Transformation-specific Decrease of Phosphorylation of 80K Protein, a Substrate of Protein Kinase C, in NIH3T3 Cells

Phosphorylation in normal and transformed NIH3T3 cells of the 80K protein, a specific substrate for protein kinase C, was compared by means of two-dimensional gel analysis. We obtained evidence that NIH3T3 cells transformed by the c- raf or H- ras oncogene maintained a decreased level of phosphorylation of the 80K protein, with or without phorbol ester (TPA)-stimulation, at all concentrations of serum tested while normal NIH3T3 cells maintained an elevated level of phosphorylation of the 80K protein. Furthermore, NIH3T3 cells transformed by N- ras , K- ras, src, mos or polyoma middle T antigen exhibited a decreased level of phosphorylation of the 80K protein. These events were confirmed by an analysis of a hormone-inducible H- ras transformant. Thus, phosphorylation of the 80K protein is inversely correlated with cellular transformation.     

Phorbol ester-mediated association of protein kinase C to the nuclear fraction in NIH 3T3 cells

Treatment of intact NIH 3T3 cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) causes a rapid redistribution (stabilization) of protein kinase C to the particulate fraction. Part of the enzyme activity stabilized to the membrane fraction in response to TPA can be recovered associated with nuclear-cytoskeletal components. An apparently pure nuclear fraction prepared from NIH 3T3 cells was found to contain 25-30% of the total membrane-associated protein kinase C activity when isolated in the presence of Ca2+. In untreated control cells, most of this activity found with the nuclear fraction can be extracted by chelators. Phorbol ester (TPA) treatment of NIH 3T3 cells induces the tight association of protein kinase C to the nucleus; this tightly bound activity is not dissociable by chelators and can be recovered only by solubilization with detergent. Nuclei purified from untreated human promyelocytic leukemic HL-60 cells contain higher amounts of chelator-stable, detergent-extractable protein kinase C activity compared with control NIH 3T3 cells. However, TPA treatment of HL-60 cells does not enhance the amount of protein kinase C found tightly associated with the nuclear fraction. Immunohistochemical studies with polyclonal antibodies directed against protein kinase C further indicate that TPA treatment of NIH 3T3 cells does significantly enhance the amount of protein kinase C found tightly associated with the nucleus and cytoskeleton, whereas exposure of HL-60 cells to TPA does not appreciably alter the amount of protein kinase C observed to be associated with the nuclear fraction. The TPA-mediated association (activation) of protein kinase C to the nuclear and cytoskeletal fractions with NIH 3T3 cells is further supported by the enhanced phosphorylation of specific endogenous proteins noted when purified nuclei and cytoskeletal preparations are incubated with [gamma-32P]ATP. These results suggest that tumor promoters may induce association (activation) of protein kinase C with different subcellular components to alter the availability of endogenous substrates. This may result in differential responses by different cell types during exposure to tumor promoters.     

Inhibition of protein kinase C and proto-oncogene expression by crocetin in NIH/3T3 cells

Crocetin, a carotenoid isolated from the seeds of Gardenia jasminoides, was found to be a potent inhibitor of tumor promotion induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in mouse skin. When mouse fibroblast NIH/3T3 cells were treated with TPA alone, protein kinase C (PKC) translocated from the cytosolic fraction to the particulate fraction. Pretreatment with 60 and 120 μM crocetin for 15 min inhibited the TPA-induced PKC activity in the particulate fraction by 50% and 66%, respectively, but did not affect the level of PKC protein. Crocetin also reduced the level of TPA-stimulated phosphorylation of cellular proteins. Cells pre-treated with crocetin (120 μM) had 55% less PKC [³H]phorbol dibutyrate-binding capacity. Suppression of TPA (100 ng/mL)-induced c-jun and c-fos gene expression was also observed in the mouse fibroblast cells pre-treated with crocetin (30, 60, and 120 μM). Our results provided a basis for understanding the inhibitory effect of crocetin on TPA-mediated tumor promotion. © 1996 Wiley-Liss, Inc.     

