A general caging method for proteins that are regulated by phosphorylation was used to study the in vivo biochemical action of cofilin and the subsequent cellular response. By acute and local activation of a chemically engineered, light-sensitive phosphocofilin mimic, we demonstrate that cofilin polymerizes actin, generates protrusions, and determines the direction of cell migration. We propose a role for cofilin that is distinct from its role as an actin-depolymerizing factor.

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The structure of the catalytically inactive mutant (C215S) of the human protein-tyrosine phosphatase 1B (PTP1B) has been solved to high resolution in two complexes. In the first, crystals were grown in the presence of bis-(para-phosphophenyl) methane (BPPM), a synthetic highaffinity low-molecular weight nonpeptidic substrate (K m ‫؍‬ 16 M), and the structure was refined to an R-factor of 18.2% at 1.9 Å resolution. In the second, crystals were grown in a saturating concentration of phosphotyrosine (pTyr), and the structure was refined to an R-factor of 18.1% at 1.85 Å. Difference Fourier maps showed that BPPM binds PTP1B in two mutually exclusive modes, one in which it occupies the canonical pTyr-binding site (the active site), and another in which a phosphophenyl moiety interacts with a set of residues not previously observed to bind aryl phosphates. The identification of a second pTyr molecule at the same site in the PTP1B͞C215S-pTyr complex confirms that these residues constitute a low-affinity noncatalytic aryl phosphate-binding site. Identification of a second aryl phosphate binding site adjacent to the active site provides a paradigm for the design of tight-binding, highly specific PTP1B inhibitors that can span both the active site and the adjacent noncatalytic site. This design can be achieved by tethering together two small ligands that are individually targeted to the active site and the proximal noncatalytic site.Protein-tyrosine phosphatases (PTPases), working in concert with protein-tyrosine kinases, regulate a vast array of cellular events, including passage through the cell cycle, proliferation and differentiation, metabolism, cytoskeletal organization, neuronal development, and the immune response (1, 2). PTPases constitute a large family of enzymes that parallel protein-tyrosine kinases in their structural diversity and complexity. A recent estimate suggests that as many as 500 PTPase genes may be encoded within the human genome (2). In vivo, PTPases catalyze the removal of the phosphoryl group from phosphotyrosine (pTyr) residue(s) in protein substrates. However, the K m value for free pTyr is three to four orders of magnitude higher than the best protein͞peptide substrates (3, 4). Several groups have demonstrated that PTPases display a range of k cat ͞K m values for pTyr-containing peptides. This sequence specificity has been exploited in the design of potent and selective PTPase inhibitors by incorporating a nonhydrolyzable pTyr analog into optimal peptide templates (5). However, owing to proteolytic susceptibility and weak partitioning across the plasma membrane, peptide-based compounds are not highly desirable for the development of medicinally effective drugs.As an initial step toward the development of selective, low-molecular weight nonpeptidic PTPase inhibitors, we investigated the active site substrate specificity of PTP1, the rat structural homologue of human protein-tyrosine phosphatase 1B (PTP1B). PTP1B (3) is the prototypical intracellular PTPase and is found in a wide varie...

Biological systems are characterized by a level of spatial and temporal organization that often lies beyond the grasp of present day methods. Light-modulated bioreagents, including analogs of low molecular weight compounds, peptides, proteins, and nucleic acids, represent a compelling strategy to probe, perturb, or sample biological phenomena with the requisite control to address many of these organizational complexities. Although this technology has created considerable excitement in the chemical community, its application to biological questions has been relatively limited. We describe the challenges associated with the design, synthesis, and use of light-responsive bioreagents, the scope and limitations associated with the instrumentation required for their application, and recent chemical and biological advances in this field.

