Death effector domain protein PEA-15 potentiates Ras activation of extracellular signal receptor-activated kinase by an adhesion-independent mechanism.

PEA-15 is a small, death effector-domain (DED)-containing protein that was recently demonstrated to inhibit tumor necrosis factor-alpha-induced apoptosis and to reverse the inhibition of integrin activation due to H-Ras. This led us to investigate the involvement of PEA-15 in Ras signaling. Surprisingly, PEA-15 activates the extracellular signal receptor-activated kinase (ERK) mitogen-activated protein kinase pathway in a Ras-dependent manner. PEA-15 expression in Chinese hamster ovary cells resulted in an increased mitogen-activated protein kinase kinase and ERK activity. Furthermore, PEA-15 expression leads to an increase in Ras guanosine 5'-triphosphate loading. PEA-15 bypasses the anchorage dependence of ERK activation. Finally, the effects of PEA-15 on integrin signaling are separate from those on ERK activation. Heretofore, all known DEDs functioned in the regulation of apoptosis. In contrast, the DED of PEA-15 is essential for its capacity to activate ERK. The ability of PEA-15 to simultaneously inhibit apoptosis and potentiate Ras-to-Erk signaling may be of importance for oncogenic processes.

The c-Abl tyrosine kinase contributes to the transient activation of MAP kinase in cells plated on fibronectin.

Previous work showed that integrin stimulation triggers activation of the c-Abl tyrosine kinase and its transient localization to focal adhesions. We now report that plating cells on fibronectin triggers association of Grb2 with c-Abl, suggesting possible involvement of c-Abl with integrin activation of the MAP kinase pathway. Expression of a kinase-defective c-Abl specifically inhibited the transient induction of Erk2 activity following cell adhesion. Together with the known ability of activated, oncogenic forms of c-Abl to activate Ras and the MAP kinase pathway, these data suggest that c-Abl contributes to the integrin induction of MAP kinase activity.

Focal adhesion kinase mediates the integrin signaling requirement for growth factor activation of MAP kinase.

The mitogen-activated protein (MAP) kinase pathway is a critical regulator of cell growth, migration, and differentiation. Growth factor activation of MAP kinase in NIH 3T3 cells is strongly dependent upon integrin-mediated adhesion, an effect that contributes to the anchorage dependence of normal cell growth. We now show that expression of constructs that constitutively activate focal adhesion kinase (FAK) rescued the defect in serum activation of MAP kinase in suspended cells without directly activating MAP kinase. Dominant negative FAK blocked both the rescue of suspended cells by the activated construct and the serum activation of MAP kinase in adherent cells. MAP kinase in FAK(-/)- mouse embryo fibroblasts was adhesion-insensitive, and reexpression of FAK restored its adhesion dependence. MAP kinase activity in ras-transformed cells is still decreased in suspension, but expression of constructs that constitutively activate FAK enhanced their anchorage-independent growth without increasing adherent growth. V-src, which activates both Ras and FAK, induced MAP kinase activation that was insensitive to loss of adhesion, and that was blocked by a dominant negative FAK. These results demonstrate that FAK mediates the integrin requirement for serum activation of MAP kinase in normal cells, and that bypassing this mechanism contributes to anchorage-independent growth in transformed cells.

Growth factor activation of MAP kinase requires cell adhesion.

The MAP kinase pathway is a major regulator of both normal and oncogenic growth. We report that activation of the MAP kinase ERK2 by serum or purified growth factors is strongly dependent on cell adhesion to extracellular matrix proteins. This effect is specific to soluble growth factors, since suspended cells still activate ERK2 in response to plating on fibronectin, and is reversible. Analysis of endogenous Ras and Raf show that these proteins are still activated by serum in suspended cells, whereas MEK activity is inhibited. Conversely, activation of ERK2 by activated mutants of Ras and Raf is still adhesion-dependent but activation by MEK is not. Consistent with these results, activated MEK enhances growth of ras-transformed cells in suspension but not when adherent. These results identify a novel synergism between cell adhesion- and growth factor-regulated pathways, and explain how oncogenic activation of MAP kinases induces both serum- and anchorage-independent growth.

Suppression of integrin activation: a novel function of a Ras/Raf-initiated MAP kinase pathway.

Rapid modulation of ligand binding affinity ("activation") is a central property of the integrin cell adhesion receptors. Using a screen for suppressors of integrin activation, we identified the small GTP-binding protein, H-Ras, and its effector kinase, Raf-1, as negative regulators of integrin activation. H-Ras inhibited the activation of integrins with three distinct alpha and beta subunit cytoplasmic domains. Suppression was not associated with integrin phosphorylation and was independent of both mRNA transcription and protein synthesis. Furthermore, suppression correlated with activation of the ERK MAP kinase pathway. Thus, regulation of integrin affinity state is a novel, transcription-independent function of a Ras-linked MAP kinase pathway that may mediate a negative feedback loop in integrin function.

Involvement of the small GTPase rho in integrin-mediated activation of mitogen-activated protein kinase.

Engagement and clustering of integrins triggers a number of intracellular signaling events, including activation of the mitogen-activated protein (MAP) kinases Erk1 and Erk2. To investigate the mechanism by which integrins mediate the activation of MAP kinases upon binding of NIH 3T3 cells to fibronectin, we assessed the effects of both inhibiting and activating the small GTPase Rho. We observed that inhibition of Rho by the Rho-specific inhibitor C3 exoenzyme or by a dominant negative Rho A (RhoN19) inhibited MAP kinase activation. Conversely, activation of Rho by expression of an activated Rho A mutant (RhoQ63L), or the Rho-specific guanine nucleotide exchange factor lbc, enhanced and partially mimicked activation of Erk2 by plating on fibronectin. These results therefore show that Rho is involved in the integrin-dependent activation of MAP kinase.

