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The advantageous chemical properties of the phosphate ester linkage were exploited early in evolution to generate the phosphate diester linkages that join neighbouring bases in RNA and DNA (Westheimer 1987 Science 235, 1173–1178). Following the fixation of the genetic code, another use for phosphate ester modification was found, namely reversible phosphorylation of the three hydroxyamino acids, serine, threonine and tyrosine, in proteins. During the course of evolution, phosphorylation emerged as one of the most prominent types of post-translational modification, because of its versatility and ready reversibility. Phosphoamino acids generated by protein phosphorylation act as new chemical entities that do not resemble any natural amino acid, and thereby provide a means of diversifying the chemical nature of protein surfaces. A protein-linked phosphate group can form hydrogen bonds or salt bridges either intra- or intermolecularly, creating stronger hydrogen bonds with arginine than either aspartate or glutamate. The unique size of the ionic shell and charge properties of covalently attached phosphate allow specific and inducible recognition of phosphoproteins by phosphospecific-binding domains in other proteins, thus promoting inducible protein–protein interaction. In this manner, phosphorylation serves as a switch that allows signal transduction networks to transmit signals in response to extracellular stimuli.

In the middle of the 20th century, animal tumor viruses were heralded as possible models for understanding human cancer. By the mid-1970s, the molecular basis by which tumor viruses transform cells into a malignant state was beginning to emerge as the first viral genomic sequences were reported and the proteins encoded by their transforming genes were identified and characterized. This was a time of great excitement and rapid progress. In 1978, prompted by the discovery from Ray Erikson’s group that the Rous sarcoma virus (RSV) v-Src–transforming protein had an associated protein kinase activity specific for threonine, my group at the Salk Institute set out to determine whether the polyomavirus middle T-transforming protein had a similar kinase activity. Here, I describe the experiments that led to the identification of a kinase activity associated with middle T antigen and our serendipitous discovery that this activity was specific for tyrosine in vitro, and how this in turn led to the fortuitous observation that the v-Src–associated kinase activity was also specific for tyrosine. Our finding that v-Src increased the level of phosphotyrosine in cellular proteins in RSV-transformed cells confirmed that v-Src is a tyrosine kinase and transforms cells by phosphorylating proteins on tyrosine. My colleague Bart Sefton and I reported these findings in the March issue of PNAS in 1980. Remarkably, all of the experiments in this paper were accomplished in less than one month.

Proline-directed phosphorylation is a posttranslational modification that is instrumental in regulating signaling from the plasma membrane to the nucleus, and its dysregulation contributes to cancer development. Protein interacting with never in mitosis A1 （Pinl）, which is overexpressed in many types of cancer, isomerizes specific phosphorylat- ed Ser/Thr-Pro bonds in many substrate proteins, including glycolytic enzyme, protein kinases, protein phosphatases, methyltransferase, lipid kinase, ubiquitin E3 ligase, DNA endonuclease, RNA polymerase, and transcription activa- tors and regulators. This Pinl-mediated isomerization alters the structures and activities of these proteins, thereby regulating cell metabolism, cell mobility, cell cycle progression, cell proliferation, cell survival, apoptosis and tumor development.

Pinkl, a mitochondrial kinase, and Parkin, an E3 ubiquitin ligase, function in mitochondrial maintenance. Pinkl accumulates on depolarized mitochondria, where it recruits Parkin to mainly induce K63-1inked chain ubiquitina- tion of outer membrane proteins and eventually mitophagy. Parkin belongs to the RBR E3 ligase family. Recently, it has been proposed that the RBR domain transfers ubiquitin to targets via a cysteine~ubiquitin enzyme intermediate, in a manner similar to HECT domain E3 ligases. However, direct evidence for a ubiquitin transfer mechanism and its importance for Parkin＇s in vivo function is still missing. Here, we report that Parkin E3 activity relies on cysteine- mediated ubiquitin transfer during mitophagy. Mutating the putative catalytic cysteine to serine （Parkin C431S） traps ubiquitin, and surprisingly, also abrogates Parkin mitochondrial translocation, indicating that E3 activity is essential for Parkin translocation. We found that Parkin can bind to K63-1inked ubiquitin chains, and that targeting K63-mimicking ubiquitin chains to mitochondria restores Parkin C431S localization. We propose that Parkin trans- location is achieved through a novel catalytic activity coupled mechanism.

Cancer-associated fibroblasts (CAFs) are a key component of the tumour microenvironment with diverse functions, including matrix deposition and remodelling, extensive reciprocal signalling interactions with cancer cells and crosstalk with infiltrating leukocytes. As such, they are a potential target for optimizing therapeutic strategies against cancer. However, many challenges are present in ongoing attempts to modulate CAFs for therapeutic benefit. These include limitations in our understanding of the origin of CAFs and heterogeneity in CAF function, with it being desirable to retain some antitumorigenic functions. On the basis of a meeting of experts in the field of CAF biology, we summarize in this Consensus Statement our current knowledge and present a framework for advancing our understanding of this critical cell type within the tumour microenvironment.This Consensus Statement highlights the importance of cancer-associated fibroblasts in cancer biology and progression, and issues a call to action for all cancer researchers to standardize assays and report metadata in studies of cancer-associated fibroblasts to advance our understanding of this important cell type in the tumour microenvironment.

