Functional Role for Protein Kinase Cβ as a Regulator of Stress-Activated Protein Kinase Activation and Monocytic Differentiation of Myeloid Leukemia Cells

The human U-937 and HL-60 myeloid leukemia cell lines proliferate autonomously in the absence of exogenous hematopoietic growth factors (6, 52). These cells, however, have retained the capacity to respond to inducers of differentiation with growth arrest and the appearance of a mature phenotype. In this context, treatment of U-937 and HL-60 cells with agents that activate protein kinase C (PKC), including 12-O-tetradecanoylphorbol-13-acetate (TPA) and phorbol-12,13-dibutyrate (PDBu), induces differentiation along the monocytic lineage. Bryostatin 1, a macrocyclic lactone, also activates PKC and induces monocytic differentiation of myeloid leukemia cells (51). While these findings have indicated that factor-independent growth of myeloid leukemia cells is reversible by activation of PKC-mediated signaling, little is known about the downstream effectors responsible for induction of the differentiated monocytic phenotype. PKC is a family of at least 12 serine/threonine protein kinase isoforms which are involved in diverse cellular responses (24, 43). The α, β, γ, δ, ɛ, μ, η, θ, and ζ forms of PKC are responsive to phorbol esters. The available evidence suggests that PKCβ is involved in TPA-induced differentiation of myeloid leukemia cells. Accordingly, TPA-resistant HL-60 cell variants are deficient in PKCβ expression (37, 42, 56, 57). Down-regulation of PKCβ expression (19) and functional defects in PKCβ (31) have also been found for TPA-resistant U-937 cell variants. In addition, defective translocation of PKCβ from the cytosol to the cell membrane has been shown for TPA-resistant variants of both U-937 and HL-60 cells (19, 64). Importantly, increased expression of PKCβ resulting from treatment with retinoic acid (64) or from transfection of the PKCβ gene (56) restores TPA inducibility of growth arrest and a differentiated monocytic phenotype. PKCβ is expressed as two isoforms, βI and βII, as a result of an alternative splicing mechanism that produces a PKCβI protein which is truncated by 50 amino acids at the carboxy terminus (32); the longer PKCβII isoform is expressed in U-937 and HL-60 cells (22, 56). Treatment of myeloid leukemia cells with TPA is associated with changes in the expression of certain early- and late-response genes. TPA down-regulates c-myc transcripts in HL-60 cells (47) and induces expression of the c-jun gene (49, 54, 61). Similar findings have been obtained with other inducers of monocytic differentiation (49), including okadaic acid, an inhibitor of phosphoserine/threonine protein phosphatases 1 and 2A (1, 25). Activation of Jun/AP-1 contributes to induction of c-jun transcription (2) and monocytic differentiation (54). The early growth response 1 (EGR-1) gene is also activated during TPA- and okadaic acid-induced monocytic differentiation (27, 29) and is necessary for the appearance of the monocytic phenotype (41). Thus, the induction of early response genes and thereby upstream signals involved in their transcriptional activation may be directly linked to the reversal of the leukemia phenotype. Members of the mitogen-activated protein kinase (MAPK) superfamily are involved in diverse cellular processes, including the induction of differentiation. Among the three related MAPK families identified to date, the extracellular signal-regulated protein kinases (ERK) have been identified as playing a role in differentiation. Activation of the MAPK kinase (MEK1) is necessary and sufficient for neuronal differentiation of PC12 rat pheochromocytoma cells (7) and for megakaryocyte differentiation of human K562 erythroleukemia cells (59). In contrast, overexpression of constitutively active MEK1 in U-937 cells results in growth inhibition but no phenotypic differentiation (15). In addition, activation of ERK by TPA in the TPA-resistant UT16 variant of U-937 cells suggests that ERK activation is not sufficient for induction of human myeloid leukemia cell differentiation (48). The stress-activated protein kinases (SAPK; also known as Jun kinases or JNK) are serine/threonine protein kinases related to the MAPK family. SAPK is activated by tumor necrosis factor, diverse DNA-damaging agents, UV light, and anisomycin (12, 28, 33). SAPK phosphorylates Ser-63 and Ser-73 of the c-Jun amino terminus and thereby activates c-Jun transcription function (12, 33). The ATF2 and Elk1 transcription factors are also phosphorylated by SAPK (18, 45, 60). Whereas TPA-induced monocytic differentiation is associated with induction of c-jun (49, 54, 61) and EGR-1 (27, 29) gene expression, SAPK-mediated activation of c-Jun, ATF2, and Elk1 and thereby early response genes is associated with the appearance of the differentiated phenotype. MEK kinase 1 (MEKK-1) (34) preferentially activates SAPK/ERK kinase 1 (SEK1) (13, 36, 38) and, consequently, SAPK (46). Of interest, Bck 1p, a MEK1 kinase homolog in yeast, functions downstream of the PKC homolog PKC 1p (35). The finding that murine MEKK-1 can also function as a downstream effector of PKC 1p and can replace Bck 1p has provided support for potential interactions between PKC and MEKK-1 (3). However, the link between events activated by TPA and the MEKK-1→SEK1→SAPK pathway is unclear. The present studies demonstrated that PKCβII is an upstream effector of TPA-induced SAPK activation. Similar findings have been obtained with other activators of PKC that induce monocytic differentiation of myeloid leukemia cells. We also showed that TPA induces the binding of PKCβII to MEKK-1 and that MEKK-1 is necessary for TPA-induced activation of SAPK.