The complexity and specificity of many forms of signal transduction are widely believed to require spatial compartmentation of protein kinase and phosphatase activities, yet existing methods for measuring kinase activities in cells lack generality or spatial or temporal resolution. We present three genetically encoded fluorescent reporters for the tyrosine kinases Src, Abl, and epidermal growth factor (EGF) receptor. The reporters consist of fusions of cyan fluorescent protein (CFP), a phosphotyrosine binding domain, a consensus substrate for the relevant kinase, and yellow fluorescent protein (YFP). Stimulation of kinase activities in living cells with addition of growth factors causes 20 -35% changes in the ratios of yellow to cyan emissions because of phosphorylationinduced changes in fluorescence resonance energy transfer (FRET). Platelet-derived growth factor (PDGF) stimulated Abl activity most strongly in actin-rich membrane ruffles, supporting the importance of this tyrosine kinase in the regulation of cell morphology. These results establish a general strategy for nondestructively imaging dynamic protein tyrosine kinase activities with high spatial and temporal resolution in single living cells. P hosphorylation is the most important way that individual proteins are posttranslationally modified to modulate their functions. To study kinase and phosphatase functions, methods are needed to image not only their localization but also their activities inside living cells. A few techniques of limited usefulness have been proposed for protein serine͞threonine kinases, as reviewed in the accompanying paper (1), but even fewer exist for tyrosine kinases. Antiphosphotyrosine antibodies (2) or incorporation of radioactive phosphate from ␥-labeled ATP are very useful in destructive assays with kinases purified from cells, but by themselves are of little or no use in unfractionated living cells. Imaging of fluorescence resonance energy transfer (FRET) from green fluorescent protein (GFP)-tagged proteins to acceptor-labeled phosphorylation-specific antibodies (3, 4) offers spatial resolution and greater specificity for the tagged substrate, but is still a destructive assay requiring membrane permeabilization or microinjection. In special cases where a natural protein or domain undergoes a significant conformational change on phosphorylation, fusion of two GFP mutants to both ends can yield phosphorylation-sensitive FRET (5). General methods are therefore still needed to nondestructively visualize the dynamics of activation of any tyrosine kinase or phosphatase in the genome. Here, we describe a class of FRET-based indicators that are genetically encoded (allowing for simple transfection rather than microinjection, and facile targeting to cellular compartments) and have a general design that can be extended in principle to most of the protein tyrosine kinases.In our design, the kinase to be monitored phosphorylates an appropriate substrate peptide sequence, whereupon the concatenated phosphoamino acid binding domain int...

cAMP-dependent protein kinase A (PKA) is important in processes requiring localized cell protrusion, such as cell migration and axonal path finding. Here, we used a membrane-targeted PKA biosensor to reveal activation of PKA at the leading edge of migrating cells. Previous studies show that PKA activity promotes protrusion and efficient cell migration. In live migrating cells, membrane-associated PKA activity was highest at the leading edge and required ligation of integrins such as alpha4beta1 or alpha5beta1 and an intact actin cytoskeleton. alpha4 integrins are type I PKA-specific A-kinase anchoring proteins, and we now find that type I PKA is important for localization of alpha4beta1 integrin-mediated PKA activation at the leading edge. Accumulation of 3' phosphorylated phosphoinositides [PtdIns(3,4,5)P(3)] products of phosphatidylinositol 3-kinase (PI3-kinase) is an early event in establishing the directionality of migration; however, polarized PKA activation did not require PI3-kinase activity. Conversely, inhibition of PKA blocked accumulation of a PtdIns(3,4,5)P(3)-binding protein, the AKT-pleckstrin homology (PH) domain, at the leading edge; hence, PKA is involved in maintaining cell polarity during migration. In sum, we have visualized compartment-specific PKA activation in migrating cells and used it to reveal that adhesion-mediated localized activation of PKA is an early step in directional cell migration.

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How might telomerase be used as a therapeutic target in people? I think that it is helpful to think of telomeres in two different contexts. In cancer cells, Telomeres and Tetrahymena: an interview with Elizabeth Blackburn Elizabeth Blackburn knows that loose ends contribute to aging and many of its associated diseases. She, together with Carol Greider and Jack Szostak, received the 2009 Nobel Prize in Physiology or Medicine for their work on the synthesis and function of telomeres, the unusual DNA sequences at the ends of chromosomes. Telomere changes are now recognized in human diseases ranging from cancer and cardiovascular disease to depression. Here, she discusses her approach to mentorship, how scientists might inform public policy, and new directions in telomere research.

Susan Lindquist is a founding editor of DMM, whose pioneering work in yeast has advanced our understanding of protein folding in disease, including Parkinson’s, Huntington’s and prion diseases. Here, she discusses her personal approach to model organism research and scientific leadership.

recently won the Nobel Prize for contributions to genetics that have catapulted the mouse to the status of the most valuable of all animal models. He has a personal story that is as rich and interesting as his science. Here, he discusses the journey that led him to gene targeting and his vision for the future.