Both tumour-promoting and non-promoting phorbol esters inhibit [125I]EGF binding and stimulate the phosphorylation of an 80 kd protein kinase C substrate protein in intact quiescent swiss 3T3 cells

Sapintoxin A (SAP A) and 12-deoxyphorbol 13-phenylacetate (DOPP), are two biologically active but non-tumour-promoting phorbol esters that potently bind to and activate the phorbol ester receptor, protein kinase C (PKC). SAP A and DOPP cause a dose-dependent increase in the phosphorylation of an 80 kd (80K) substrate protein for PKC in Swiss 3T3 cells. A similar dose-response effect was seen with sapintoxin D (SAP D), the stage 2 promoting analogue of 12-O-tetradecanoylphorbol-13-acetate and the complete promoter phorbol 12,13-dibutyrate (PDB). The doses resulting in a half maximal phosphorylation of this protein (Ka) were 20 nM (SAP A), 45 nM (DOPP), 23 nM (SAP D) and 37 nM (PDB). Both non-promoting and promoting phorbol esters induced a dose-dependent inhibition of [125I]epidermal growth factor (EGF) binding to its receptor in Swiss 3T3 cells. The doses required for 50% inhibition of binding (Ki) were: 8 nM (SAP A), 16 nM (DOPP), 14 nM (SAP D) and 17 nM (PDB). The results clearly demonstrate that induction of phosphorylation of the 80K phosphoprotein and inhibition of [125I]EGF binding in Swiss 3T3 cells following exposure to phorbol esters is independent of the tumour-promoting activity of these compounds. The fact that SAP A, DOPP, SAP D and PDB are mitogenic for a variety of cell types and that exposure to these compounds leads to 80K phosphorylation and inhibition of [125I]EGF binding, suggests that these early biological events may play a role in the mitogenic response induced by these compounds.     

Inhibitory effects of curcumin on protein kinase C activity induced by 12-O-tetradecanoyl-phorbol-13-acetate in NIH 3T3 cells

Curcumin is a dietary pigment responsible for the yellow colorof curry. It is a potent inhibitor of tumor promotion inducedby 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in mouse skin.When mouse fibroblast cells (NIH 3T3) were treated with TPAalone, protein kinase C (PKC) translocated from the cytosolicfraction to the particulate fraction. Treatment with 15 or 20µm curcumin for 15 min inhibited TPA-induced PKC activityin particulate fraction by 26 or 60% and did not affect thelevel of PKC protein. Curcumin also inhibited PKC activity inboth cytosolic and particulate fractions in vitro by competingwith phosphatidylserine. However, the inhibitory effect of curcuminwas reduced after preincubation with the thiol compounds. Thesefindings suggest that the suppression of PKC activity may contributeto the molecular mechanism of inhibition of TPA-induced tumorpromotion by curcumin.     

Constitutive phosphorylation of a Rac GAP MgcRacGAP is implicated in v-Src-induced transformation of NIH3T3 cells

MgcRacGAP plays critical roles in cell division through regulating Rho family small GTPases. As we previously reported, phosphorylation of MgcRacGAP on serine 387 (S387) is induced by Aurora B kinase at the midbody during cytokinesis, which is a critical step of cytokinesis. Phosphorylation of S387-MgcRacGAP converts it from RacGAP to RhoGAP, leading to completion of cytokinesis. Here we show that MgcRacGAP is prominently phosphorylated on S387 even in the interphase of v-Src-transformed NIH3T3 cells in the cytoplasm, but not in the interphase of parental NIH3T3 or H-RasV12-transformed NIH3T3 cells. Interestingly, levels of phosphorylation on S387 (pS387) correlated with soft agar colony-forming abilities of v-Src-transformed NIH3T3 cells. Expression of a phosphorylation-mimic mutant MgcRacGAP-S387D enhanced colony formation of v-Src-transformed NIH3T3 cells. Surprisingly, a Rac1 inhibitor but not kinase inhibitors including Aurora B kinase inhibitor specifically inhibited phosphorylation of S387-MgcRacGAP in v-Src-transformed NIH3T3 cells, suggesting the v-Src-induced pathological positive feedback mechanisms towards Rac1 activation using pS387-MgcRacGAP. These results indicated the difference in the mechanisms between v-Src- and H-RasV12-induced transformation, and should shed some light on pathological roles of disordered phosphorylation of MgcRacGAP at S387 in v-Src-induced cell transformation. ( Cancer Sci 2009; 100: 1675–1679)     

