DATA MINING

Data Mining

Abstract

Due to heterodimerization and a variety of stimulating ligands, the ErbB receptor system is both diverse and flexible, which proves particularly advantageous to the aberrant signaling of cancer cells. However, specific mechanisms of how a particular receptor contributes to generating the flexibility that leads to aberrant growth regulation have not been well described. We compared the utilization of ErbB2 in response to epidermal growth factor (EGF) and heregulin stimulation in colon carcinoma cells. Anti-ErbB2 monoclonal antibody 2C4 blocked heregulin-stimulated phosphorylation of ErbB2 and ErbB3; activation of mitogen-activated protein kinase (MAPK), phosphatidylinositol 3'-kinase (PI3K), and Akt; proliferation; and anchorage-independent growth. 2C4 blocked EGF-mediated phosphorylation of ErbB2 and inhibited PI3K/Akt and anchorage-independent growth but did not affect ErbB1 or MAPK. Immunoprecipitations showed that ErbB3 and Grb2-associated binder (Gab) 1 were phosphorylated and associated with PI3K activity after heregulin treatment and that Gab1 and Gab2, but not ErbB3, were phosphorylated and associated with PI3K activity after EGF treatment. These data show that monoclonal antibody 2C4 inhibited all aspects of heregulin signaling as well as anchorage-independent and monolayer growth. Furthermore, we identify ErbB2 as a critical component of EGF signaling to the Gab1/Gab2-PI3K-Akt pathway and anchorage-independent growth, but EGF stimulation of MAPK and monolayer growth can occur efficiently without the contribution of ErbB2.

Table of Contents

Data Mining4

Introduction4

Materials And Methods6

Reagents.7

Cell Stimulation and Lysis.7

Immunoprecipitations.8

Immunoblotting.8

PI3K Assay.9

Cell Monolayer Growth.10

Soft Agar Assay.10

Results10

Monoclonal Antibody 2C4 Can Inhibit Heregulin- and EGF-Stimulated Phosphorylation of ErbB2.11

Activation of Erk and Akt after 2C4 Treatment and Growth Factor Stimulation.12

Coimmunoprecipitation of Distinct Phospho-Tyrosine-Containing Proteins after EGF and Heregulin Treatment.13

Identification of Gab1/Gab2 and ErbB3/Gab1 as Modulators of PI3K for EGF and Heregulin, Respectively.14

2C4 Inhibits EGF and Heregulin Stimulated PI3K Activity.16

2C4 Inhibits Heregulin-Stimulated Cell Proliferation and EGF- and Heregulin-Stimulated Anchorage-Independent Growth.16

Discussion17

References23

Appendix32

Data Mining

Introduction

The ErbB family of tyrosine kinase receptors (ErbB1-4) plays an important role in the progression of numerous types of malignancies (1 , 2) . The complexity of available ligands, receptors, and oligomerization provide ErbB receptor signaling with a great deal of flexibility and diversity, enabling cells to respond to environmental stimuli. Given the extensive capabilities of this signaling system, it is not surprising that the aberrant regulation of ErbB family signaling is frequently associated with several histological types of cancer in a manner that is not dependent on receptor overexpression (3, 4, 5) .

These diverse signals originate when ErbB family receptors are trans-phosphorylated at specific tyrosine residues in their COOH termini after ligand binding. This phosphorylation can occur in the context of homodimers, such as ErbB1-ErbB1, or heterodimers, such as ErbB1-ErbB2. Various signaling molecules and adaptor proteins are then recruited to these docking sites, and downstream signals are transmitted (2) . For example, activation of the mitogen-activated protein kinase (MAPK) pathway can occur when the adaptor molecule Grb2 directly binds ErbB receptors via an SH2 domain (6 , 7) , or when Shc, which itself has Grb2 binding sites, binds ErbB receptors via a phospho-tyrosine binding domain (8) . Grb2 can then activate MAPKs by initiating a cascade involving SOS, ras, raf, and MAP/ERK kinase (9) ...