Phorbol 12-myristate 13-acetate inhibits granulocyte-macrophage colony stimulating factor-induced protein tyrosine phosphorylation in a human factor-dependent hematopoietic cell line.

Y Kanakura,B Druker,J Dicarlo,S A Cannistra,J D Griffin

doi:10.1016/s0021-9258(18)52462-5

Abstract

The human myeloid cell line MO7 requires either granulocyte-macrophage colony stimulating factor (GM-CSF) or interleukin 3 (IL-3) for proliferation. We have previously shown that both GM-CSF and IL-3 transiently induce tyrosine phosphorylation of a number of proteins, including two cytosolic proteins, p93 and p70, which are maximally phosphorylated 5-15 min after addition of growth factor to factor-deprived cells. GM-CSF-induced proliferation of MO7 cells was found to be inhibited by two activators of protein kinase C, phorbol 12-myristate 13-acetate (PMA) and bryostatin-1. PMA did not affect surface expression or affinity of the GM-CSF receptor but significantly inhibited GM-CSF- or IL-3-induced tyrosine phosphorylation of p93 and p70. In contrast, PMA augmented GM-CSF-induced tyrosine phosphorylation of another protein, p42. Pretreatment of cells with sodium orthovanadate to inhibit protein tyrosine phosphatases (PTPase) partially reversed the inhibitory effects of PMA. These results suggest that one aspect of GM-CSF and IL-3 signal transduction, protein tyrosine phosphorylation, can be inhibited by a mechanism which does not involve receptor down-regulation, and may involve either receptor down-regulation, and may involve either inhibition of a receptor-activated tyrosine kinase, activation of a protein tyrosine phosphatase, or both. This mechanism could be important in exerting control of proliferation of some types of hematopoietic cells.

Highlights

Thehuman myeloid cell line M07 requires either sine phosphorylation of a number of proteins, including two granulocyte-macrophage colony stimulating factor (GM-CSF)or interleukin 3 (IL-3)for proliferation
These results suggest that one aspect of GM-CSF liferation of these myeloidcells, we have investigated the and IL-3 signal transduction, protein tyrosine phos- effects of phorbol 12-myristate13-acetate (PMA) on GM-CSF and IL-3-induced protein tyrophorylation, can be inhibited by a mechanism which sine phosphorylation in M07 cells
In some experi- by GM-CSF or IL-3-"07 cells were cultured for 72 h with ments, PMA or sodium orthovanadate (10 p ~ w)ere added for the control medium orGM-CSF (10 ng/ml), with or without PMA

Summary

RESULTS

M 0 7 cells were washed free of serum and growth factors and incubated in serum-free RPMI-1640 containing0.5% bovine serum albu- P M A Is Inhibitory to the Growth of M 0 7 Cells Stimulated min for 6-18 h a t 37 "C to factor deprive the cells. With alkalinephosphatase-conjugated tec) diluted 1:2,000 in TBST,washed the blots were anti-mouse IgG three times in incubated 2 h After PMA treatment, cells were suspended in 300 p1 of binding CSF-induced phosphorylation of p93 was normal, and no buffer consisting of RPMI-1640 with bovine serum albumin Bindinwgas performed for 4h a t 4 "Cto prevent the possibility of delayed changes in GM-CSF receptor expression induced by initial exposure to PMA Under these conditions, binding was essentially 80% complete and nonspecific binding was typically in the range of 5-20%. Were obtained with IL-3 (10 ng/ml) (data not shown)

Vanadate Control

Treatment of normal blood monocytes or immature marrow

The results presented here indicate that inhibits