Chiharu Itagaki

Cell Surface Labeling and Mass Spectrometry Reveal Diversity of Cell Surface Markers and Signaling Molecules Expressed in Undifferentiated Mouse Embryonic Stem Cells

Although interactions between cell surface proteins and extracellular ligands are key to initiating embryonic stem cell differentiation to specific cell lineages, the plasma membrane protein components of these cells are largely unknown. We describe here a group of proteins expressed on the surface of the undifferentiated mouse embryonic stem cell line D3. These proteins were identified using a combination of cell surface labeling with biotin, subcellular fractionation of plasma membranes, and mass spectrometry-based protein identification technology. From 965 unique peptides carrying biotin labels, we assigned 324 proteins including 235 proteins that have putative signal sequences and/or transmembrane segments. Receptors, transporters, and cell adhesion molecules were the major classes of proteins identified. Besides known cell surface markers of embryonic stem cells, such as alkaline phosphatase, the analysis identified 59 clusters of differentiation-related molecules and more than 80 components of multiple cell signaling pathways that are characteristic of a number of different cell lineages. We identified receptors for leukemia-inhibitory factor, interleukin 6, and bone morphogenetic protein, which play critical roles in the maintenance of undifferentiated mouse embryonic stem cells. We also identified receptors for growth factors/cytokines, such as fibroblast growth factor, platelet-derived growth factor, ephrin, Hedgehog, and Wnt, which transduce signals for cell differentiation and embryonic development. Finally we identified a variety of integrins, cell adhesion molecules, and matrix metalloproteases. These results suggest that D3 cells express diverse cell surface proteins that function to maintain pluripotency, enabling cells to respond to various external signals that initiate differentiation into a variety of cell types.

Rb plays a role in survival of Abl-dependent human tumor cells as a downstream effector of Abl tyrosine kinase.

The retinoblastoma (Rb) gene product is a tumor suppressor that is mutated or inactivated in many types of human cancers. Although Rb is known to be an upstream negative regulator of Abl protein tyrosine kinase, we propose here that Rb also functions as a downstream effector of Abl that plays a positive role in survival of Abl-dependent human tumor cells, including Bcr/Abl-positive chronic myelogenous leukemia (CML). We show that Rb is constitutively phosphorylated at tyrosine in Abl-dependent tumor cells, and that Abl phosphorylates Rb specifically at Y805 within the C-terminal domain of the molecule. We also show that ectopic expression of Rb induces apoptosis in Abl-dependent tumor cells by inhibiting the Abl tyrosine kinase activity, and that Rb-induced apoptosis is compromised by Abl-catalysed phosphorylation of Rb at Y805. Furthermore, the silencing of endogenous Rb by RNA interference induced apoptosis in Abl-dependent tumor cells. Thus, our findings suggest that Abl-catalysed tyrosine phosphorylation of Rb is necessary for survival of Abl-dependent human tumor cells, and raises the possibility that this phosphorylated Rb can be a molecular target for cancer therapy aimed at inducing apoptosis of Abl-dependent tumor cells, such as Bcr/Abl-positive CML.

INTERACTION OF TYROSINE HYDROXYLASE AND 14-3-3 PROTEINS IN PC12 CELLS

The 14–3–3 protein is a family of acidic, dimeric proteins distributed widely among eukaryotic cells. This protein family binds to a variety of proteins in a phosphorylation-dependent manner and participates in the regulation of cell proliferation, differentiation and function. To date, more than 70 proteins, localized in the cell membrane to nuclei, are reported to bind with the 14–3–3 family. These proteins include at least 19 membrane components (receptors, ion channels and effectors), 22 signal transduction kinases and phosphatases, 5 cytoskelton proteins, 18 nuclear proteins including transcription factors, 10 metabolic enzymes, and others. We previously showed, using PC12nnr5 cells transfected with myc-tagged 14–3–3β and η) isoforms, that 14–3–3s form a complex with tyrosine hydroxylase (TH) particularly when the hydroxylase is phosphorylated at Ser-19 by endogeneous Ca2+, calmodulin-dependent protein kinase II1. In this study we have employed proteomic techniques for systematic characterization of PC12 proteins and identified the TH protein as a major constituent of the proteins that are co-immunoprecipitated with 14–3–3. We have also shown that the TH protein is predominantly associated with 14–3-3e and η isoforms, but not with ζ and θ isoforms. These results indicate that the activity of TH is regulated through phosphorylation-dependent interaction with limited members of the 14–3–3 family.