Type 3 protein kinase C localization to the nuclear envelope of phorbol ester-treated NIH3T3 cells

K. L. Leach, E. A. Powers, V. A. Ruff, S. Jaken, Scott H Kaufmann

Research output: Contribution to journalArticle

163 Citations (Scopus)

Abstract

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 stained. 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.

Original languageEnglish (US)
Pages (from-to)685-695
Number of pages11
JournalJournal of Cell Biology
Volume109
Issue number2
StatePublished - Jan 1 1989
Externally publishedYes

Fingerprint

Nuclear Envelope
Phorbol Esters
Protein Kinase C
Staining and Labeling
Acetates
NIH 3T3 Cells
Nuclear Proteins
Cell Nucleus
Isoenzymes
Epitopes
Western Blotting

ASJC Scopus subject areas

  • Cell Biology

Cite this

Type 3 protein kinase C localization to the nuclear envelope of phorbol ester-treated NIH3T3 cells. / Leach, K. L.; Powers, E. A.; Ruff, V. A.; Jaken, S.; Kaufmann, Scott H.

In: Journal of Cell Biology, Vol. 109, No. 2, 01.01.1989, p. 685-695.

Research output: Contribution to journalArticle

Leach, K. L. ; Powers, E. A. ; Ruff, V. A. ; Jaken, S. ; Kaufmann, Scott H. / Type 3 protein kinase C localization to the nuclear envelope of phorbol ester-treated NIH3T3 cells. In: Journal of Cell Biology. 1989 ; Vol. 109, No. 2. pp. 685-695.
@article{62f477f5afd342b48df130145ae4dbcd,
title = "Type 3 protein kinase C localization to the nuclear envelope of phorbol ester-treated NIH3T3 cells",
abstract = "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 stained. 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.",
author = "Leach, {K. L.} and Powers, {E. A.} and Ruff, {V. A.} and S. Jaken and Kaufmann, {Scott H}",
year = "1989",
month = "1",
day = "1",
language = "English (US)",
volume = "109",
pages = "685--695",
journal = "Journal of Cell Biology",
issn = "0021-9525",
publisher = "Rockefeller University Press",
number = "2",

}

TY - JOUR

T1 - Type 3 protein kinase C localization to the nuclear envelope of phorbol ester-treated NIH3T3 cells

AU - Leach, K. L.

AU - Powers, E. A.

AU - Ruff, V. A.

AU - Jaken, S.

AU - Kaufmann, Scott H

PY - 1989/1/1

Y1 - 1989/1/1

N2 - 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 stained. 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.

AB - 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 stained. 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.

UR - http://www.scopus.com/inward/record.url?scp=0024318707&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0024318707&partnerID=8YFLogxK

M3 - Article

C2 - 2668302

AN - SCOPUS:0024318707

VL - 109

SP - 685

EP - 695

JO - Journal of Cell Biology

JF - Journal of Cell Biology

SN - 0021-9525

IS - 2

ER -