The 28-kDa protein whose phosphorylation is induced by protein kinase C activators in MCF-7 cells belongs to the family of low molecular mass heat shock proteins and is the estrogen-regulated 24-kDa protein.
We have previously reported the presence of a 28-kDa protein in human mammary adenocarcinoma MCF-7 cells, whose phosphorylation by phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and permeant diacylglycerol 1,2-dioctanoyl-sn-glycerol was correlated to growth arrest induced by the protein kinase C (PKC) activators. We now investigate the possible identity of this protein with the estrogen-regulated "24-kDa" protein shown as related to the mammalian heat shock protein 27 (Fuqua, S. A. W., Blum-Salingaros, M., and McGuire, W. L. (1989) Cancer Res 49, 4126-4129). 32P-Labeled 28-kDa protein from TPA-treated MCF-7 cells was immunoprecipitated with a 24-kDa-specific monoclonal antibody. Immunoblots from cell extracts fractionated by two-dimensional isoelectric focusing/SDS-polyacrylamide gel electrophoresis demonstrated that TPA induced the conversion of a 28-kDa isoform "a" (pI 6.7) to a more acidic isoform "b" (pI 6.2). Two-dimensional gel analysis of [3H]leucine-labeled MCF-7 cell extracts demonstrated that conversely to TPA, which induced only phosphorylation of 28-kDa protein, heat shock induced both synthesis (increase of isoform a) and phosphorylation (conversion of isoforms a to b) of the protein. 32P labeling of MCF-7 cells allowed demonstration of the presence of an extra phosphoisoform "c" (pI 5.9) upon TPA as well as heat shock treatment. When cells were pretreated with the bisindolylmaleimide GF109203X, a selective inhibitor of PKC, the heat shock-induced phosphorylation was unchanged, while the TPA effect was almost abolished, suggesting that the heat shock-activated protein kinase was very likely different from PKC. However, peptide mapping of the 28-kDa phosphoprotein suggested identical sites of phosphorylation upon TPA and heat shock stimulation. Partial amino acid sequencing of the 28-kDa protein revealed identity with both the 24-kDa protein and the mammalian HSP27. The fact that estrogens and PKC, respectively, regulate expression and phosphorylation of this 24/28-kDa protein strongly argues for its key role in MCF-7 cell proliferation and differentiation.
Faucher
C
C
Institut National de la Santé et de la Recherche Médicale, Unité 133, Faculté de Médecine, Toulouse, France.
Capdevielle
J
J
Canal
I
I
Ferrara
P
P
Mazarguil
H
H
McGuire
W L
WL
Darbon
J M
JM
D000595:Amino Acid Sequence; D002454:Cell Differentiation; D002455:Cell Division; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004967:Estrogens / Q000502:physiology*; D055551:HSP27 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007525:Isoelectric Focusing; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009363:Neoplasm Proteins; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011960:Receptors, Estrogen; D017386:Sequence Homology, Amino Acid; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D004967:Estrogens; D055551:HSP27 Heat-Shock Proteins; C057725:HSPB1 protein, human; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D009363:Neoplasm Proteins; D010750:Phosphoproteins; D011960:Receptors, Estrogen; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate