Induction of a cytoplasmic activator of DNA synthesis in lymphocytes is mediated through a membrane-associated protein kinase.

We have shown previously that cytoplasmic extracts from actively dividing lymphoid cells are capable of inducing DNA synthesis in isolated nuclei. One of the factors involved in this activity, ADR, appears to be a greater than 90 kDa heat-labile protease. Cytoplasmic extracts prepared from nonproliferating lymphocytes express little to no ADR activity. However, ADR activity can be generated in these extracts by brief exposure to a membrane-enriched fraction of spontaneously proliferating, leukemic human T lymphoblastoid (MOLT-4) cells. This suggests that ADR activity is present in the resting cytoplasm in an inactive or precursor form. This in vitro generation of ADR activity can be inhibited in a dose-dependent manner by the isoquinolinesulfonamide derivative, H-7 (1-(5-isoquinoline-sulfonyl)-2-methylpiperazine dihydrochloride), an inhibitor of both cyclic adenosine monophosphate (cAMP)-dependent protein kinases and protein kinase C (PKC). However, more specific inhibitors of cAMP-dependent protein kinases, including N-[( 2-methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H8) and N-(2-gua-nidinoethyl)-5-isoquinolinesulfonamide (HA-1004), had little to no effect on the in vitro generation of ADR activity. Furthermore, membranes from MOLT-4 cells depleted of PKC by long-term exposure (24 h) to phorbol esters and calcium ionophores were unable to induce ADR activity in resting peripheral blood lymphocytes extracts. The results of these studies suggest 1) ADR activity is present in resting cell cytoplasm in an inactive or precursor form; and 2) ADR activity can be induced in this resting cytoplasm through a mechanism involving a membrane-associated protein kinase, possibly PKC. The ability of alkaline phosphatase to deplete the activity of preformed ADR suggests the possibility that ADR itself is phosphoprotein.

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Roscovitine, a specific inhibitor of cyclin-dependent protein kinases, reversibly inhibits chromatin condensation during in vitro maturation of porcine oocytes

Zygote ◽

10.1017/s0967199401001356 ◽

2001 ◽

Vol 9 (4) ◽

pp. 309-316 ◽

Cited By ~ 21

Author(s):

Carsten Krischek ◽

Burkhard Meinecke

Keyword(s):

Map Kinase ◽

Protein Kinases ◽

Chromatin Condensation ◽

Specific Inhibitor ◽

Histone H1 ◽

Dependent Manner ◽

Free Medium ◽

Concentration Dependent Manner ◽

Dependent Protein

In the present study the effects of roscovitine on the in vitro nuclear maturation of porcine oocytes were investigated. Roscovitine, a specific inhibitor of cyclin-dependent protein kinases, prevented chromatin condensation in a concentration-dependent manner. This inhibition was reversible and was accompanied by non-activation of p34cdc2/histone H1 kinase. It also decreased enzyme activity of MAP kinase, suggesting a correlation between histone H1 kinase activation and the onset of chromatin condensation. The addition of roscovitine (50 μM) to extracts of metaphase II oocytes revealed that the MAP kinase activity was not directly affected by roscovitine, which indicates a possible link between histone H1 and MAP kinase. Chromatin condensation occurred between 20 and 28 h of culture of cumulus-oocyte complexes (COCs) in inhibitor-free medium (germinal vesicle stage I, GV1: 74.6% and 13.7%, respectively). Nearly the same proportion of chromatin condensation was detected in COCs incubated initially in inhibitor-free medium for 20-28 h and subsequently in roscovitine-supplemented medium (50 μM) for a further 2-10 h (GV I: 76.2% and 18.8%, respectively). This observation indicates that roscovitine prevents chromatin condensation even after an initial inhibitor-free cultivation for 20 h. Extending this initial incubation period to ≥22 h led to an activation of histone H1 and MAP kinase and increasing proportions of oocytes exhibiting chromatin condensation in the presence of roscovitine. It is concluded that histone H1 kinase is involved in the induction of chromatin condensation during in vitro maturation of porcine oocytes.

