To ensure the fidelity of chromosome segregation, the spindle checkpoint blocks the ubiquitin ligase activity of APC/C(Cdc20) in response to a single chromatid not properly attached to the mitotic spindle. Here we show that HeLa cells depleted for Bub1 by RNA interference are defective in checkpoint signaling. Bub1 directly phosphorylates Cdc20 in vitro and inhibits the ubiquitin ligase activity of APC/C(Cdc20) catalytically. A Cdc20 mutant with all six Bub1 phosphorylation sites removed is refractory to Bub1-mediated phosphorylation and inhibition in vitro. Upon checkpoint activation, Bub1 itself is hyperphosphorylated and its kinase activity toward Cdc20 is stimulated. Ectopic expression of the nonphosphorylatable Cdc20 mutant allows HeLa cells to escape from mitosis in the presence of spindle damage. Therefore, Bub1-mediated phosphorylation of Cdc20 is required for proper checkpoint signaling. We speculate that inhibition of APC/C(Cdc20) by Bub1 in a catalytic fashion may partly account for the exquisite sensitivity of the spindle checkpoint.

The reproducibility of conventional two-dimensional (2D) gel electrophoresis can be improved using differential in-gel electrophoresis (DIGE) Proteomics (1) includes the systematic cataloging of protein expression on a large scale, providing complementary information to that obtained from mRNA profiling by microarray (2, 3). Such studies could lead to the molecular characterization of cellular events associated with cancer progression, cellular signaling, and developmental stages (4 -7). Proteomics studies of clinical tumor samples have led to the identification of cancer-specific protein markers, which provide a basis for developing new methods for early diagnosis and early detection and clues to understand the molecular characterization of cancer progression (5, 8 -10).,A mainstay of conventional proteomics is high resolution 2D 1 gel electrophoresis (11, 12) followed by protein identification using mass spectrometry (13-15). The state of the art 2D gel system can be loaded with a few milligrams of protein and separates thousands of protein spots (5, 16). Although the technique has been widely used and successfully applied in a variety of biological systems, several technical limitations exist. Because of subtle changes in experimental conditions, the protein expression patterns on a single 2D gel usually cannot be fully duplicated, which makes it difficult to find the proteins changed between gels and to quantify the changes in protein expression. Although a comparison of protein expression profiles from regular 2D gel electrophoresis can be carried out with the assistance of various software programs, it typically requires some computerized justification of 2D gel images so that two images can be superimposed and compared. These difficulties limit the speed and accuracy of quantitation of protein spots in 2D gel electrophoresis.The differential in-gel electrophoresis (DIGE) technique recently introduced by Amersham Biosciences, Inc. is aimed at improving reproducibility. The concept of DIGE was originally developed by Minden and colleagues (17). To analyze the samples in DIGE, two pools of protein extracts are labeled covalently with fluorescent cyanine dyes, Cy3 and Cy5, re-

Phosphorylation is a major regulatory mechanism controlling circadian clocks. In the Neurospora circadian clock, the PER-ARNT-SIM (PAS) domain-containing transcription factor, WHITE COLLAR (WC)-1 , acts both as the blue light photoreceptor of the clock and as a positive element in the circadian negative feedback loop in constant darkness, by activating the transcription of the frequency (frq) gene. To understand the role of WC-1 phosphorylation, five in vivo WC-1 phosphorylation sites, located immediately downstream of the WC-1 zinc finger DNA binding domain, were identified by tandem mass spectrometry using biochemically purified endogenous WC-1 protein. Mutations of these phosphorylation sites suggest that they are major WC-1 phosphorylation sites under constant conditions but are not responsible for the light-induced hyperphosphorylation of WC-1. Although phosphorylation of these sites does not affect the light function of WC-1, strains carrying mutations of these sites show short period, low amplitude, or arrhythmic conidiation rhythms in constant darkness. Furthermore, normal or slightly higher levels of frq mRNA and FRQ proteins were observed in a mutant strain containing mutations of all five sites despite its low WC-1 levels. Together, these data suggest that phosphorylation of these sites negatively regulates the function of WC-1 in the circadian negative feedback loop and is important for the function of the Neurospora circadian clock.Eukaryotic circadian oscillators consist of autoregulatory transcription/translation-based negative feedback loops (1, 2). In these negative feedback loops, the positive elements activate the transcription of the negative elements, whereas the negative elements inhibit their own transcription by inhibiting the activity of the positive elements. In the Neurospora circadian negative feedback loop, such as those in Drosophila and mammals, the positive element is a heterodimeric complex made of two PER-ARNT-SIM (PAS) 1 domain-containing transcription factors, WHITE COLLAR (WC)-1 and WC-2. In the dark, the two WC proteins form a heterodimer through their PAS domains and activate the transcription of the frequency (frq) gene by directly binding to its promoter (3-7). When the amount of FRQ protein reaches a certain level, the homodimeric FRQ in complex with FRH, a FRQ-interacting RNA helicase, represses frq transcription by interacting with the WC complex and preventing its binding to the frq promoter, thus closing the negative feedback loop (3, 8 -14).In addition to their essential role in the circadian negative feedback loop in the dark, WC-1 and WC-2 are required for all known light responses in Neurospora, including the entrainment of the circadian clock (4, 7, 15-21). WC-1 binds to chromophore FAD through its photosensory LOV (light, oxygen, or voltage) domain, a specialized PAS domain, and functions as the blue light photoreceptor for light responses (22)(23)(24). Light triggers the formation of a large WC complex and its binding to the promoters of light-inducible genes ...

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...

WNK (with no lysine [K]) protein kinases were named for their unique active site organization. Mutations in WNK1 and WNK4 cause a familial form of hypertension by undefined mechanisms. Here, we report that WNK1 selectively binds to and phosphorylates synaptotagmin 2 (Syt2) within its calcium binding C2 domains. Endogenous WNK1 and Syt2 coimmunoprecipitate and colocalize on a subset of secretory granules in INS-1 cells. Phosphorylation by WNK1 increases the amount of Ca2+ required for Syt2 binding to phospholipid vesicles; mutation of threonine 202, a WNK1 phosphorylation site, partially prevents this change. These findings suggest that phosphorylation of Syts by WNK1 can regulate Ca2+ sensing and the subsequent Ca2+-dependent interactions mediated by Syt C2 domains. These findings provide a biochemical mechanism that could lead to the retention or insertion of proteins in the plasma membrane. Interruption of this regulatory pathway may disturb membrane events that regulate ion balance.