A lineage-specific protein kinase crucial for myeloid maturation

Abstract

To identify genes involved in macrophage development, we used the differential display technique and compared the gene expression profiles for human myeloid HL-60 leukemia cell lines susceptible and resistant to macrophage maturation. We identified a gene coding for a protein kinase, protein kinase X (PRKX), which was expressed in the maturation-susceptible, but not in the resistant, cell line. The expression of the PRKX gene was found to be induced during monocyte, macrophage, and granulocyte maturation of HL-60 cells. We also studied the expression of the PRKX gene in 12 different human tissues and transformed cell lines and found that, among these tissues and cell types, the PRKX gene is expressed only in blood. Among the blood cell lineages, the PRKX gene is specifically expressed in macrophages and granulocytes. Antisense inhibition of PRKX expression blocked terminal development in both the leukemic HL-60 cells and normal peripheral blood monocytes, implying that PRKX is a key mediator of macrophage and granulocyte maturation. Using the HL-60 cell variant deficient in protein kinase C-␤ (PKC-␤) and several stable PKC-␤ transfectants, we found that PRKX gene expression is under control of PKC-␤; hence PRKX is likely to act downstream of this PKC isozyme in the same signal transduction pathway leading to macrophage maturation.

Smad6 is a protein kinase X phosphorylation substrate and is required for HL-60 cell differentiation

“…To determine in which cellular compartment PrKX and Smad6 reside during macrophage differentiation, we separated the nuclear and cytoplasmic fractions of HL-60 cells that were either untreated or treated for 6 or 24 h with PMA. Western blotting of the resultant lysates (Figure 4) demonstrated that PrKX was induced by PMA as has been reported previously (Semizarov et al, 1998). In contrast, Smad6 was constitutively expressed and its levels did not change as a result of PMA treatment.…”

“…PrKX activity during HL-60 differentiation D Glesne and E Huberman deficient in PMA-resistant HL-60 cells (Semizarov et al, 1998). We determined that PrKX gene expression was induced during differentiation of HL-60 cells into macrophages by PMA, into monocytes by 1, 25-dihydroxy vitamin D3 and into granulocytes by ATRA as well as in peripheral blood granulocytes but not in lymphocytes (Semizarov et al, 1998).…”

“…We have previously demonstrated that PrKX is essential for macrophage differentiation of HL-60 cells by using an antisense oligonucleotide-based knockout approach (Semizarov et al, 1998). To substantiate this conclusion by an independent method, and to query the requirement for Smad6 expression for this differentiation, we undertook an RNA interference (RNAi) approach.…”

“…Using this system, a number of differentiation mediators have been identified. For example, as a result of PMA binding and activation of PKC-b (Tonetti et al, 1994), a signaling process is initiated, which involves the induction of protein kinase X (PrKX) gene expression (Semizarov et al, 1998), generation of soluble tumor necrosis factor (TNF)-a and its interaction with TNF receptor II, secretion and activation of collagenase matrix metalloproteinase-9 (Xie et al, 1998), production and cell-surface display of a5b1 integrin, and generation, release and deposition onto the culture dish of fibronectin that serves as a cell attachment matrix (Laouar et al, 1999). While inhibitory experiments employing either antisense oligonucleotides or neutralizing antibodies have allowed us to establish a temporal order for many of these events, direct interactors for some of these components have not been determined.…”

“…Protein kinase X, which is a key component of PKC-b signaling that leads to macrophage differentiation (Semizarov et al, 1998), was first identified as a putative protein kinase by its homology to cyclic AMP (cAMP)-dependent protein kinases and the Drosophila melanogaster DC2 protein kinase (Melendez et al, 1995). Its gene was localized to a region of the human Xchromosome frequently involved in XY-chromosome rearrangement aberrations (Klink et al, 1995).…”