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Ca2+ and phospholipid-dependent protein kinase (protein kinase C) activity is not necessarily required for secretion by human neutrophils

Blood ◽

10.1182/blood.v68.4.810.bloodjournal684810 ◽

1986 ◽

Vol 68 (4) ◽

pp. 810-817

Author(s):

KJ Balazovich ◽

JE Smolen ◽

LA Boxer

Keyword(s):

Protein Kinase ◽

Phorbol Esters ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Soluble Form ◽

Human Neutrophils ◽

Dependent Manner ◽

Dependent Protein Kinase ◽

Dependent Protein ◽

Dose Dependent

Ca2+-dependent and phospholipid-dependent protein kinase (PKC) is a receptor for and is activated by phorbol esters. This enzyme is reportedly involved in the mechanism of superoxide anion (O2-) production and the release of intracellular granule contents from human neutrophils. As previously reported by others, we found that greater than 75% of the total cellular PKC activity existed in a soluble form in untreated neutrophils and that this activity was enhanced in a dose- dependent manner by phorbol 12-myristate 13-acetate (PMA) and by phorbol 12,13-dibutyrate (PDBu). Furthermore, mezerein, an analogue of PMA that is thought to be a competitive inhibitor, did not activate PKC, and on the contrary, inhibited PMA-stimulated activity in a dose- dependent manner. Pretreatment of intact neutrophils with PMA or PDBu caused the “translocation” of PKC activity to the insoluble cell fraction; PKC translocation was not detected after mezerein stimulation at any of the tested concentrations. Neither did mezerein cause an increase in intracellular Ca2+, as monitored by Quin 2 fluorescence. Both phorbol esters and mezerein stimulated intact neutrophils to generate O2- and release lysosomal enzymes into the extracellular medium. Finally sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis demonstrated key differences in the patterns of endogenous phosphoproteins of neutrophils stimulated with phorbol as compared with mezerein. We therefore suggest that PKC activation may not be the only pathway required to elicit neutrophil responses.

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

Biochemical Journal ◽

10.1042/bj3520483 ◽

2000 ◽

Vol 352 (2) ◽

pp. 483-490 ◽

Cited By ~ 25

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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Identification of a 42-kilodalton phosphotyrosyl protein as a serine(threonine) protein kinase by renaturation

Molecular and Cellular Biology ◽

10.1128/mcb.10.6.3020-3026.1990 ◽

1990 ◽

Vol 10 (6) ◽

pp. 3020-3026

Author(s):

J E Ferrell ◽

G S Martin

Keyword(s):

Protein Kinase ◽

Sodium Dodecyl Sulfate ◽

Tyrosine Phosphorylation ◽

Protein Kinases ◽

Phorbol Esters ◽

Sodium Dodecyl ◽

Deae Cellulose ◽

Serum Stimulation ◽

Dodecyl Sulfate

We have surveyed fibroblast lysates for protein kinases that might be involved in mitogenesis. The assay we have used exploits the ability of blotted, sodium dodecyl sulfate-denatured proteins to regain enzymatic activity after guanidine treatment. About 20 electrophoretically distinct protein kinases could be detected by this method in lysates from NIH 3T3 cells. One of the kinases, a 42-kilodalton serine(threonine) kinase (PK42), was found to possess two- to fourfold-higher in vitro activity when isolated from serum-stimulated cells than when isolated from serum-starved cells. This kinase comigrated on sodium dodecyl sulfate-gels with a protein (p42) whose phosphotyrosine content increased in response to serum stimulation. The time courses of p42 tyrosine phosphorylation and PK42 activation were similar, reaching maximal levels within 10 min and returning to basal levels within 5 h. Both p42 tyrosine phosphorylation and PK42 activation were stimulated by low concentrations of phorbol esters, and the responses of p42 and PK42 to TPA were abolished by chronic 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. Chronic TPA treatment had less effect on serum-induced p42 tyrosine phosphorylation and PK42 activation. PK42 and p42 bound to DEAE-cellulose, and both eluted at a salt concentration of 250 mM. Thus, PK42 and p42 comigrate and cochromatograph, and the kinase activity of PK42 correlates with the tyrosine phosphorylation of p42. These findings suggest that PK42 and p42 are related or identical, that PK42 is activated by tyrosine phosphorylation, and that this tyrosine phosphorylation can be regulated by protein kinase C.

