Integrins are cell-surface receptors that mediate and coordinate cellular responses to the extracellular matrix (ECM). Cellular signalling pathways can regulate cell adhesion by altering the affinity and avidity of integrins for ECM. The Ras family of small G proteins, which includes H-ras, R-ras and Rap, are important elements in cellular signalling pathways that control integrin function.

Cell cycle progression is dependent on the nuclear localization and transcriptional effects of activated extracellular signal-regulated kinase (ERK)1 and ERK2 mitogen-activated protein (MAP) kinases (ERK1/2). Phosphoprotein enriched in astrocytes (PEA-15) binds ERK1/2 and inhibits their nuclear localization, thus blocking cell proliferation. Here, we report that phosphorylation of PEA-15 blocks its interaction with ERK1/2 in vitro and in vivo and that phosphorylation of both Ser 104 and Ser 116 is required for this effect. Using phosphomimetic and nonphosphorylatable mutants of PEA-15, we found that PEA-15 phosphorylation abrogates its capacity to block the nuclear localization and transcriptional activities of ERK1/2; this phosphorylation therefore enables the proliferation of cells that express high levels of PEA-15. Additionally, we report that PEA-15 phosphorylation can modulate nontranscriptional activities of ERK1/2, such as the modulation of the affinity of integrin adhesion receptors. Finally, we used a novel anti-phospho-specific PEA-15 antibody to establish that PEA-15 is phosphorylated in situ in normal mammary epithelium. These results define a novel posttranslational mechanism for controlling the subcellular localization of ERK1/2 and for specifying the output of MAP kinase signaling.

Activation of Raf-1 suppresses integrin activation, potentially through the activation of extracellular signalregulated kinases 1 and 2 (ERK1/2). However, bulk ERK1/2 activation does not correlate with suppression. PEA-15 reverses suppression of integrin activation and binds ERK1/2. Here we report that PEA-15 reversal of integrin suppression depends on its capacity to bind ERK1/2, indicating that ERK1/2 function is indeed required for suppression. Mutations in either the death effector domain or C-terminal tail of PEA-15 that block ERK1/2 binding abrogated the reversal of integrin suppression. Furthermore, we used ERK/p38 chimeras and site-directed mutagenesis to identify ERK1/2 residues required for binding PEA-15. Mutations of residues that precede the ␣G helix and within the mitogenactivated protein kinase insert blocked ERK2 binding to PEA-15, but not activation of ERK2. These ERK2 mutants blocked the ability of PEA-15 to reverse suppression of integrin activation. Thus, PEA-15 regulation of integrin activation depends on its binding to ERK1/2. To directly test the role of ERK1/2 localization in suppression, we enforced membrane association of ERK1 and 2 by joining a membrane-targeting CAAX box sequence to them. Both ERK1-CAAX and ERK2-CAAX were membrane-localized and suppressed integrin activation. In contrast to suppression by membrane-targeted Raf-CAAX, suppression by ERK1/2-CAAX was not reversed by PEA-15. Thus, ERK1/2 are the Raf effectors for suppression of integrin activation, and PEA-15 reverses suppression by binding ERK1/2.

The rapid modulation of ligand-binding affinity ("activation") is a central property of the integrin family of cell adhesion receptors. The Ras family of small GTP-binding proteins and their downstream effectors are key players in regulating integrin activation. H-Ras can suppress integrin activation in fibroblasts via its downstream effector kinase, Raf-1. In contrast, to H-Ras, a closely related small GTP-binding protein R-Ras has the opposite activity, and promotes integrin activation. To gain insight into the regulation of integrin activation by Ras GTPases, we created a series of H-Ras/R-Ras chimeras. We found that a 35-amino acid stretch of H-Ras was required for full suppressive activity. Furthermore, the suppressive chimeras were weak activators of the ERK1/2 MAP kinase pathway, suggesting that the suppression of integrin activation may be independent of the activation of the bulk of ERK MAP kinase. Additional data demonstrating that the ability of H-Ras or Raf-1 to suppress integrin activation was unaffected by inhibition of bulk ERK1/2 MAP kinase activation supported this hypothesis. Thus, the suppression of integrin activation is a Raf kinase induced regulatory event that can be mediated independently of bulk activation of the ERK MAP-kinase pathway. INTRODUCTIONInteractions of integrin cell adhesion receptors with their extracellular ligands are important for cell migration, growth, and survival (Schwartz et al., 1995;Schwartz, 1997;Clark et al., 1998). A characteristic feature of many integrins is their ability to alter their affinity for ligands in response to intracellular signals, a process termed "activation" (Hughes and Pfaff, 1998).Presently, the signal transduction cascades controlling integrin activation are incompletely understood (Hughes and Pfaff, 1998). However, several observations suggest that members of the Ras family of small GTP-binding proteins and their downstream effectors are critically involved in the regulation of integrin activation (Zhang et al., 1996;Hughes et al., 1997;Reedquist et al., 2000). Activated H-Ras can suppress the activation of certain ␤1 and ␤3 integrins in fibroblasts via its effector serine/threonine kinase Raf-1 (Hughes et al., 1997). This activity of H-Ras is implicated in the control of cell morphology, cell movement, and assembly of the extracellular matrix (Hughes et al., 1997;Brenner et al., 2000). The suppressive activity of H-Ras does not require protein synthesis or mRNA transcription, and furthermore, suppression can be reversed by MAP kinase phosphatase 1 (Hughes et al., 1997). Thus, suppression appears to be mediated by a MAP kinase and correlates with activation of the ERK1/2 MAP kinase pathway.In contrast, to H-Ras other closely related small GTPbinding proteins, such as R-Ras and Rap1, have the opposite activity, promoting rather than suppressing integrin activation (Zhang et al., 1996;Osada et al., 1999;Caron et al., 2000;Reedquist et al., 2000;Shimizu, 2000). In CHO cells, activated R-Ras can antagonize the Ras/Raf suppressor pathway, and in...

Background: Somatic cell mutants can be informative in the analysis of a wide variety of cellular processes. The use of map-based positional cloning strategies in somatic cell hybrids to analyze genes responsible for recessive mutant phenotypes is often tedious, however, and remains a major obstacle in somatic cell genetics. To fulfill the need for more efficient gene mapping in somatic cell mutants, we have developed a new DNA microarray comparative genomic hybridization (array-CGH) method that can rapidly and efficiently map the physical location of genes complementing somatic cell mutants to a small candidate genomic region. Here we report experiments that establish the validity and efficacy of the methodology.