Identification of lamin B and histones as 1,25-dihydroxyvitamin D3-regulated nuclear phosphoproteins in HL-60 cells.
1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3)-induced differentiation of HL-60 leukemia cells is accompanied by a number of cellular changes including regulation of oncogene expression and induction of terminal differentiation. We investigated the mechanism by which 1,25-(OH)2D3 induces these changes. We detected 10 nuclear phosphoproteins, designated p66, p45, p36, p33, p32, p27, p22, p19, p18 and p17, that show alterations in phosphorylation within 6-40 h of 1,25-(OH)2D3 treatment. When phosphorylation reactions were performed with isolated nuclei (in vitro), three of these proteins were phosphorylated in a calcium and phospholipid dependent manner: p66, p36, and p19 P66 was phosphorylated in response to 1,25-(OH)2D3 and purified in a manner similar to that used for nuclear lamins. Western blot analysis of 2-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels confirmed its identity as lamin B. Phosphorylation of p17 and p18 decreased following 1,25-(OH)2D3 treatment. We separated p17 and p18 by SDS-PAGE and obtained N-terminal amino acid sequence to identify these phosphorproteins as histones H2b and H3, respectively. P19 and p22 were both DNA-cellulose binding proteins whose phosphorylation was altered by 1,25-(OH)2D3 treatment. Increased phosphorylation of p27 was detected using 2-dimensional SDS-PAGE. Phosphorylation of nuclear proteins in the intact cell (in vivo), revealed increases in p66, p45, p36, and p33 phosphorylation and a decrease in p17 phosphorylation following 1,25-(OH)2D3 treatment. We detected an increase in phosphorylation of p32, which was extracted with salt from nuclei and migrated on SDS-PAGE similar to histone H1. Thus, we have identified 1,25-(OH)2D3-sensitive nuclear phosphoproteins, including lamin B and several histones. We have also detected and characterized several less abundant nuclear DNA binding phosphoproteins whose phosphorylation was affected by 1,25-(OH)2D3.
Martell
R E
RE
Department of Pharmacology, University of Michigan Medical Center, Ann Arbor 48109-0626.
Strahler
J R
JR
Simpson
R U
RU
D001345:Autoradiography; D002117:Calcitriol / Q000502:physiology*; D002848:Chromatography, DEAE-Cellulose; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D006657:Histones / Q000378:metabolism*; D006868:Hydrolysis; D034921:Lamin Type B; D034882:Lamins; D009687:Nuclear Proteins / Q000378:metabolism*; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D014407:Tumor Cells, Cultured
D006657:Histones; D034921:Lamin Type B; D034882:Lamins; D009687:Nuclear Proteins; D010750:Phosphoproteins; D002117:Calcitriol