Key Points Protein kinases and proteases are both major classes of cellular enzymes; the kinome and the degradome each include more than 500 genes. Direct interactions between kinases and proteases are frequent, and more than 125 examples of kinases that undergo regulated processing by one or more proteases are currently known. Targets for proteases include kinases in all the major branches of the kinome, as well as atypical protein kinases. Conversely, approximately 150 proteases in all the main families in the degradome are known to be phosphorylated by one or more kinases. Bidirectional kinase–protease interactions have an important role in many cellular processes, including transmembrane signalling, apoptosis and cell migration. Prominent examples include kinase–caspase crosstalk in apoptosis and interactions between deubiquitylases or γ-secretase and receptor tyrosine kinases in cell proliferation and invasion. Kinase–protease interactions are implicated as causal events in disease, and have a particularly important role in cancer. Cell migration and invasion are hallmarks of metastatic cancer, and many kinase–protease interactions are involved in activating these processes in a dysregulated fashion in cancer cells. Clinical implications of kinase–protease crosstalk are emerging. Interplay between ERBB family receptor tyrosine kinases and a disintegrin and metalloproteinase (ADAM) proteases results in the shedding of ERBB2 soluble forms that blunt the efficiency of ERBB2-specific monoclonal antibody therapy in breast cancer. A clinical trial in patients with breast cancer is underway using a combination of an ADAM10 and ADAM17 inhibitor with an ERBB2-specific antibody. Kinase–protease connections are being uncovered at a rapid pace, and their roles in cancer will undoubtedly become increasingly important and offer new opportunities for clinical intervention. Many proteases are regulated by phosphorylation, and many kinases are regulated by proteolytic cleavage. This Review examines kinase–protease interactions and their functional effects in cancer in depth, revealing the enormous diversity and complexity of this crosstalk. Kinases and proteases are responsible for two fundamental regulatory mechanisms — phosphorylation and proteolysis — that orchestrate the rhythms of life and death in all organisms. Recent studies have highlighted the elaborate interplay between both post-translational regulatory systems. Many intracellular or pericellular proteases are regulated by phosphorylation, whereas multiple kinases are activated or inactivated by proteolytic cleavage. The functional consequences of this regulatory crosstalk are especially relevant in the different stages of cancer progression. What are the clinical implications derived from the fertile dialogue between kinases and proteases in cancer?

Ubiquitylation is a critical post-translational modification that controls a wide variety of processes in eukaryotes. Ubiquitin chains of different topologies are specialized for different cellular functions and control the stability, activity, interaction properties, and localization of many different proteins. Recent work has highlighted a role for branched ubiquitin chains in the regulation of cell signaling and protein degradation pathways. Similar to their unbranched counterparts, branched ubiquitin chains are remarkably diverse in terms of their chemical linkages, structures, and the biological information they transmit. In this review, we discuss emerging themes related to the architecture, synthesis, and functions of branched ubiquitin chains. We also describe methodologies that have recently been developed to identify and decode the functions of these branched polymers.

How metabolism is reprogrammed during neuronal differentiation is unknown. We found that the loss of hexokinase (HK2) and lactate dehydrogenase (LDHA) expression, together with a switch in pyruvate kinase gene splicing from PKM2 to PKM1, marks the transition from aerobic glycolysis in neural progenitor cells (NPC) to neuronal oxidative phosphorylation. The protein levels of c-MYC and N-MYC, transcriptional activators of the HK2 and LDHA genes, decrease dramatically. Constitutive expression of HK2 and LDHA during differentiation leads to neuronal cell death, indicating that the shut-off aerobic glycolysis is essential for neuronal survival. The metabolic regulators PGC-1α and ERRγ increase significantly upon neuronal differentiation to sustain the transcription of metabolic and mitochondrial genes, whose levels are unchanged compared to NPCs, revealing distinct transcriptional regulation of metabolic genes in the proliferation and post-mitotic differentiation states. Mitochondrial mass increases proportionally with neuronal mass growth, indicating an unknown mechanism linking mitochondrial biogenesis to cell size. Structures called mitochondria act like the batteries of cells, and use several different metabolic processes to release energy. For example, neurons rely on a metabolic process called oxidative phosphorylation, while neural progenitor cells (which develop, or differentiate, into neurons) use a process called aerobic glycolysis instead. Little is known about why neurons prefer to use oxidative phosphorylation to provide them with energy, and it is also not clear why problems that affect this process are often seen in neurological disorders and neurodegenerative diseases. Zheng, Boyer et al. have now used human neural progenitor cells to explore the metabolic changes that occur as these cells develop into neurons. It appears that the loss of two metabolic enzymes, called hexokinase and lactate dehydrogenase, marks the transition from aerobic glycolysis to oxidative phosphorylation. In addition, the instructions to produce an enzyme called pyruvate kinase are altered or “alternatively spliced” when progenitor cells differentiate, which in turn changes the structure of the enzyme. The levels of the proteins that activate and regulate the production of these three metabolic enzymes also decrease dramatically during this transition. Further experiments showed that neurons that produce hexokinase and lactate dehydrogenase while they differentiate die, which means that neurons must shut off aerobic glycolysis in order to survive. The amounts of two proteins that regulate metabolism (called PGC-1α and ERRγ) increase significantly when a neuron differentiates. This sustains a constant level of activity for several metabolic and mitochondrial genes as neural progenitor cells differentiate to form neurons. Zheng, Boyer et al. also found that neurons build more mitochondria as they grow; this suggests that an unknown mechanism exists that links the creation of mitochondria to the size of the neuron. Zheng, Boyer et al. have mainly focused on how much of each metabolic enzyme is produced inside cells, but these levels may not completely reflect the actual level of enzyme activity. The next steps are therefore to investigate whether any other processes or modifications play a part in regulating the enzymes. Further investigation is also needed to determine the effects of changes in mitochondrial structure that occur as a neuron develops from a neural progenitor cell.

