scholarly journals Type 3 protein kinase C localization to the nuclear envelope of phorbol ester-treated NIH 3T3 cells.

1989 ◽  
Vol 109 (2) ◽  
pp. 685-695 ◽  
Author(s):  
K L Leach ◽  
E A Powers ◽  
V A Ruff ◽  
S Jaken ◽  
S Kaufmann
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cell Surface ◽  
Nuclear Envelope ◽  
Immunofluorescent Staining ◽  
Regulatory Domain ◽  
3T3 Cells ◽  
Kinase C ◽  
Type 3 ◽  
Nih 3T3

We have examined the immunocytochemical localization of protein kinase C (PKC) in NIH 3T3 cells using mAbs that recognize Type 3 PKC. In control cells, the immunofluorescent staining was similar with mAbs directed to either the catalytic or the regulatory domain of PKC. Type 3 PKC localized in a diffuse cytoplasmic pattern, while the nuclei were apparently unstained. Cytoskeletal components also were Treatment of the cells with phorbol 12-myristate 13-acetate (PMA) resulted in a redistribution of PKC with a specific increase in nuclear PKC. Compared to control cells, the staining with the anticatalytic domain mAbs changed markedly, covering the entire cell surface. In contrast, the staining by the antiregulatory domain mAb did not cover the cell surface and the nuclei remained unstained; these results suggest that PKC activation leads to a conformational change of the regulatory domain such that the epitope recognized by the antiregulatory domain mAb is not readily accessible. We have demonstrated by three criteria that PMA treatment specifically increased PKC in the nucleus: (a) immunofluorescent staining in isolated nuclei increased; (b) Western blots showed that our mAbs detected only one protein, the 82-kD PKC, whose level increased in nuclear lysates from PMA-treated cells; and (c) PKC activity increased in nuclear lysates. In fractionation studies we demonstrated that PKC specifically localized to the nuclear envelope fraction. These results demonstrate that PMA activation leads to a rapid redistribution of Type 3 PKC to the nuclear envelope, and suggests that this isozyme may play a role in mediating PKC-induced changes in gene expression.

Role of nuclear protein kinase C in the mitogenic response to platelet-derived growth factor

1990 ◽  
Vol 96 (1) ◽  
pp. 107-114
Author(s):  
A.P. Fields ◽  
G. Tyler ◽  
A.S. Kraft ◽  
W.S. May
Keyword(s):  
Protein Kinase C ◽  
Growth Factor ◽  
Protein Kinase ◽  
Nuclear Envelope ◽  
Platelet Derived Growth Factor ◽  
3T3 Cells ◽  
Mitogenic Response ◽  
3T3 Fibroblasts ◽  
Kinase C ◽  
Nih 3T3

We have assessed the involvement of nuclear envelope protein phosphorylation in the mitogenic response to platelet-derived growth factor (PDGF) in NIH/3T3 fibroblasts. We find that stimulation of quiescent NIH/3T3 cells with PDGF or with the mitogenic protein kinase C (PKC) activators phorbol 12-myristate 13-acetate (PMA) or bryostatin 1 (bryo) leads to rapid, dose-dependent phosphorylation of several nuclear envelope polypeptides. The predominant nuclear envelope targets for mitogen-induced phosphorylation are immunologically identified as the nuclear envelope lamins. All three lamin species (A, B and C) are phosphorylated in response to PMA or bryo, while lamins A and C are preferentially phosphorylated in response to PDGF. Phosphopeptide mapping and phosphoamino acid analysis indicate that similar serine sites on the lamins are phosphorylated in response to PDGF, PMA and bryo. Both mitogenicity and lamina phosphorylation induced by these mitogens can be inhibited by the selective PKC inhibitor staurosporine at 2 nM. Treatment of quiescent NIH/3T3 cells with PDGF, PMA or bryo leads to rapid translocation of PKC to the nuclear envelope. These data indicate that rapid nuclear events, including translocation of cytosolic PKC to the nuclear membrane and lamina phosphorylation, may play a role in the transduction of the mitogenic signals of PDGF from the cytoplasm to the nucleus in NIH/3T3 fibroblasts.

scholarly journals Both the Catalytic and Regulatory Domains of Protein Kinase C Chimeras Modulate the Proliferative Properties of NIH 3T3 Cells

1997 ◽  
Vol 272 (45) ◽  
pp. 28793-28799 ◽  
Author(s):  
Péter Ács ◽  
Qiming J. Wang ◽  
Krisztina Bögi ◽  
Adriana M. Marquez ◽  
Patricia S. Lorenzo ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
3T3 Cells ◽  
Regulatory Domains ◽  
Kinase C ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

scholarly journals Insulin and vasopressin elicit inhibition of cholera-toxin-stimulated adenylate cyclase activity in both hepatocytes and the P9 immortalized hepatocyte cell line through an action involving protein kinase C

1995 ◽  
Vol 312 (3) ◽  
pp. 769-774 ◽  
Author(s):  
L Zeng ◽  
M D Houslay
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Cell Line ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
3T3 Cells ◽  
Kinase C ◽  
Nih 3T3