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The role of protein kinase C in LHRH-induced LH and FSH release and LHRH self-priming in rat anterior pituitary glands in vitro

Journal of Endocrinology ◽

10.1677/joe.0.1160231 ◽

1988 ◽

Vol 116 (2) ◽

pp. 231-239 ◽

Cited By ~ 24

Author(s):

M. S. Johnson ◽

R. Mitchell ◽

G. Fink

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Phorbol Esters ◽

Polymyxin B ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Kinase C ◽

Pituitary Glands

ABSTRACT We have investigated the role of protein kinase C (PKC) in LHRH-induced LH and FSH secretion and LHRH priming. Hemipituitary glands from pro-oestrous rats were incubated with agents known to affect PKC and with or without LHRH, during which time the secretion of gonadotrophins was measured. Phorbol esters and phospholipase C, activators of PKC, released LH and FSH in a concentration-dependent manner and potentiated the LHRH-induced secretion of gonadotrophins in parallel with their ability to release these hormones alone. Inhibitors of PKC had either no effect on LH release (1-(5-isoquinolinesulphonyl)-2-methylpiperazine hydrochloride) or they augmented LHRH-induced gonadotrophin release (polymyxin B and 8-(N,N-diethylamino) octyl-3,4,5-trimethoxybenzoate). Neither the activators nor the inhibitors of PKC, when present with LHRH, caused any change in LHRH priming, even though the activators alone produced a release of gonadotrophins that showed a temporal pattern similar to that produced by LHRH priming. The profiles of effects on LH and FSH secretion were always qualitatively similar. These results show that PKC may be involved in general regulation of gonadotrophin release but that it is not important in acute responses to LHRH nor in LHRH self-priming. J. Endocr. (1988) 116, 231–239

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ATR-Dependent Phosphorylation of DNA-Dependent Protein Kinase Catalytic Subunit in Response to UV-Induced Replication Stress

Molecular and Cellular Biology ◽

10.1128/mcb.00048-06 ◽

2006 ◽

Vol 26 (20) ◽

pp. 7520-7528 ◽

Cited By ~ 92

Author(s):

Hirohiko Yajima ◽

Kyung-Jong Lee ◽

Benjamin P. C. Chen

Keyword(s):

Protein Kinase ◽

Uv Irradiation ◽

Cellular Response ◽

Replication Stress ◽

Dominant Negative ◽

Catalytic Subunit ◽

Dependent Manner ◽

Dependent Protein Kinase ◽

Dependent Protein

ABSTRACT Phosphorylation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) upon ionizing radiation (IR) is essential for cellular radioresistance and nonhomologous-end-joining-mediated DNA double-strand break repair. In addition to IR induction, we have previously shown that DNA-PKcs phosphorylation is increased upon camptothecin treatment, which induces replication stress and replication-associated double-strand breaks. To clarify the involvement of DNA-PKcs in this process, we analyzed DNA-PKcs phosphorylation in response to UV irradiation, which causes replication stress and activates ATR (ATM-Rad3-related)/ATM (ataxia-telangiectasia mutated) kinases in a replication-dependent manner. Upon UV irradiation, we observed a rapid DNA-PKcs phosphorylation at T2609 and T2647, but not at S2056, distinct from that induced by IR. UV-induced DNA-PKcs phosphorylation occurs specifically only in replicating cells and is dependent on ATR kinase. Inhibition of ATR activity via caffeine, a dominant-negative kinase-dead mutant, or RNA interference led to the attenuation of UV-induced DNA-PKcs phosphorylation. Furthermore, DNA-PKcs associates with ATR in vivo and is phosphorylated by ATR in vitro, suggesting that DNA-PKcs could be the direct downstream target of ATR. Taken together, these results strongly suggest that DNA-PKcs is required for the cellular response to replication stress and might play an important role in the repair of stalled replication forks.

