|SWI/SNF chromatin remodeling enzyme ATPases promote cell proliferation in normal mammary epithelial cells.|
Cohet, N; Stewart, KM; Mudhasani, R; Asirvatham, AJ; Mallappa, C; Imbalzano, KM; Weaver, VM; Imbalzano, AN; Nickerson, JA
Journal of cellular physiology
The ATPase subunits of the SWI/SNF chromatin remodeling enzymes, Brahma (BRM) and Brahma-related gene 1 (BRG1), can induce cell cycle arrest in BRM and BRG1 deficient tumor cell lines, and mice heterozygous for Brg1 are pre-disposed to breast tumors, implicating loss of BRG1 as a mechanism for unregulated cell proliferation. To test the hypothesis that loss of BRG1 can contribute to breast cancer, we utilized RNA interference to reduce the amounts of BRM or BRG1 protein in the nonmalignant mammary epithelial cell line, MCF-10A. When grown in reconstituted basement membrane (rBM), these cells develop into acini that resemble the lobes of normal breast tissue. Contrary to expectations, knockdown of either BRM or BRG1 resulted in an inhibition of cell proliferation in monolayer cultures. This inhibition was strikingly enhanced in three-dimensional rBM culture, although some BRM-depleted cells were later able to resume proliferation. Cells did not arrest in any specific stage of the cell cycle; instead, the cell cycle length increased by approximately 50%. Thus, SWI/SNF ATPases promote cell cycle progression in nonmalignant mammary epithelial cells.
|Activation of an S6 kinase from human placenta by autophosphorylation.|
Dennis, P B and Masaracchia, R A
J. Biol. Chem., 268: 19833-41 (1993)
A number of protein kinases have been shown to undergo autophosphorylation, but few have demonstrated a coordinate increase or decrease in enzymatic activity as a result. Described here is a novel S6 kinase isolated from human placenta which autoactivates through autophosphorylation in vitro. This S6/H4 kinase, purified in an inactive state, exhibited a molecular mass of 60 kDa as estimated by SDS-polyacrylamide gel electrophoresis. The 60-kDa protein underwent autophosphorylation, was labeled by 8-azido-[alpha-32P]ATP, and reacted with an antibody to the conserved APE domain of the cAMP-dependent protein kinase. The protein did not cochromatograph with p70 S6 kinase and did not cross-react with an anti-p70 kinase antibody. The synthetic peptide S6-21, histone H4, and myelin basic protein were phosphorylated by the purified S6/H4 kinase. Mild digestion of the inactive S6/H4 kinase with trypsin generated a 40-kDa fragment, as determined by SDS-polyacrylamide gel electrophoresis. The trypsin treatment was necessary, but not sufficient, to fully activate the kinase. Subsequent incubation of the trypsin-treated S6 kinase with MgATP resulted in the rapid autophosphorylation of the 40-kDa fragment along with a coordinate increase in kinase activity. The autophosphorylation of the 40-kDa protein was positively correlated with MgATP incubation time and an increase in activity toward the S6-21 peptide, histone H4, and myelin basic protein. Taken together, these data support the hypothesis that this previously uncharacterized S6 kinase belongs to a unique family of protein kinases which utilize autophosphorylation as part of their in vivo activation mechanism.