Incubation of hepatocytes or the SV40-DNA-immortalized hepatocyte P9 cell line with cholera toxin led to a time-dependent activation of adenylate cyclase activity, which occurred after a defined lag period. When added together with cholera toxin, each of the hormones insulin and vasopressin was capable of attenuating the maximum stimulatory effect achieved by cholera toxin over a period of 60 min through a process which could be blocked by the compounds staurosporine and chelerythrine. Attenuating effects on cholera-toxin-stimulated adenylate cyclase activity could also be elicited by using either the protein kinase C (PKC)-stimulating phorbol ester PMA (phorbol 12-myristate 13-acetate) or the protein phosphatase inhibitor okadaic acid. Alkaline phosphatase treatment of membranes reversed the inhibitory effect of PMA. Cholera toxin also stimulated the adenylate cyclase activity of intact CHO (Chinese-hamster ovary) and NIH-3T3 cells, but this activity was insensitive to the addition of PMA. Overexpression of various PKC isoforms in CHO cell lines did not confer sensitivity to inhibition by PMA upon cholera-toxin-stimulated adenylate cyclase activity. Rather, overexpression of the gamma isoform of PKC allowed PMA to stimulate adenylate cyclase activity in CHO cells. It is suggested that the PKC-mediated phosphorylation of a membrane protein attenuates cholera-toxin-stimulated adenylate cyclase activity in hepatocytes and P9 cells. The cellular selectivity of such an action may be due to the target for this inhibitory action of PKC being a particular isoform of adenylate cyclase which provides the major activity in hepatocytes and P9 cells, but not in either CHO or NIH-3T3 cells.

Effect of a dominant inhibitory Ha-ras mutation on mitogenic signal transduction in NIH 3T3 cells

1990 ◽  
Vol 10 (10) ◽  
pp. 5314-5323
Author(s):  
H Cai ◽  
J Szeberényi ◽  
G M Cooper
Keyword(s):  
Signal Transduction ◽  
Protein Kinase C ◽  
Growth Factor ◽  
Protein Kinase ◽  
Dna Synthesis ◽  
3T3 Cells ◽  
Kinase C ◽  
Mitogenic Signal Transduction ◽  
Nih 3T3 ◽  
Mitogenic Signal

We used a dominant inhibitory mutation of c-Ha-ras which changes Ser-17 to Asn-17 in the gene product p21 [p21(Asn-17)Ha-ras] to investigate ras function in mitogenic signal transduction. An NIH 3T3 cell line [NIH(M17)] was isolated that displayed inducible expression of the mutant Ha-ras gene (Ha-ras Asn-17) via the mouse mammary tumor virus long terminal repeat and was growth inhibited by dexamethasone. The effect of dexamethasone induction on response of quiescent NIH(M17) cells to mitogens was then analyzed. Stimulation of DNA synthesis by epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA) was completely blocked by p21(Asn-17) expression, and stimulation by serum, fibroblast growth factor, and platelet-derived growth factor was partially inhibited. However, the induction of fos, jun, and myc by EGF and TPA was not significantly inhibited in this cell line. An effect of p21(Asn-17) on fos induction was, however, demonstrated in transient expression assays in which quiescent NIH 3T3 cells were cotransfected with a fos-cat receptor plasmid plus a Ha-ras Asn-17 expression vector. In this assay, p21(Asn-17) inhibited chloramphenicol acetyltransferase expression induced by EGF and other growth factors. In contrast to its effect on DNA synthesis, however, Ha-ras Asn-17 expression did not inhibit fos-cat expression induced by TPA. Conversely, downregulation of protein kinase C did not inhibit fos-cat induction by activated ras or other oncogenes. These results suggest that ras proteins are involved in at least two parallel mitogenic signal transduction pathways, one of which is independent of protein kinase C. Although either pathway alone appears to be sufficient to induce fos, both appear to be necessary to induce the full mitogenic response.

Induction of a mitogen-responsive gene after expression of the Ha-ras oncogene in NIH 3T3 fibroblasts

1989 ◽  
Vol 9 (11) ◽  
pp. 5207-5214
Author(s):  
A K Werenskiold ◽  
S Hoffmann ◽  
R Klemenz
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Transcriptional Level ◽  
3T3 Cells ◽  
Serum Factors ◽  
Kinase C ◽  
Conditional Expression ◽  
T1 Gene ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

A cDNA clone, T1, has been isolated whose corresponding mRNA was transiently expressed at highly elevated levels after conditional expression of the Ha-ras(EJ) gene and after mitogenic activation of quiescent NIH 3T3 cells. Glucocorticoid hormone stimulated substantial T1 expression as well but only in proliferating cells. At least two different signaling pathways participate in the regulation of the T1 gene: a protein kinase C-dependent signal is involved in the response of proliferating NIH 3T3 cells to glucocorticoid in the absence but not the presence of p21ras, whereas a protein kinase C-independent mechanism mediates the response to serum factors. Treatment of cells with the protein kinase inhibitor 2-aminopurine blocked induction of expression of the T1 gene. T1 mRNA accumulation is regulated at the transcriptional level.

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