selective inhibitor of calpain. Both spastic and tonic basilar arteries were effectively dilated by H-7. Calphostin C caused only slight dilation of spastic basilar arteries but moderate dilation of tonic basilar arteries. Dilation in response to calpeptin was remarkable in the spastic basilar arteries but slight in the tonic basilar arteries. The doses of calphostin C and calpeptin required to obtain maximum effect were markedly lower in the tonic model than in the spastic model. The spastic and tonic models had a similar dose-dependent response to H-7 but quite a different response to calphostin C or calpeptin, suggesting a difference in the function of protein kinase C and calpain in the two models. Furthermore, the effect of calphostin C on the reversal of vasospasm was increased significantly after topical treatment with calpeptin. It is suggested that the majority of the catalytic domain of protein kinase C is dissociated from the regulatory domain, probably by a limited proteolysis with calpain, and is markedly activated in vasospasm.

Abstract P191: Calpain-Generated Free PKCα Catalytic Domains Induce HDAC5 Nuclear Export and Regulate Cardiac Gene Transcription

2011 ◽  
Vol 109 (suppl_1) ◽  
Author(s):  
Yan Zhang ◽  
Scot Matkovich ◽  
Abhinav Diwan ◽  
Min-Young Kang ◽  
Gerald W Dorn
Keyword(s):  
Gene Expression ◽  
Protein Kinase ◽  
Nuclear Export ◽  
Kinase Inhibitor ◽  
Proteolytic Processing ◽  
Full Length ◽  
Independent Effect ◽  
Independent Manner ◽  
Cardiac Gene

Receptor-mediated activation of protein kinase (PK) C is a central pathway regulating cell growth, homeostasis, and programmed death. Recently, we showed that calpain-mediated proteolytic processing of PKC in ischemic myocardium activates PKC signaling in a receptor-independent manner by releasing a persistent and constitutively active free catalytic C-terminal fragment, PKCα-CT. This unregulated kinase provokes cardiomyopathy, but the mechanisms remain unclear. We examined hypothesis that PKCα-CT has transcriptional activity. Using immunoblot analysis and confocal microscopy, we found that PKCα-CT localized in part to nuclei and spontaneously induced cytosolic relocalization HDAC5 of the transcriptional regulator. Co- expression of calpain 1 with full length PKCα can generate PKCα-CT and produced the same HDAC5 cytosolic relocalization, whereas full length PKCα alone had no such effect. HDAC5 cytosolic relocalization induced by PKCα-CT was abolished by the protein kinase inhibitor GO6976, but not by PKD inhibitor CID 755673. The in vivo relevance of these findings was examined in transgenic mice expressing PKCα and PKCα-CT. To assess the consequence on gene expression, we performed global transcriptome profiling by Affymetrix microarrays and mRNA sequencing. The two techniques substantially agreed. Compared to control hearts, 621 mRNAs were regulated at least 1.3 fold in PKCα-CT hearts (P< 0.001), only 59 in full-length PKCα hearts. MEF2-dependent inflammatory pathway genes which are putative HDAC targets were upregulated in PKCα-CT heart: 15 MEF2 target mRNAs were upregulated in PKCα-CT hearts (p<0.001), only one in PKCα hearts. These results reveal that PKCα-CT is a potent regulator of pathological cardiac gene expression by localizing to nuclei and directly promoting nuclei-cytoplasmic shuttling of HDAC5. Receptor-independent effect of PKCα-CT and HDAC phosphorylation in ischemic hearts has broad ramifications for understanding and preventing the pathological transcriptional stress response.

scholarly journals Ammonia inhibits energy metabolism in astrocytes in a rapid and glutamate dehydrogenase 2-dependent manner

2020 ◽  
Vol 13 (10) ◽  
pp. dmm047134
Author(s):  
Leonie Drews ◽  
Marcel Zimmermann ◽  
Philipp Westhoff ◽  
Dominik Brilhaus ◽  
Rebecca E. Poss ◽  
...  
Keyword(s):  
Amino Acids ◽  
Energy Metabolism ◽  
Glutamate Dehydrogenase ◽  
Mitochondrial Respiration ◽  
Tca Cycle ◽  
Dependent Manner ◽  
Independent Manner ◽  
The Tca Cycle ◽  
Branched Chain ◽  
Tca Cycle Intermediates

ABSTRACTAstrocyte dysfunction is a primary factor in hepatic encephalopathy (HE) impairing neuronal activity under hyperammonemia. In particular, the early events causing ammonia-induced toxicity to astrocytes are not well understood. Using established cellular HE models, we show that mitochondria rapidly undergo fragmentation in a reversible manner upon hyperammonemia. Further, in our analyses, within a timescale of minutes, mitochondrial respiration and glycolysis were hampered, which occurred in a pH-independent manner. Using metabolomics, an accumulation of glucose and numerous amino acids, including branched chain amino acids, was observed. Metabolomic tracking of 15N-labeled ammonia showed rapid incorporation of 15N into glutamate and glutamate-derived amino acids. Downregulating human GLUD2 [encoding mitochondrial glutamate dehydrogenase 2 (GDH2)], inhibiting GDH2 activity by SIRT4 overexpression, and supplementing cells with glutamate or glutamine alleviated ammonia-induced inhibition of mitochondrial respiration. Metabolomic tracking of 13C-glutamine showed that hyperammonemia can inhibit anaplerosis of tricarboxylic acid (TCA) cycle intermediates. Contrary to its classical anaplerotic role, we show that, under hyperammonemia, GDH2 catalyzes the removal of ammonia by reductive amination of α-ketoglutarate, which efficiently and rapidly inhibits the TCA cycle. Overall, we propose a critical GDH2-dependent mechanism in HE models that helps to remove ammonia, but also impairs energy metabolism in mitochondria rapidly.

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