Signaling pathways invoke interplays between forward signaling and feedback to drive robust cellular response. In this study, we address the dynamics of growth factor signaling through profiling of protein phosphorylation and gene expression, demonstrating the presence of a kinetically defined cluster of delayed early genes that function to attenuate the early events of growth factor signaling. Using epidermal growth factor receptor signaling as the major model system and concentrating on regulation of transcription and mRNA stability, we demonstrate that a number of genes within the delayed early gene cluster function as feedback regulators of immediate early genes. Consistent with their role in negative regulation of cell signaling, genes within this cluster are downregulated in diverse tumor types, in correlation with clinical outcome. More generally, our study proposes a mechanistic description of the cellular response to growth factors by defining architectural motifs that underlie the function of signaling networks.

The erbB-2͞HER2 oncogene is overexpressed in a significant fraction of human carcinomas of the breast, ovary, and lung in a manner that correlates with poor prognosis. Although the encoded protein resembles several receptors for growth factors, no high affinity ligand of ErbB-2 has so far been fully characterized. However, several lines of evidence have raised the possibility that ErbB-2 can augment signal transduction initiated by binding of certain growth factors to their direct receptors. Here, we contrasted these two models of ErbB-2 function: First, examination of a large series of epidermal growth factor (EGF)-like ligands and neuregulins, including virus-encoded ligands as well as related motifs derived from the precursor of EGF, failed to detect interactions with ErbB-2 when this protein was singly expressed. Second, by using antibodies that block inter-ErbB interactions and cells devoid of surface ErbB-2, we learned that signaling by all ligands examined, except those derived from the precursor of EGF, was enhanced by the oncoprotein. These results imply that ErbB-2 evolved as a shared receptor subunit of all ErbB-specific growth factors. Thus, oncogenicity of ErbB-2 in human epithelia may not rely on the existence of a specific ligand but rather on its ability to act as a coreceptor for multiple stroma-derived growth factors.Cellular growth and fate determination are controlled by a large variety of extracellular ligands and specific cell surface receptors. The largest family of such receptors is that of the growth factor receptors with intrinsic tyrosine kinase activity (1). Type-1 tyrosine kinase receptors, also known as ErbB͞ HER proteins, comprise one of the better-characterized subfamilies of growth factor receptors, of which the epidermal growth factor (EGF) receptor (ErbB-1) is the prototype (reviewed in ref.2). The four ErbB members form homo-and heterodimeric complexes on binding of EGF-like or neuregulin (NRG) ligands, and, thereby, their kinase activity is stimulated and intracellular signals are generated. Constitutive stimulation of these pathways through autocrine or other mechanisms is associated with several types of human cancer (3). Most relevant is the frequent overexpression, often as a result of gene amplification, of ErbB-2͞HER2 in breast, ovary, lung, and other types of epithelial cancers (reviewed in refs. 4 and 5). In some tissues, this overexpression was correlated with poorer prognosis and a more aggressive tumor phenotype (6).Although ErbB-2 shares extensive structural homology with other ErbBs both along the catalytic intracellular domain and in the extracellular putative ligand binding region, many attempts to identify stimulatory ligands specific to ErbB-2 have so far failed. For example, detection of an activity that enhances ErbB-2 phosphorylation led to molecular cloning of the Neu differentiation factor (NDF) and heregulin, two of a dozen isoforms of NRG1, all of which bind to ErbB-3 and ErbB-4 (7). Nevertheless, several observations imply that ErbB-2 homodime...

ErbB-2 is an orphan receptor that belongs to a family of tyrosine kinase receptors for either epidermal growth factor (EGF) or Neu di erentiation factor (NDF/ neuregulin). Because overexpression of the erbB-2 proto-oncogene is frequently associated with an aggressive clinical course of certain human adenocarcinomas, the encoded protein is an attractive target for immunotherapy. Indeed, certain monoclonal antibodies (mAbs) to ErbB-2 e ectively inhibit tumor growth in animal models and in clinical trials, but the underlying mechanism is incompletely understood. To study this question, we generated a large battery of mAbs to ErbB-2, that were classi®ed epitopically. Whereas most antibodies stimulated tyrosine phosphorylation of ErbB-2, their anti-tumor e ect correlated with its accelerated endocytic degradation. One group of tumor-inhibitory mAbs (Class II mAbs) was elicited by the most antigenic site of ErbB-2, and inhibited in trans binding of NDF and EGF to their direct receptors. The inhibitory e ect was due to acceleration of ligand dissociation, and it resulted in the reduction of the ability of ErbB-2 to transactivate the mitogenic signals of NDF and EGF. These results identify two potential mechanisms of antibody-induced therapy: acceleration of ErbB-2 endocytosis by homodimerization and blocking of heterodimerization between ErbB-2 and growth factor receptors.

Sprouty proteins are evolutionarily conserved negative feedback regulators of multiple receptor tyrosine kinases. Mammalian versions of these proteins differentially regulate signaling induced by the fibroblast and the epidermal growth factors (FGF and EGF, respectively). Herein we show that, although both growth factors elevate expression of Sprouty-2, FGF-and not EGFinduced activation of the Erk/MAPK pathway is inhibited by Sprouty-2. Attenuation of FGF-signaling is accompanied by the induction of Sprouty-2 phosphorylation on the amino-terminal as well as carboxyl-terminal tyrosine residues, which are less effectively modified upon EGF treatment. Mutagenesis of carboxyl-terminal tyrosines, especially a newly identified phosphorylation site, tyrosine 227, impaired the inhibitory activity of Sprouty-2. These results attribute a novel role for carboxyl-terminal tyrosine residues and yet unidentified phosphotyrosine-binding proteins in the differential regulation of Sprouty-2 activity.

Successful progression through the cell cycle requires the coupling of mitotic spindle formation to DNA replication. In this report we present evidence suggesting that, in Saccharomyces cerevisiae, the CDC40 gene product is required to regulate both DNA replication and mitotic spindle formation. The deduced amino acid sequence of CDC40 (455 amino acids) contains four copies of a beta-transducin-like repeat. Cdc40p is essential only at elevated temperatures, as a complete deletion or a truncated protein (deletion of the C-terminal 217 amino acids in the cdc40-1 allele) results in normal vegetative growth at 23 degrees C, and cell cycle arrest at 36 degrees C. In the mitotic cell cycle Cdc40p is apparently required for at least two steps: (1) for entry into S phase (neither DNA synthesis, nor mitotic spindle formation occurs at 36 degrees C and (2) for completion of S-phase (cdc40::LEU2 cells cannot complete the cell cycle when returned to the permissive temperature in the presence of hydroxyurea). The role of Cdc40p as a regulatory protein linking DNA synthesis, spindle assembly/maintenance, and maturation promoting factor (MPF) activity is discussed.