A tyrosine-specific protein kinase inhibitor, alpha-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamamide, blocks the phosphorylation of tyrosine kinase substrate in intact cells

Inhibition by alpha-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamamide (ST 638) of tyrosine-specific protein kinase was examined using epidermal growth factor (EGF)-treated A431 cells at the concentration of 25 to 100 microM. ST 638 had negligible effects on the growth and morphology of A431 cells and on EGF binding to its receptor, and subsequent down-regulation of the receptor. ST 638 specifically inhibited EGF-induced phosphorylation of tyrosine residues of whole cell proteins in a dose-dependent manner without affecting the phosphorylation of serine and threonine residues. ST 638 greatly inhibited the EGF-induced phosphorylation of lipocortin I at 25 microM, and yet had a negligible effect on the EGF-induced phosphorylation of EGF receptor. Neither the amount of [35S]methionine-labeled lipocortin I nor the serine/threonine phosphorylation level of fodrin beta-subunit was affected by the same concentration of ST 638. These results indicate that the phosphorylation of lipocortin I is not relevant to the transformation of A431 cells. In cell lines transformed by src or fgr oncogene encoding tyrosine kinase, ST 638 also inhibited phosphorylation of calpactin I (p36) without affecting that of the oncogene products. Two-dimensional polyacrylamide gel electrophoresis showed that ST 638 specifically inhibited the EGF-induced phosphorylation and dephosphorylation of cellular proteins in A431 cells.     

Activation of protein kinase C and specific phosphorylation of a Mr 90,000 membrane protein of promotable BALB/3T3 and C3H/10T1/2 cells by tumor promoters

The activation of protein kinase C and protein phosphorylation by tumor promoters were examined using quiescent cultures of BALB/3T3 and C3H/10T1/2 cells, because in these cells tumor promoters enhance chemically induced transformation and also induce DNA synthesis and ornithine decarboxylase. The cytosol and membrane fractions were partially purified, and the activity of protein kinase C was assayed. In quiescent cells, protein kinase C activity was found only in the cytosol fraction. Treatment with 100 ng of 12-O-tetradecanoylphorbol-13-acetate or teleocidin B per ml caused rapid translocation of protein kinase C from the cytosol to the membrane fraction. The activity in the cytosol disappeared almost completely after 15 min when the activity in the membrane reached a peak. The membrane activity gradually decreased to the control level after 6 h, while no activity reappeared in the cytosol within 6 h. Under these circumstances, a membrane protein with a molecular weight of 90,000 and pl of 4.0-4.4 (termed p90) was specifically phosphorylated, possibly by the activated protein kinase C, in both cell-free and intact-cell systems. On treatment of quiescent BALB/3T3 cells with 100 ng of 12-O-tetradecanoylphorbol-13-acetate, p90 phosphorylation increased 2-fold in 1 min, reaching a peak in 15 min of 3.4-fold the initial value. The phosphorylation of p90 increased with increase in the concentrations of 12-O-tetradecanoylphorbol-13-acetate between 0.1 and 10 ng/ml and reached a plateau at 10 ng/ml. p90 phosphorylation also occurred on exposure of the cells to non-phorbol ester tumor promoters (mezerein and teleocidin B) and growth factors, such as platelet-derived growth factor and fibroblast growth factor. p90 was not immunoprecipitated by antibody against the insulin receptor. Phosphorylation of p90 occurred at a serine residue. The present study suggests that activation of protein kinase C and phosphorylation of p90 by it are early events leading to tumor promotion.     

