Retinoylation of vimentin in the human myeloid leukemia cell line HL60.

N Takahashi,T.R Breitman

doi:10.1016/s0021-9258(17)37548-8

Abstract

Retinoylation (retinoic acid acylation) is a posttranslational modification of proteins occurring in many eukaryotic cell lines. The widespread occurrence of retinoylation suggests that it may play a role in many effects of retinoic acid (RA) on cells. The regulatory subunits of cyclic AMP-dependent protein kinase are retinoylated in the human myeloid leukemia cell line HL60 (Takahashi, N., Liapi, C., Anderson, W. B., and Breitman, T. R. (1991) Arch. Biochem. Biophys. 290, 293-302), and cytokeratins are retinoylated in normal human keratinocytes (Takahashi, N., Jetten, A. M., and Breitman, T. R. (1991) Biochem. Biophys. Res. Commun. 180, 393-400). We show, in this study, that the intermediate filament protein vimentin is retinoylated in HL60 cells during a 24-h exposure to 100 nM [3H]RA. We found that a retinoylated protein of M(r) 55,000 coeluted on anion exchange chromatography and comigrated on either one- or two-dimensional polyacrylamide gel electrophoresis with a protein that also was stained on immunoblots by an anti-vimentin antibody. About 50% of the [3H]RA was released from this M(r) 55,000 retinoylated protein after hydrolysis with either NH2OH (1 M, pH 10) or CH3OH, 0.1 M KOH. These results indicated that a large fraction of the RA was bound to vimentin by an ester bond. Both the M(r) 55,000 retinoylated protein and immunoreactive vimentin were associated with cell nuclei isolated by two procedures. They were detached during exposure to a nonionic detergent buffer, suggesting that they are bound to the nuclear envelope. These results indicate that retinoylation is a new modification of vimentin that may be an early event in RA-induced differentiation of HL60 cells.

Highlights

They were detached during exposure to a nonionic de- The widespread occurrence of retinoylation suggests that it tergent buffer, suggesting that they are bound to the may play a role in manyeffects of RAon cells
We found that the regulatory early event in RA-induced differentiation-0of cells. subunits of cyclicAMP-dependent protein kinase typeIs and I1 are retinoylated in both HL60 (28) and MCF-7 cells (26) and that cytokeratins are retinoylated in normal human keratino
How retinoylation affects the function of these proeloid leukemia cell line HL60 to cells with many functional and teins is under investigation in ourlaboratory

Summary

MATERIALS AND METHODS

Cific high affinity binding sites for RA and some of its metabolites. It was generally accepted that the actions. Cells-In this study, we used the human myeloid leukemia cell lines HL60 (29), HLGOMRI (30),a mutant that is more sensitiveto RA than HL60 cells, and HLGOEU-res (31),a mutant that is resistant to differentiation by RA. Payment of page charges.This article must be hereby marked (v/v) fetal bovine serum (Life Technologies, Inc.). Cell cultures were “advertisement”in accordance with 18 U.S.C.Section 1734 solelyto incubated at 37 “C in a humidified atmosphere of 5% CO, in air and indicate this fact. Incorporationof RA intoHL60 Proteins-Exponentially growing cells amide gel electrophoresis; RAR, retinoic acid nuclear receptor.

Retinoylation of Vimentin

BASE ACID

RESULTS

DISCUSSION