SUMMARY Although most tissues in an organism are genetically identical, the biochemistry of each is optimized to fulfill its unique physiological roles, with important consequences for human health and disease. Each tissue’s unique physiology requires tightly regulated gene and protein expression coordinated by specialized, phosphorylation-dependent intracellular signaling. To better understand the role of phosphorylation in maintenance of physiological differences among tissues, we performed proteomic and phosphoproteomic characterizations of nine mouse tissues. We identified 12,039 proteins, including 6296 phosphoproteins harboring nearly 36,000 phosphorylation sites. Comparing protein abundances and phosphorylation levels revealed specialized, interconnected phosphorylation networks within each tissue while suggesting that many proteins are regulated by phosphorylation independently of their expression. Our data suggest that the ‘typical’ phosphoprotein is widely expressed, yet displays variable, often tissue-specific phosphorylation that tunes protein activity to the specific needs of each tissue. We offer this dataset as an online resource for the biological research community.

Cyclin D1 is a component of the core cell cycle machinery1. Abnormally high levels of cyclin D1 are detected in many human cancer types2. To elucidate the molecular functions of cyclin D1 in human cancers, here we performed a proteomic screen for cyclin D1 protein partners in several types of human tumors. Analyses of cyclin D1-interactors revealed a network of DNA repair proteins, including RAD51, a recombinase that drives the homologous recombination process3. We found that cyclin D1 directly binds RAD51, and that cyclin D1-RAD51 interaction is induced by radiation. Like RAD51, cyclin D1 is recruited to DNA damage sites in a BRCA2-dependent fashion. Reduction of cyclin D1 levels in human cancer cells impaired recruitment of RAD51 to damaged DNA, impeded the homologous recombination-mediated DNA repair, and increased sensitivity of cells to radiation in vitro and in vivo. This effect was seen in cancer cells lacking the retinoblastoma protein, which do not require D-cyclins for proliferation4, 5. These findings reveal an unexpected function of a core cell cycle protein in DNA repair and suggest that targeting cyclin D1 may be beneficial also in retinoblastoma-negative cancers which are currently thought to be oblivious to cyclin D1 inhibition.

HFE and transferrin receptor 2 (TFR2) are membrane proteins integral to mammalian iron homeostasis and associated with human hereditary hemochromatosis. Here we demonstrate that HFE and TFR2 interact in cells, that this interaction is not abrogated by disease-associated mutations of HFE and TFR2, and that TFR2 competes with TFR1 for binding to HFE. We propose a new model for the mechanism of iron status sensing that results in the regulation of iron homeostasis.All mammalian cells have an absolute requirement for iron. Both cellular iron deficiency and iron-overload are pathological and iron concentration in cells and body fluids is tightly regulated. Chronic malaccumulation of iron in tissues results in systemic iron-overload diseases that are collectively called hemochromatosis. Hereditary hemochromatosis in humans is linked to mutations in several genes namely HFE, transferrin receptor 2 (TFR2), hemojuvelin (HJV), and hepcidin (HAMP) (reviewed in Ref. 1).Hepcidin, a hepatocyte-derived soluble factor, negatively regulates intestinal absorption of dietary iron and the release of recycled iron into circulation from macrophages (2). Also, hepcidin expression responds to body iron status (3). Hepcidin, therefore, plays a pivotal role as a regulator of whole-body iron homeostasis. Since disruption of HFE, TFR2, or HJV causes decreased hepcidin production these gene products appear to be involved in the upstream regulation of hepcidin (reviewed in Ref. 4).HFE, an atypical major histocompatibility complex class I molecule, associates with transferrin receptor 1 (TFR1), 3 a type II transmembrane glycoprotein that is the primary effector of cellular iron uptake (5). TFR2 is a homolog of TFR1 and, like TFR1, can bind and internalize diferric-transferrin (Fe 2 -TF) (6). However, while TFR1 is widely expressed, TFR2 is expressed predominantly in hepatocytes, hematopoietic cells, and duodenal crypt cells, overlapping with HFE expression (6, 7). This and other differences in transcriptional regulation, Fe 2 -TF binding affinities and gene deletion phenotypes suggest that TFR1 and TFR2 have distinct roles in iron homeostasis. While TFR1 is a key mediator of iron uptake, TFR2 is postulated to play a regulatory role in whole-body iron homeostasis. Since hepcidin is produced predominantly by hepatocytes, it is likely that these cells express molecular determinants of iron sensing. Serum transferrin deficiency in hpx mice is associated with low hepcidin levels despite parenchymal iron loading, a phenotype corrected by transferrin administration (8). Also, elevated serum Fe 2 -TF stabilizes TFR2 protein in liver (9), an effect recapitulated in vitro (9, 10). Circulating Fe 2 -TF is therefore likely to be an iron signal sensed by hepatocyte membrane proteins that regulate hepcidin production and TFR2 may be part of the regulatory system sensing transferrin saturation.Previous investigators hypothesized that HFE and TFR2 might belong together in such a regulatory pathway. An earlier study, however, demonstrated that soluble, purifi...