Transfection of constitutively active mitogen-activated protein/extracellular signal-regulated kinase kinase confers tumorigenic and metastatic potentials to NIH3T3 cells

Cellular growth and differentiation are controlled by multiple extracellular signals, many of which activate extracellular signal-regulated kinase (ERK)/mitogen-activated protein (MAP) kinases. Components of the MAP kinase pathways also cause oncogenic transformation in their constitutively active forms. Moreover, expression of activated ras can confer metastatic potential upon some cells. Activation of MAP kinases requires phosphorylation of both Thr and Tyr in the catalytic domain by a family of dual-specificity kinases, called MEKs (MAP kinase/ERK kinase). MEK1 is activated by phosphorylation at Ser218 and Ser222 by Raf. Mutation of these two sites to acidic residues, specifically [Asp218], [Asp218, Asp222], and [Glu218, Glu222], results in constitutively active MEK1. Using these mutant variants of MEK1, we showed previously that transfection of NIH/3T3 or Swiss 3T3 cells causes morphological transformation and increases growth on soft agar, independent of ERK activity. The transformed cell lines show increased expression of matrix metalloproteinases 2 and 9 and cathepsin L, proteinases that have been implicated in the metastatic process. We tested NIH3T3 cells transfected with the [Asp218] or [Asp218, Asp222] for metastatic potential after i.v. injection into athymic mice. Parental 3T3 cells formed no tumors grossly or histologically. However, all MEK1 mutant transformants formed macroscopic metastases. Thus, like activated Ras, MEK1 can confer both tumorigenic and metastatic potential upon NIH3T3 cells. These results refine the mechanism through which ras could confer tumorigenic and metastatic potential (ie., the critical determinants of tumorigenic and metastatic potential are downstream of MEK1).     

Polyamine metabolism and interconversion in NIH 3T3 and ras-transfected NIH 3T3 cells

The effect of transfection of NIH 3T3 cells by the human ras (c-Ha-ras-1) oncogene on uptake, interconversion, and excretion of polyamines was studied. Uptake and interconversion of spermidine were higher in the ras-transfected cells. Acetylpolyamines were excreted into the medium by the ras-transfected cells, whereas they were retained by NIH 3T3 cells. In addition to acetylpolyamines, some unknown polyamine conjugates occurred in the ras-transfected cells.     

Oncogenic H-Ras up-regulates expression of Ku80 to protect cells from gamma-ray irradiation in NIH3T3 cells

The Ras activation contributes to radioresistance, but the mechanism is unclear. This article shows that the expression of the dominant-positive H-Ras increased the Ku80 level, which is one of the key enzymes involved in repairing dsDNA breaks (DSB). After exposing the cells to ionizing radiation and analyzing them using an electrophoretic mobility shift assay and pulsed-field gel electrophoresis, it was found that activated H-Ras expression in NIH3T3 cells increases the DNA-binding activity of Ku80 and increases the DSB repair activity. Ku80 small interfering RNA expression was shown to reduce the oncogenic H-Ras-mediated increase in the DSBs and suppress the oncogenic H-Ras-mediated resistance of the cells to gamma-ray irradiation, whereas Ku80 overexpression in the NIH3T3 cells significantly increased the radioresistance. These results suggest that the Ku80 expression induced by oncogenic H-Ras seems to play an important role in protecting cells against gamma-ray irradiation.     

Omega-3 fatty acids decrease protein kinase expression in human breast cancer cells

We report that 5-day exposure to physiological concentrations of eicosapentaenoic and docosahexaenoic acids resulted in a strong decrease in expression of the RI regulatory subunit of protein kinase A and the PKC- isozyme of protein kinase C in the human breast cancer cell line MDA-MB-231.     

Effect of microinjected catalytic fragment of protein kinase C on morphological change in Swiss 3T3 cells

Although phorbol esters can enhance formation of an active, catalytic domain of protein kinase C (PKC) in intact cells, little is known about the actual importance of the proteolytic pathway in mediating cellular responses to the phorbol esters or other PKC activators. To explore this issue, we examined the effect of microinjected catalytic fragment of PKC on Swiss 3T3 cell morphology. In contrast to the dramatic, rapid response upon phorbol ester treatment, catalytic fragment microinjected in the presence of bovine serum albumin or normal goat immunoglobulin G as carrier protein had no effect. A morphological response similar but not identical to the effect of phorbol ester treatment was obtained, however, if catalytic fragment was microinjected in the presence of normal rabbit immunoglobulin G rather than the usual carrier proteins. The normal rabbit immunoglobulin by itself was inactive. Although the mechanism remains undefined, normal rabbit immunoglobulin but not other carrier proteins modulated PKC activity in vitro. We conclude that the generation of free catalytic fragment of PKC cannot account for the morphological response of Swiss 3T3 cells to the phorbol esters; secondary factors may, however, potentiate its action.     