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In vitro phosphorylation of ciliary dyneins by protein kinases from Paramecium

Journal of Cell Science ◽

10.1242/jcs.106.4.1369 ◽

1993 ◽

Vol 106 (4) ◽

pp. 1369-1376 ◽

Cited By ~ 2

Author(s):

C.E. Walczak ◽

D.L. Nelson

Keyword(s):

Protein Kinase ◽

Protein Kinases ◽

Sucrose Gradient ◽

Dependent Protein Kinase ◽

Cgmp Dependent Protein Kinase ◽

Camp Dependent Protein Kinase ◽

Ciliary Protein ◽

Dependent Protein ◽

Camp And Cgmp

Paramecium dyneins were tested as substrates for phosphorylation by cAMP-dependent protein kinase, cGMP-dependent protein kinase, and two Ca(2+)-dependent protein kinases that were partially purified from Paramecium extracts. Only cAMP-dependent protein kinase caused significant phosphorylation. The major phosphorylated species was a 29 kDa protein that was present in both 22 S and 12 S dyneins; its phosphate-accepting activity peaked with 22 S dynein. In vitro phosphorylation was maximal at five minutes, then decreased. This decrease in phosphorylation was inhibited by the addition of vanadate or NaF. The 29 kDa protein was not phosphorylated by a heterologous cAMP-dependent protein kinase, the bovine catalytic subunit. Phosphorylation of dynein did not change its ATPase activity. In sucrose gradient fractions from the last step of dynein purification, phosphorylation by an endogenous kinase occurred. This phosphorylation could not be attributed to the small amounts of cAMP- and cGMP-dependent protein kinases known to be present, nor was it Ca(2+)-dependent. This previously uncharacterized ciliary protein kinase used casein as an in vitro substrate.

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Melatonin Induces Autophagy via Reactive Oxygen Species-Mediated Endoplasmic Reticulum Stress Pathway in Colorectal Cancer Cells

Molecules ◽

10.3390/molecules26165038 ◽

2021 ◽

Vol 26 (16) ◽

pp. 5038

Author(s):

Kian Chung Chok ◽

Rhun Yian Koh ◽

Ming Guan Ng ◽

Pei Ying Ng ◽

Soi Moi Chye

Keyword(s):

Colorectal Cancer ◽

Endoplasmic Reticulum ◽

Protein Kinase ◽

Er Stress ◽

Mammalian Target Of Rapamycin ◽

Adenosine Monophosphate ◽

Specific Protein ◽

Dependent Manner ◽

Stress Pathway

Even though an increasing number of anticancer treatments have been discovered, the mortality rates of colorectal cancer (CRC) have still been high in the past few years. It has been discovered that melatonin has pro-apoptotic properties and counteracts inflammation, proliferation, angiogenesis, cell invasion, and cell migration. In previous studies, melatonin has been shown to have an anticancer effect in multiple tumors, including CRC, but the underlying mechanisms of melatonin action on CRC have not been fully explored. Thus, in this study, we investigated the role of autophagy pathways in CRC cells treated with melatonin. In vitro CRC cell models, HT-29, SW48, and Caco-2, were treated with melatonin. CRC cell death, oxidative stress, and autophagic vacuoles formation were induced by melatonin in a dose-dependent manner. Several autophagy pathways were examined, including the endoplasmic reticulum (ER) stress, 5′–adenosine monophosphate-activated protein kinase (AMPK), phosphoinositide 3-kinase (PI3K), serine/threonine-specific protein kinase (Akt), and mammalian target of rapamycin (mTOR) signaling pathways. Our results showed that melatonin significantly induced autophagy via the ER stress pathway in CRC cells. In conclusion, melatonin demonstrated a potential as an anticancer drug for CRC.

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Possible Intracellular Regulators of Female Sexual Maturation

Physiological Research ◽

10.33549/physiolres.932838 ◽

2015 ◽

pp. 379-386 ◽

Cited By ~ 6

Author(s):

A. KOLESAROVA ◽

A. V. SIROTKIN ◽

M. MELLEN ◽

S. ROYCHOUDHURY

Keyword(s):

