Association of PKC delta and active Src in PMA-treated MCF-7 human breast cancer cells.
Phorbol ester treatment of MCF-7 cells led to the tyrosine phosphorylation and activation of PKC delta. However, through Western blot analysis and in vitro immunecomplex kinase assays, we detected a differential localization of tyrosine-phosphorylated PKC delta and catalytically active PKC delta. Catalytically active PKC delta was concentrated in Triton X-100 solubilized-membrane fractions while tyrosine-phosphorylated PKC delta was localized to the cytosol fraction. Phorbol ester treatment of MCF-7 cells stimulated both the time-dependent in vivo association of Src with PKC delta, evidenced in Src immunoprecipitates by the co-immunoprecipitation of PKC delta, and activation of Src, evidenced in Src immunoprecipitates as an increase in reactivity with a Src antibody (clone 28) reactive only with active Src (dephosphorylated on residue 530) and in Src and PKC delta immunoprecipitates by an increase in Src kinase activity. While our data are consistent with reports in the literature showing the activator/stimulus-dependent tyrosine phosphorylation of PKC delta, our data show that the tyrosine phosphorylation of PKC delta is not essential for kinase activity. These results are the first to demonstrate an in vivo association between PKC delta and active Src in the absence of over-expression of either PKC delta or Src, and support the association of Src and PKC delta towards a physiological function.
Shanmugam
M
M
Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611, USA.
Krett
N L
NL
Peters
C A
CA
Maizels
E T
ET
Murad
F M
FM
Kawakatsu
H
H
Rosen
S T
ST
Hunzicker-Dunn
M
M
D001943:Breast Neoplasms; D002457:Cell Extracts; D002458:Cell Fractionation; D004789:Enzyme Activation; D055551:HSP27 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D008934:Mitogens / Q000494:pharmacology*; D018832:Molecular Chaperones; D009363:Neoplasm Proteins / Q000378:metabolism; D015688:Oncogene Protein pp60(v-src) / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051745:Protein Kinase C-delta; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D013997:Time Factors; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism
D002457:Cell Extracts; D055551:HSP27 Heat-Shock Proteins; C057725:HSPB1 protein, human; D006360:Heat-Shock Proteins; D007527:Isoenzymes; D008934:Mitogens; D018832:Molecular Chaperones; D009363:Neoplasm Proteins; D014443:Tyrosine; D015688:Oncogene Protein pp60(v-src); C497258:PRKCD protein, human; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D013755:Tetradecanoylphorbol Acetate