Scrape-loading of Swiss 3T3 cells with ras protein rapidly activates protein kinase C in the absence of phosphoinositide hydrolysis

Scrape-loading has been used to analyse the biochemical function of purified p21ras protein. We have shown that scrape-loading oncogenic p21ras into quiescent Swiss 3T3 cells causes morphological transformation of 90% of the cell population within 15 h. Since large numbers of cells can be loaded with p21ras, early induced biochemical changes can be analysed. In this way we have shown that oncogenic p21ras causes rapid activation of protein kinase C five minutes after introduction of protein, but that ras protein fails to stimulate measurable inositol phosphate formation. It appears, therefore, that the stimulation of protein kinase C activity is due to a ras induced increase in diacylglycerol from a source other than inositol phospholipids. Efficient stimulation of DNA synthesis by oncogenic p21ras only occurs in the presence of insulin. This stimulation of DNA synthesis by ras is absolutely dependent on functional protein kinase C activity.     

Role of protein kinase C in Adriamycin-induced erythroid differentiation of K562 cells

Summary Modulators of protein kinase C (PKC) were used to investigate the role of this enzyme during Adriamycin-induced erythroid differentiation of K562 cells. Adriamycin (0.1 M) induced erythroid differentiation in 60%±10% of K562 cells. Phorbol myristate-12-acetate, an activator of protein kinase C, was strongly anti-proliferative to K562 cells (IC₅₀, 8 nM) but did not induce erythroid differentiation. Staurosporine inhibited PKC from K562 cells (IC₅₀, 8 nM) and blocked Adriamycin-induced erythroid differentiation, but only at concentrations marginally below those that inhibited proliferation (IC₅₀, 81 nM). 1-(5-Isoquinolinylsulphonyl)-2-methylpiperazine (H-7) inhibited K562 PKC (IC₅₀, 26 M) but reduced Adriamycin-induced differentiation by <50% at concentrations of up to 600 M. These data argue against a major role for PKC during Adriamycin-induced erythroid differentiation in K562 cells.     

Role of protein kinase C in phosphorylation of vinculin in adriamycin-resistant HL-60 leukemia cells

In response to phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA), HL-60 cells differentiate to macrophage-like cells and exhibit the ability to phosphorylate vinculin in vitro. Adriamycin-resistant HL-60 (HL-60/ADR) cells similarly demonstrate this characteristic without prior treatment with TPA. Since protein kinase C (PK-C) is a cellular TPA receptor, we have examined the role of this enzyme in the inherent ability of HL-60/ADR cells to phosphorylate vinculin. DEAE-cellulose chromatography of cell extracts revealed that HL-60/ADR cells contained 2-fold more PK-C than did the parental cell line. All PK-C activity was found in the cytosol of wild type HL-60 cells, whereas 85% of PK-C activity was cytosolic and 15% was membrane-bound in HL-60/ADR cells. After a 2-day treatment with 10 nM TPA, PK-C activity was reduced 80-90% in both cell lines regardless of its intracellular distribution. Immunoblotting of cell extracts from HL-60/ADR cells or HL-60 cells following treatment with TPA revealed increased levels of a 52-kDa species of similar mass to M-kinase. Coincident with these changes after TPA treatment was a reduction in Ca2+ and phospholipid-independent phosphorylation of vinculin in vitro in extracts from HL-60/ADR cells, whereas HL-60 cells exhibited an elevation of this phosphoprotein. The phosphorylation of vinculin in TPA-treated HL-60 cells or untreated HL-60/ADR cells was blocked by antibodies to protein kinase C. These results suggest that it is not the absolute level of protein kinase C but rather the proteolytic activation of PK-C to a Ca2+ and phospholipid-independent form which is associated with the utilization of vinculin as an endogenous substrate.