The 41-kDa and 43-kDa mitogen-activated protein (MAP) kinases play a pivotal role in the mitogenic signal transduction pathway and are essential components of the MAP kinase cascade, which includes MAP kinase kinase (MEK) and Raf-1. As aberrant activation of signal transducing molecules such as Ras and Raf-1 has been linked with cancer, we examined whether constitutive activation of the 41-/43-kDa MAP kinases is associated with the neoplastic phenotype of 138 tumor cell lines and 102 primary tumors derived from various human organs. Constitutive activation of the MAP kinases was observed in 50 tumor cell lines (36.2%) in a rather tissue-speci®c manner: cell lines derived from pancreas, colon, lung, ovary and kidney showed especially high frequencies with a high degree of MAP kinase activation, while those derived from brain, esophagus, stomach, liver and of hematopoietic origin showed low frequencies with a limited degree of MAP kinase activation. We also detected constitutive activation of the 41-/43-kDa MAP kinases in a relatively large number of primary human tumors derived from kidney, colon and lung tissues but not from liver tissue. Many tumor cells, in which point mutations of ras genes were detected, showed constitutive activation of MAP kinases, however, there were also many exceptions to this observation. In contrast, the activation of the 41-/43-kDa MAP kinases was accompanied by the activation of Raf-1 in the majority of tumor cells and was completely associated with the activation of MEK and p90 rsk in all the tumor cells examined. These results suggest that the constitutive activation of 41-/43-kDa MAP kinases in tumor cells is not due to the disorder of MAP kinases themselves, but is due to the disorder of Raf-1, Ras, or some other signaling molecules upstream of Ras.

Bone morphogenetic proteins (BMPs), 1 members of the transforming growth factor-␤ superfamily, were originally identified by their novel activity to induce cartilage and bone formation in ectopic extraskeletal sites in vivo (1, 2). A number of studies have demonstrated that BMPs play essential roles in bone formation and bone cell differentiation; e.g. BMPs stimulate proteoglycan synthesis, alkaline phosphatase activity, collagen synthesis, and osteocalcin expression in chondroblasts/ osteoblasts (3-5). However, expression of BMPs (6 -8) and their receptors (9) in many tissues other than bone suggests that they are also involved in the regulation of many biological processes unrelated to bone formation. In accordance with this possibility, increasing evidence suggests a regulatory role for BMPs in early vertebrate embryogenesis such as in mesoderm induction, limb development, and hematopoietic formation (10 -14). Moreover, BMPs have drawn attention as possible regulators of central nervous system development; BMPs are expressed in multiple central nervous system regions throughout development (15)(16)(17). In this respect, we have recently demonstrated that BMP-2 acts as a neurotrophic factor; BMP-2 induces the neuronal differentiation of rat pheochromocytoma PC12 cells (18) and also promotes the survival and differentiation of striatal GABAergic neurons in culture (19).BMPs transduce their signals by binding to two different types of serine/threonine kinase receptor, type I and type II (20 -22). Upon ligand binding, type I and type II receptors form heteromeric receptor complexes. Then type I receptors are phosphorylated by type II receptors, and subsequent activation of the catalytic activity of type I receptor kinase is essential for BMP signaling. Although phosphorylation of Smad proteins by the activated type I receptor kinase was suggested to play an important role in the mediation of BMP signaling (20 -22), the precise cytoplasmic signaling pathway for BMPs through which it elicits its effects on the regulation of wide variety of biological processes remains largely unknown.We have recently shown that BMP-2 has the capacity to induce the neuronal differentiation of PC12 cells. Unlike NGF, however, BMP-2 did not induce the activation of MEK or 41-/ 43-kDa MAP kinases (ERK2 and ERK1, respectively) in these cells (18). MEK and ERK1/2 constitute a protein kinase cascade (the ERK cascade), which is one of the major cytoplasmic signaling pathways commonly activated in a wide variety of cells by diverse extracellular stimuli (23)(24)(25)(26). Activation of the cascade elicits a wide array of physiological responses such as cell division, differentiation, and secretion (23-26), and it has been reported that the activation of the cascade is necessary and sufficient for PC12 cell neuronal differentiation (27,28). However, our previous findings, which showed a clear distinction between the requirement for the activation of MEK and ERK1/2 and the ability of BMP-2 to induce PC12 cell neuronal differentiation (18), hav...

