Msx2-deficient mice exhibit progressive hair loss, starting at P14 and followed by successive cycles of wavelike regrowth and loss. During the hair cycle, Msx2 deficiency shortens anagen phase, but prolongs catagen and telogen. Msx2-deficient hair shafts are structurally abnormal. Molecular analyses suggest a Bmp4/Bmp2/Msx2/Foxn1 acidic hair keratin pathway is involved. These structurally abnormal hairs are easily dislodged in catagen implying a precocious exogen. Deficiency in Msx2 helps to reveal the distinctive skin domains on the same mouse. Each domain cycles asynchronously -although hairs within each skin domain cycle in synchronized waves. Thus, the combinatorial defects in hair cycling and differentiation, together with concealed skin domains, account for the cyclic alopecia phenotype.
A major goal of the Alliance for Cellular Signaling is to elaborate the components of signal transduction networks in model cell systems, including murine B lymphocytes. Due to the importance of protein phosphorylation in many aspects of cell signaling, the initial efforts have focused on the identification of phosphorylated proteins. In order to identify serine-and threonine-phosphorylated proteins on a proteome-wide basis, WEHI-231 cells were treated with calyculin A, a serine/threonine phosphatase inhibitor, to induce high levels of protein phosphorylation. Proteins were extracted from whole-cell lysates and digested with trypsin. Phosphorylated peptides were then enriched using immobilized metal affinity chromatography and identified by liquid chromatography-tandem mass spectrometry. A total of 107 proteins and 193 phosphorylation sites were identified using these methods. Knowledge about covalent modifications and their regulation is essential for the understanding of protein function. Regulation of protein activity is often modulated by reversible phosphorylation, and information about specific sites of phosphorylation is vital for understanding cellular signaling pathways. Two-dimensional gel electrophoresis is still the most common method used for detecting large-scale changes in phosphorylation (1). However, this method is time consuming and has a number of significant limitations. Although mass spectrometry is a sensitive tool for the identification of phosphopeptides, their detection within a complex peptide mixture can be limited by weak ionization of phosphopeptides (2). Due to the low stoichiometry of phosphorylation and the low abundance of signaling proteins within cells, enrichment of the phosphoproteins or phosphopeptides is often necessary. The ability to isolate phosphopeptides by immobilized metal affinity chromatography (IMAC) 1 was first recognized by Andersson and Porath in 1986 (3). Recently, the use of IMAC in combination with mass spectrometry has allowed the identification of hundreds of phosphorylation sites in yeast (4) and Arabidopsis plasma membrane proteins (5).An important goal of the Alliance for Cellular Signaling (AfCS) is the global analysis of ligand-induced changes in protein phosphorylation (6). Important steps in this process are the identification of phosphoproteins present in the AfCS model cell systems and the determination of their sites of phosphorylation. This information will be used to generate probes to obtain quantitative information on the effects of ligands on phosphorylation of specific sites. Based on recent progress in the application of IMAC and mass spectrometry, we used this approach to identify phosphoproteins and their phosphorylation sites in the murine WEHI-231 B lymphoma cell line. EXPERIMENTAL PROCEDURESProtein Test Samples-One test sample (prepared at a ratio of 1:1:5) contained 1 pmol of a synthetic phosphotyrosine (p-Tyr) phosphopeptide (m/z 1127, DRVpYIHPF), a tryptic digest of 1 pmol of ␣-casein (containing three phosphopeptides: m/z 1467, TV...
The formation of a simple columnar epithelium in the uterus is essential for implantation. Perturbation of this developmental process by exogenous estrogen, such as diethylstilbestrol (DES), results in uterine metaplasia that contributes to infertility. The cellular and molecular mechanism underlying this transformation event is not well understood. Here we use a combination of global gene expression analysis and a knockout mouse model to delineate genetic pathways affected by DES. Global gene expression profiling experiment revealed that neonatal DES treatment alters uterine cell fate, particularly in the luminal epithelium by inducing abnormal differentiation, characterized by the induction of stratified epithelial markers including members of the small proline-rich protein family and epidermal keratins. We show that Msx2, a homeodomain transcription factor, functions downstream of DES and is required for the proper expression of several genes in the uterine epithelium including Wnt7a, PLAP, and K2.16. Finally, Msx2-/- uteri were found to exhibit abnormal water trafficking upon DES exposure, demonstrating the importance of Msx2 in tissue responsiveness to estrogen exposure. Together, these results indicate that developmental exposure to DES can perturb normal uterine development by affecting genetic pathways governing uterine differentiation.
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