Rac is required for v-Abl tyrosine kinase to activate mitogenesis.

BACKGROUND: The v-abl oncogene of the Abelson murine leukemia virus (A-MuLV) encodes a cytoplasmic tyrosine kinase that can associate with phosphoinositide 3-kinase, Shc and Grb2, and activate the pathway that leads from Ras to mitogen-activated protein (MAP) kinase. Despite the link to these mitogenic signal transducers, v-Abl does not stimulate cell growth in general. Only a subset of NIH3T3 cells, represented by the P-3T3 subclone, can respond to the mitogenic effects of v-Abl, whereas the majority of NIH3T3 cells respond to the growth-inhibitory effects of v-Abl. This restricted mitogenic function suggests that v-Abl may rely on additional signal transducers--which are not required for the physiological mitogenic response--to stimulate DNA synthesis. RESULTS: A dominant-negative mutant of the small GTP-binding protein Rac (Rac N17, containing an asparagine residue at position 17) was found to block v-Abl-induced activation of two mitogenic enhancer elements. Activation of the serum response element by v-Abl was inhibited by Rac N17 and Ras N17, whereas activation of the TPA response element was inhibited by Rac N17 but not by Ras N17. The Rac N17 mutant also compromized the ability of v-Abl to activate the MAP-kinase, Erk2, and the stress-activated protein kinase, JNK1. Activation of endogenous Rac was indicated by the ability of v-Abl to stimulate pinocytosis. P-3T3 cells transformed by v-abl could proliferate without serum, but became serum-dependent when Rac N17 was expressed. However, Rac N17 did not inhibit the morphological change or anchorage-independent growth of cells transformed with v-Abl. CONCLUSIONS: The activation of the mitogenic program by v-Abl tyrosine kinase requires not only Ras but also Rac. Multiple pathways are activated by v-Abl in transformed cells. The availability of some of these pathways may determine whether v-Abl can activate mitogenesis.

The human leukemia oncogene bcr-abl abrogates the anchorage requirement but not the growth factor requirement for proliferation.

Proliferation of normal cells in a multicellular organism requires not only growth factors but also the proper attachment to the extracellular matrix. A hallmark of neoplastic transformation is the loss of anchorage dependence which usually accompanies the loss of growth factor requirement. The Bcr-Abl tyrosine kinase of human leukemias is shown here to abrogate only the anchorage, not the growth factor, requirement. Bcr-Abl-transformed cells grow in soft agar but do not proliferate in serum-free media. Bcr-Abl does not activate the mitogenic pathway, as indicated by its inability to induce enhancers such as the serum response element or the tetradecanoyl phorbol acetate response element (TRE). However, Bcr-Abl can alleviate the anchorage requirement for the induction of the TRE enhancer; i.e., it allows serum to activate the TRE in detached cells. This activity is dependent on the association of an active Bcr-Abl tyrosine kinase with the actin filaments. Despite its association with the adapter protein Grb2, Bcr-Abl's effect on the TRE enhancer is not blocked by dominant negative Ras or Raf. The finding that Bcr-Abl tyrosine kinase abrogates only anchorage dependence may have important implications on the pathogenesis of chronic myelogenous leukemia.

Oncogenic v-Abl tyrosine kinase can inhibit or stimulate growth, depending on the cell context.

The v-abl oncogene of Abelson murine leukemia virus (A-MuLV) induces two opposite phenotypes in NIH3T3 cells. In the majority of cells, v-abl causes a growth arrest at the G1 phase of the cell cycle; while in a minority of cells, v-abl abrogates the requirement for growth factors. Using temperature sensitive mutants, it can be demonstrated that v-Abl tyrosine kinase is required for growth inhibition or stimulation. The two phenotypes are not caused by mutations or differences in the expression of v-Abl, but are dependent on the cell context. Two stable subclones of NIH3T3 cells have been isolated that exhibit similar morphology and growth characteristics. However, upon infection with A-MuLV, the 'positive' cells become serum- and anchorage-independent, whereas the 'negative' cells become arrested in G1. The positive phenotype is dominant, shown by cell fusion, and treatment with 5-azacytidine converts the negative cells to the positive phenotype. Activation of v-Abl tyrosine kinase induces the serum-responsive genes in the positive but not in the negative cells. Transactivation of the c-fos promoter by v-Abl in transient assays is also restricted to the positive cells. These results show that v-Abl tyrosine kinase is not an obligatory activator of growth, but requires a permissive cellular context to manifest its mitogenic function.

Differential expression of type-specific c-abl mRNAs in mouse tissues and cell lines.

The mammalian c-abl proto-oncogene produces mRNAs with 5' heterogeneity from two distinct promoters and the alternative splicing of variable 5' exons. By using quantitative RNase protection assays, the relative abundance of two major c-abl mRNAs, type I and type IV, in several mouse tissues and cell lines has been determined. Our results demonstrate that the level of type IV c-abl mRNA is rather constant, whereas that of the type I mRNA varies over a 10-fold range in different tissues and cell types. This finding has interesting implications for the function of the two c-abl proteins.