Transforming growth factor-beta (TGF-beta) is a potent growth inhibitor of a variety of epithelial cell types. The primary signaling mechanism involved in mediating this and other cellular effects of TGF-beta is still unknown. We report here that both TGF-beta 1 and TGF-beta 2 resulted in a rapid activation of mitogen-activated protein kinase (MAPK) p44mapk, occurring within 5-10 min of growth factor addition. This effect occurred in exponentially proliferating cultures of intestinal epithelial (IEC) 4-1 cells under conditions in which DNA synthesis was inhibited by 95% to 98%. Furthermore, TGF-beta 2 induced a sustained activation of p44mapk under these conditions, lasting for at least 90 min after initial growth factor treatment. Another TGF-beta-sensitive epithelial cell line (CCL 64) displayed a similar rapid increase in p44mapk activity when treated with TGF-beta 1. In contrast, in IEC 4-6 cells that are resistant to TGF-beta effects on growth and DNA synthesis, TGF-beta 2 treatment did not result in an activation of p44mapk. In contrast to the results in proliferating cultures, treatment of quiescent cultures of IEC 4-1 cells with TGF-beta 2 resulted in no significant change in either DNA synthesis or p44mapk activity within 15 min of TGF-beta addition. In contrast, addition of the growth-stimulatory combination of factors (epidermal growth factor + insulin + transferrin = EIT) to quiescent and proliferating IEC 4-1 cells stimulated DNA synthesis and resulted in a sustained activation of p44mapk. Together, our results suggest an association between activation of p44mapk and both TGF-beta-mediated growth inhibition and EIT-mediated growth stimulation. This suggests that the specificity for the cellular effects of growth factors may not occur at the level of MAPK activation per se, but rather at downstream events that include phosphorylation of distinct transcriptional complexes and activation of a select assortment of genes. With regard to TGF-beta specifically, we have proposed a model to explain how activation of p44mapk may be associated with a growth-inhibitory response.

The metastasis-suppressive activity of Nm23-H1 was previously correlated with its in vitro histidine protein kinase activity, but physiological substrates have not been identified. We hypothesized that proteins that interact with histidine kinases throughout evolution may represent partners for Nm23-H1 and focused on the interaction of Arabidopsis "two-component" histidine kinase ERS with CTR1. A mammalian homolog of CTR1 was previously reported to be c-Raf; we now report that CTR1 also exhibits homology to the kinase suppressor of Ras (KSR), a scaffold protein for the mitogen-activated protein kinase (MAPK) cascade. Nm23-H1 co-immunoprecipitated KSR from lysates of transiently transfected 293T cells and at endogenous protein expression levels in MDA-MB-435 breast carcinoma cells. Autophosphorylated recombinant Nm23-H1 phosphorylated KSR in vitro. Phosphoamino acid analysis identified serine as the major target, and two peaks of Nm23-H1 phosphorylation were identified upon high performance liquid chromatography analysis of KSR tryptic peptides. Using site-directed mutagenesis, we found that Nm23-H1 phosphorylated KSR serine 392, a 14-3-3-binding site, as well as serine 434 when serine 392 was mutated. Phosphorylated MAPK but not total MAPK levels were reduced in an nm23-H1 transfectant of MDA-MB-435 cells. The data identify a complex in vitro histidine-to-serine protein kinase pathway, which may contribute to signal transduction and metastasis.Metastasis suppressor genes are credentialed by their ability to suppress metastatic potential in vivo upon injection of a transfected tumor cell line, without a concomitant reduction in primary tumor size (reviewed in Ref. 1). The nm23 gene family was described by its reduced expression in highly metastatic murine melanoma cell lines, as compared with related, tumorigenic but less metastatic cell lines (2) and consists of eight family members (reviewed in Ref.3). In a recent review, 18 of 24 studies found a significant relationship between decreased Nm23 expression in primary human breast carcinomas and an aspect of aggressive clinical course (patient survival, lymph node metastasis, etc.), although Nm23 expression does not represent an independent prognostic or predictive factor (4). Ten transfection experiments have shown that nm23-transfected, metastatically competent cell lines are 40 -98% less metastatic in vivo than control transfectants (5-14). For nm23-H1, the most studied member, overexpression in human MDA-MB-435 breast carcinoma cells reduced colonization in soft agar, both unstimulated and transforming growth factor-␤-stimulated (6), and invasion/motility to a variety of chemoattractants (15-17). Nm23-H1 breast carcinoma transfectants exhibited morphological (ascinus formation) and biosynthetic aspects of differentiation in three-dimensional culture (18), and this finding is supported by similar studies in neural cells (19 -25).Despite extensive work, the biochemical mechanism of action whereby Nm23-H1 suppresses the metastatic potential of cancer cells is unk...

We have previously demonstrated that growth inhibition of untransformed intestinal epithelial cells by transforming growth factor ␤ 1 (TGF␤) and TGF␤ 2 was associated with a rapid activation of both Ras and extracellular signal-regulated kinase 1 (Erk1) In order to determine whether the activation of Ras by TGF␤ was required for the growth inhibitory effect of TGF␤, we examined TGF␤ 2 effects on Cdk2-associated histone H1 kinase activity, cyclin A protein expression levels, and DNA synthesis in two intestinal epithelial cell clones transfected with RasN17. In cells expressing RasN17, we observed a 50% reversal of the inhibition of Cdk2 activity, a 78% reversal of the down-regulation of cyclin A protein expression, and a 21% reversal of the inhibition of DNA synthesis by TGF␤. Collectively, these results indicate that Ras activation is obligatory for TGF␤-mediated activation of Erk1, whereas it is partially required for the growth inhibitory effect of TGF␤.(The transforming growth factor-␤ (TGF␤) 1 family currently consists of three mammalian secreted polypeptides (TGF␤ 1 , TGF␤ 2 , and TGF␤ 3 ) that regulate cellular growth, morphogenesis, differentiation, and adhesion (3). TGF␤ exerts these cellular effects through a heteromeric complex of the type I (RI) and type II (RII) TGF␤ receptors, each containing serine/threonine kinase domains that interact in a phosphorylation-dependent manner (4, 5). However, little is currently known regarding TGF␤ regulation of cytoplasmic components that are rapidly activated after receptor interaction with ligand. Twohybrid screens have indicated that immunophilin FKBP-12 and farnesyltransferase-␣ specifically bind RI and that a novel protein, termed TGF␤-receptor interacting protein-1, interacts with RII (6 -9). The functional significance of these receptor interacting proteins has yet to be elucidated. We have reported direct evidence for the rapid activation of cytoplasmic signaling components by TGF␤ in a mammalian cell system. That is, we have shown that both Ras and Erk1 are rapidly activated by TGF␤ 1 and TGF␤ 2 in TGF␤-sensitive epithelial cells but not in TGF␤-resistant cells (1, 2). These effects occurred in asynchronous cultures of epithelial cells under conditions where DNA synthesis was inhibited by 95-98% (2). The recent identification of the interaction between RI and farnesyltransferase-␣ mentioned above (7, 8) suggests a potential upstream mechanism for the activation of Ras in the TGF␤ signaling pathway. The only other cytoplasmic signaling events that have been shown to be modulated by TGF␤ in untransformed epithelial cells are an activation of protein phosphatase 1 (10), an involvement of protein kinase C in early TGF␤ responses (11,12), and an association of phospholipase C with the elevation of gene expression by TGF␤ (12).In contrast to the effects of TGF␤ on cytoplasmic signaling components, a direct association between TGF␤ modulation of nuclear cell cycle components and the growth inhibitory effects of TGF␤ has been demonstrated. The G 1 cell cycle event...

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