Protein phosphorylation events during T cell receptor (TCR) signaling control the formation of complexes among proteins proximal to the TCR, the activation of kinase cascades, and the activation of transcription factors; however, the mode and extent of the influence of phosphorylation in coordinating the diverse phenomena associated with T cell activation are unclear. Therefore, we used the human Jurkat T cell leukemia cell line as a model system and performed large-scale quantitative phosphoproteomic analyses of TCR signaling. We identified 10,665 unique phosphorylation sites, of which 696 showed TCR-responsive changes. In addition, we analyzed broad trends in phosphorylation data sets to uncover underlying mechanisms associated with T cell activation. We found that, upon stimulation of the TCR, phosphorylation events extensively targeted protein modules involved in all of the salient phenomena associated with T cell activation: patterning of surface proteins, endocytosis of the TCR, formation of the F-actin cup, inside-out activation of integrins, polarization of microtubules, production of cytokines, and alternative splicing of messenger RNA. Further, case-by-case analysis of TCR-responsive phosphorylation sites on proteins belonging to relevant functional modules together with network analysis allowed us to deduce that serine-threonine (S-T) phosphorylation modulated protein-protein interactions (PPIs) in a system-wide fashion. We also provide experimental support for this inference by showing that phosphorylation of tubulin on six distinct serine residues abrogated PPIs during the assembly of microtubules. We propose that modulation of PPIs by stimulus-dependent changes in S-T phosphorylation state is a widespread phenomenon applicable to many other signaling systems.

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The ventral striatum (VS) is considered a key region that flexibly updates recent changes in reward values for habit learning. However, this update process may not serve to maintain learned habitual behaviors, which are insensitive to value changes. Here, using fMRI in humans and single-unit electrophysiology in macaque monkeys we report another role of the primate VS: that the value memory subserving habitual seeking is stably maintained in the VS. Days after object-value associative learning, human and monkey VS continue to show increased responses to previously rewarded objects, even when no immediate reward outcomes are expected. The similarity of neural response patterns to each rewarded object increases after learning among participants who display habitual seeking. Our data show that long-term memory of high-valued objects is retained as a single representation in the VS and may be utilized to evaluate visual stimuli automatically to guide habitual behavior.

Identification and characterization of the nuclear proteome is important for detailed understanding of multiple signaling events in eukaryotic cells. Toward this goal, we extensively characterized the nuclear proteome of human T leukemia cells by sequential extraction of nuclear proteins with different physicochemical properties using three buffer conditions. This large scale proteomic study also tested the feasibility and technical challenges associated with stable isotope labeling by amino acids in cell culture (SILAC) to uncover quantitative changes during apoptosis. Analyzing proteins from three nuclear fractions extracted from naïve and apoptotic cells generated 780,530 MS/MS spectra that were used for database searching using the SEQUEST algorithm. This analysis resulted in the identification and quantification of 1,174 putative nuclear proteins. A number of known nuclear proteins involved in apoptosis as well as novel proteins not known to be part of the nuclear apoptotic machinery were identified and quantified. Consistent with SILACbased quantifications, immunofluorescence staining of nucleus, mitochondria, and some associated proteins from both organelles revealed a dynamic recruitment of mitochondria into nuclear invaginations during apoptosis. Molecular & Cellular Proteomics 5:1131-1145, 2006.Comprehensive understanding of the biology of the nucleus will require complete identification of the proteome of this important organelle. The nucleus of the cell is where a number of key enzymatic processes occur, and most of these processes are critical for cellular homeostasis: DNA synthesis, DNA replication and repair, transcription, higher order chromatin organization, and gene and chromosomal silencing (1-8). A comprehensive list of proteins that reside in the nucleus, as well as the proteins that shuttle between multiple subcellular compartments and nucleus, has not been completed to date (9 -11). In addition, different classes of proteins are sequestered in the nucleus based on their affinity to DNA, nuclear matrix, nuclear membrane, nucleolus, specialized nuclear bodies such as speckles and PIKA (polymorphic interphase karyosomal association), or Cajal bodies (coiled bodies) (12-15). Thus, large scale identification of nuclear proteins from human cells and characterization of their associated physicochemical properties will likely provide insights into the biology of the nucleus.We have been interested in identifying proteins that control apoptosis in the nucleus. Apoptosis or programmed cell death is a process essential for the development and maintenance of cellular homeostasis of higher eukaryotes (16). One of the hallmarks of apoptosis is rapid chromatin condensation and DNA fragmentation (17). The exact mechanisms that control rapid DNA fragmentation and chromatin condensation in the nucleus are not fully understood, although some of the proteins that are crucial for these events have been identified (18 -25). For example, the caspase-activated DNase (CAD/DFF40) (18, 19), lamin protease (Caspase 6...

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