Identification of lamin B and histones as 1,25-dihydroxyvitamin D3-regulated nuclear phosphoproteins in HL-60 cells

• Published in: The Journal of biological chemistry Vol. 267; no. 11; pp. 7511 - 7519
• Main Authors: MARTELL, R. E, STRAHLER, J. R, SIMPSON, R. U
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 15-04-1992
• Subjects: Autoradiography; Biological and medical sciences; Calcitriol - physiology; Cell physiology; Chromatography, DEAE-Cellulose; Electrophoresis, Gel, Two-Dimensional; Electrophoresis, Polyacrylamide Gel; Fundamental and applied biological sciences. Psychology; Histones - metabolism; Hydrolysis; Lamin Type B; Lamins; Molecular and cellular biology; Nuclear Proteins - metabolism; Peptide Mapping; Phosphoproteins - metabolism; Phosphorylation; Responses to growth factors, tumor promotors, other factors; Tumor Cells, Cultured; Human; Phosphorylation; Cholecalciferol(1,25-dihydroxy); Phosphoproteins; DNA binding protein; Two dimensional electrophoresis; Histone; Nuclear protein
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)42547-1

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.