The 41-/43-kDa mitogen-activated protein (MAP) 1 kinase pathway, also called the extracellular signal-regulated kinase (ERK) pathway, is activated in a variety of cell types by diverse extracellular stimuli and is among the most thoroughly studied of signaling pathways that connect different membrane receptors to the nucleus (1, 2). Activation of the ERK pathway involves the activation of Ras at the plasma membrane, and the sequential activation of a series of protein kinases. Initially, Ras interacts with and activates Raf-1, which in turn activates MAP kinase/ERK kinase (MEK)-1 and -2 by serine phosphorylation. MEK-1/2 then catalyze the phosphorylation of 41-and 43-kDa MAP kinases (ERK2 and ERK1, respectively) on tyrosine and threonine residues, and these activated MAP kinases can phosphorylate cytoplasmic and nuclear targets. The ERK pathway participates in a wide range of cellular programs including proliferation, differentiation, and movement (1, 2).Aberrant activation of signal transducing proteins has been linked with cancer. For example, constitutively active mutants of Ras (3) and Raf-1 (4) have been observed in several human tumors, and constitutively active mutants of MEK-1 have been shown to transform mammalian cells (5, 6). We recently examined whether constitutive activation of the ERK pathway is associated with the neoplastic phenotype of human tumor cells. Constitutive activation of ERKs and MEK was observed in a relatively large number of tumors; tumor cells derived from pancreas, colon, lung, ovary, and kidney tissues showed especially high frequencies (30 -50%) and a high degree of kinase activation (7,8). Activation of the ERKs is also associated with prostate cancer progression (9). The precise cause of constitutive activation of the ERK pathway in many of these tumor cells remains unclear. However, such high frequencies of ERK/ MEK activation in human tumors indicate that specific inhibitors might be developed against these protein kinases for cancer therapy, especially for treatment of tumors showing constitutive activation of the ERK pathway.In the present study, we have examined the effect of blockade of the ERK pathway on the proliferation of human tumor cells. We utilized small molecule inhibitors of this pathway, PD98059 (10) and U0126 (11), which specifically inhibit MEK activity. Our results demonstrate that these MEK inhibitors induce a remarkable G 1 cell cycle arrest, followed by a modest apoptotic response, in tumor cells in which the ERK pathway is constitutively activated. Up-regulation of the CDK inhibitor p27Kip1 was observed in these G 1 -arrested tumor cells.

Hepatocyte growth factor (HGF) markedly induced the spreading, dissociation and scattering of Madin ± Darby canine kidney epithelial cells (MDCK) and human stomach adenocarcinoma cells (TMK1). Scattering of MDCK and TMK1 cells was induced by 12-Otetradecanoyl-phorbol-13-acetate (PMA) and epidermal growth factor (EGF), respectively. In all these agentstimulated cells, rapid activation of Raf-1, MAP kinase/ ERK kinase (MEK), 41/43 kDa MAP kinases and p90 rsk was commonly observed. In contrast, PMA neither induced the scattering nor activation of all these kinases in TMK1 cells. Pretreatment of MDCK and TMK1 cells with 2-(2-amino-3-methoxyphenyl) choromone (AMPC), a speci®c inhibitor of MEK, selectively inhibited the HGF-, PMA-and EGF-stimulated activities of MEK, 41/43 kDa MAP kinases and p90 rsk in a dose dependent manner. AMPC-pretreatment, however, did not aect HGF-, PMA-or EGF-induced activation of Raf-1, nor HGF-induced activation of phosphatidylinositol 3-kinase in these cells. Importantly, HGF-, PMA-and EGFinduced scattering of MDCK and TMK1 cells was inhibited at doses of AMPC similar to those that gave comparable levels of inhibition of the activities of MEK, 41/43 kDa MAP kinases and p90 rsk . These results suggest that activation of the 41/43 kDa MAP kinase signaling pathway is required for the motility response of MDCK and TMK1 cells induced by agents such as HGF, PMA and EGF.

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