Protein–protein interactions, essential for understanding how a cell functions, are predicted using a new method that combines protein structure with other computationally and experimentally derived clues. Protein interactions predicted The analysis of protein-interaction networks is essential to an understanding of the regulatory processes in a living cell. Many methods have been developed with a view to predicting protein–protein interactions (PPIs) at a genome-wide level, although the differences obtained using these approaches suggest that there are still factors unaccounted for. Barry Honig and colleagues have developed a new way of predicting PPIs that is based on the proteins' three-dimensional structures and functional data. Tests of several predictions of the new algorithm, known as PREPPI, confirm the accuracy of the results. The genome-wide identification of pairs of interacting proteins is an important step in the elucidation of cell regulatory mechanisms 1 , 2 . Much of our present knowledge derives from high-throughput techniques such as the yeast two-hybrid assay and affinity purification 3 , as well as from manual curation of experiments on individual systems 4 . A variety of computational approaches based, for example, on sequence homology, gene co-expression and phylogenetic profiles, have also been developed for the genome-wide inference of protein–protein interactions (PPIs) 5 , 6 . Yet comparative studies suggest that the development of accurate and complete repertoires of PPIs is still in its early stages 7 , 8 , 9 . Here we show that three-dimensional structural information can be used to predict PPIs with an accuracy and coverage that are superior to predictions based on non-structural evidence. Moreover, an algorithm, termed PrePPI, which combines structural information with other functional clues, is comparable in accuracy to high-throughput experiments, yielding over 30,000 high-confidence interactions for yeast and over 300,000 for human. Experimental tests of a number of predictions demonstrate the ability of the PrePPI algorithm to identify unexpected PPIs of considerable biological interest. The surprising effectiveness of three-dimensional structural information can be attributed to the use of homology models combined with the exploitation of both close and remote geometric relationships between proteins.

Exosomes, extracellular nanovesicles that carry nucleic acids, lipids, and proteins, have been the subject of several studies to assess their ability to transfer functional cargoes to cells. We recently characterized extracellular nanovesicles released from glioblastoma cells that carry active Ras in complex with proteins regulating exosome biogenesis. Here, we investigated whether a functional transfer of Ras from exosomes to other cells can initiate intercellular signaling. We observed that treatment of serum-starved, cultured glioblastoma cells with exogenous glioblastoma exosomes caused a significant increase in cellular viability over time. Moreover, we detected fluorescent signal transfer from lipophilic dye-labeled exogenous glioblastoma exosomes into cultured glioblastoma cells. To probe possible signaling from cell-to-cell, we utilized bimolecular luciferase complementation to examine the ability of K-Ras in exosomes to interact with the Raf-Ras Binding domain (Raf-RBD) expressed in a recipient cell line. Although the K-Ras/Raf-RBD interaction was readily detectable upon co-expression in a single cell line, or following lysis of co-cultured cell lines separately expressing K-Ras and RBD, bearing in mind the limitations of our assay, we were unable to detect the interaction in the intact, co-cultured cell lines or upon treatment of the Raf-RBD-expressing cells with exosomes containing K-Ras. Furthermore, HA-Tag-BFP fused to the K-Ras hypervariable region and CAAX sequence failed to be transferred at significant levels from extracellular vesicles into recipient cells, but remained detectable in the cell supernatants even after 96 hours of culture of naïve cells with extracellular vesicles. We conclude that if transfer of functional K-Ras from extracellular vesicles into the cytoplasm of recipient cells occurs, it must do so at an extremely low efficiency and therefore is unlikely to initiate Ras-ERK MAP kinase pathway signaling. These results suggest that studies claiming functional transfer of protein cargoes from exosomes should be interpreted with caution.