Protein Kinase ◽

Protein Kinases ◽

Granulosa Cells ◽

Sexual Maturation ◽

Cyclin B1 ◽

Nuclear Antigen ◽

Dependent Protein Kinase ◽

Significant Difference ◽

Dependent Protein

Protein kinases, transcription factors and other apoptosis- and proliferation-related proteins can regulate reproduction, but their involvement in sexual maturation remains to be elucidated. The general aim of the in vivo and in vitro experiments with porcine ovarian granulosa cells was to identify possible intracellular regulators of female sexual maturation. For this purpose, proliferation (expression of proliferating cell nuclear antigen – PCNA, mitogen-activated protein kinases – ERK 1,2 related MAPK and cyclin B1), apoptosis (expression of the apoptotic protein Bax and apoptosis regulator Bcl-2 protein), expression of some protein kinases (cAMP dependent protein kinase – PKA, cGMP-dependent protein kinase – PKG, tyrosine kinase – TK) and cAMP responsive element binding protein 1 (CREB-1) was examined in granulosa cells isolated from ovaries of immature and mature gilts. Expression of PCNA, ERK1,2 related MAPK, cyclin B1, Bcl-2, Bax, PKA, CREB-1, TK and PKG in porcine granulosa cells were detected by immunocytochemistry. Sexual maturation was associated with significant increase in the expression of Bcl-2, Bax, PKA, CREB-1 and TK and with decrease in the expression of ERK1,2 related MAPK, cyclin B1 and PKG in granulosa cells. No significant difference in PCNA expression was noted. The present data obtained from in vitro study indicate that sexual maturation in females is influenced by puberty-related changes in porcine ovarian signaling substances: increase in Bcl-2, Bax, PKA, CREB-1, TK and decrease in ERK1,2 related MAPK, cyclin B1 and PKG. It suggests that these signaling molecules could be potential regulators of porcine sexual maturation.

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Phosphodiesterase 3A binds to 14-3-3 proteins in response to PMA-induced phosphorylation of Ser428

Biochemical Journal ◽

10.1042/bj20051103 ◽

2005 ◽

Vol 392 (1) ◽

pp. 163-172 ◽

Cited By ~ 39

Author(s):

Mercedes Pozuelo Rubio ◽

David G. Campbell ◽

Nicholas A. Morrice ◽

Carol Mackintosh

Keyword(s):

Protein Kinase ◽

Metal Ion ◽

Mitogen Activated Protein Kinase ◽

Dependent Manner ◽

Cell Extracts ◽

Mitogen Activated Protein ◽

Dependent Protein ◽

Pkc Protein Kinase C

PDE3A (phosphodiesterase 3A) was identified as a phosphoprotein that co-immunoprecipitates with endogenous 14-3-3 proteins from HeLa cell extracts, and binds directly to 14-3-3 proteins in a phosphorylation-dependent manner. Among cellular stimuli tested, PMA promoted maximal binding of PDE3A to 14-3-3 proteins. While p42/p44 MAPK (mitogen-activated protein kinase), SAPK2 (stress-activated protein kinase 2)/p38 and PKC (protein kinase C) were all activated by PMA in HeLa cells, the PMA-induced binding of PDE3A to 14-3-3 proteins was inhibited by the non-specific PKC inhibitors Ro 318220 and H-7, but not by PD 184352, which inhibits MAPK activation, nor by SB 203580 and BIRB0796, which inhibit SAPK2 activation. Binding of PDE3A to 14-3-3 proteins was also blocked by the DNA replication inhibitors aphidicolin and mimosine, but the PDE3A–14-3-3 interaction was not cell-cycle-regulated. PDE3A isolated from cells was able to bind to 14-3-3 proteins after in vitro phosphorylation with PKC isoforms. Using MS/MS of IMAC (immobilized metal ion affinity chromatography)-enriched tryptic phosphopeptides and phosphospecific antibodies, at least five sites on PDE3A were found to be phosphorylated in vivo, of which Ser428 was selectively phosphorylated in response to PMA and dephosphorylated in cells treated with aphidicolin and mimosine. Phosphorylation of Ser428 therefore correlated with 14-3-3 binding to PDE3A. Ser312 of PDE3A was phosphorylated in an H-89-sensitive response to forskolin, indicative of phosphorylation by PKA (cAMP-dependent protein kinase), but phosphorylation at this site did not stimulate 14-3-3 binding. Thus 14-3-3 proteins can discriminate between sites in a region of multisite phosphorylation on PDE3A. An additional observation was that the cytoskeletal cross-linker protein plectin-1 coimmunoprecipitated with PDE3A independently of 14-3-3 binding.

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