In metastatic rat mammary adenocarcinoma cells, cell motility can be induced by epidermal growth factor. One of the early events in this process is the massive generation of actin barbed ends, which elongate to form filaments immediately adjacent to the plasma membrane at the tip of the leading edge. As a result, the membrane moves outward and forms a protrusion. To test the involvement of ADF/cofilin in the stimulus-induced barbed end generation at the leading edge, we inhibited ADF/cofilin's activity in vivo by increasing its phosphorylation level using the kinase domain of LIM-kinase 1 (GFP-K). We report here that expression of GFP-K in rat cells results in the near total phosphorylation of ADF/cofilin, without changing either the G/F-actin ratio or signaling from the EGF receptor in vivo. Phosphorylation of ADF/cofilin is sufficient to completely inhibit the appearance of barbed ends and lamellipod protrusion, even in the continued presence of abundant G-actin. Coexpression of GFP-K, together with an active, nonphosphorylatable mutant of cofilin (S3A cofilin), rescues barbed end formation and lamellipod protrusion, indicating that the effects of kinase expression are caused by the phosphorylation of ADF/cofilin. These results indicate a direct role for ADF/cofilin in the generation of the barbed ends that are required for lamellipod extension in response to EGF stimulation.

Single-cell analysis has revealed that transcription is dynamic and stochastic, but tools are lacking that can determine the mechanism operating at a single gene. Here we utilize single-molecule observations of RNA in fixed and living cells to develop a single-cell model of steroid-receptor mediated gene activation. We determine that steroids drive mRNA synthesis by frequency modulation of transcription. This digital behavior in single cells gives rise to the well-known analog dose response across the population. To test this model, we developed a light-activation technology to turn on a single steroid-responsive gene and follow dynamic synthesis of RNA from the activated locus.DOI: http://dx.doi.org/10.7554/eLife.00750.001

Protein-tyrosine phosphatases (PTPases) form a large family of enzymes that serve as key regulatory components in signal transduction pathways. Defective or inappropriate regulation of PTPase activity leads to aberrant tyrosine phosphorylation, which contributes to the development of many human diseases including cancers and diabetes. For example, recent gene knockout studies in mice identify PTP1B as a promising target for anti-diabetes/obesity drug discovery. Thus, there is intense interest in obtaining specific and potent PTPase inhibitors for biological studies and pharmacological development. However, given the highly conserved nature of the PTPase active site, it is unclear whether selectivity in PTPase inhibition can be achieved. We describe a combinatorial approach that is designed to target both the active site and a unique peripheral site in PTP1B. Compounds that can simultaneously associate with both sites are expected to exhibit enhanced affinity and specificity. We also describe a novel affinitybased high-throughput assay procedure that can be used for PTPase inhibitor screening. The combinatorial library/high-throughput screen protocols furnished a small molecule PTP1B inhibitor that is both potent (K i ‫؍‬ 2.4 nM) and selective (little or no activity against a panel of phosphatases including Yersinia PTPase, SHP1, SHP2, LAR, HePTP, PTP␣, CD45, VHR, MKP3, Cdc25A, Stp1, and PP2C). These results demonstrate that it is possible to acquire potent, yet highly selective inhibitors for individual members of the large PTPase family of enzymes.The initiation, propagation, and termination of signaling events controlling many cellular processes are determined by the level of tyrosine phosphorylation. Phosphotyrosine level, in turn, is maintained in an exquisite balance by the reciprocal activities of protein-tyrosine kinases and protein-tyrosine phosphatases (PTPases).

Protein-tyrosine phosphatase 1B (PTP1B) has been implicated as an important regulator in several signaling pathways including those initiated by insulin and leptin. Potent and specific PTP1B inhibitors could serve as useful tools in elucidating the physiological functions of PTP1B and may constitute valuable therapeutics in the treatment of several human diseases. We have determined the crystal structure of PTP1B in complex with compound 2, the most potent and selective PTP1B inhibitor reported to date. The structure at 2.15-Å resolution reveals that compound 2 simultaneously binds to the active site and a unique proximal noncatalytic site formed by Lys-41, Arg-47, and Asp-48. The structural data are further corroborated by results from kinetic analyses of the interactions of PTP1B and its site-directed mutants with compound 2 and several of its variants. Although many of the residues important for interactions between PTP1B and compound 2 are not unique to PTP1B, the combinations of all contact residues differ between PTP isozymes, which provide a structural basis for potent and selective PTP1B inhibition. Our data further suggest that potent, yet highly selective, PTP1B inhibitory agents can be acquired by targeting the area defined by residues Lys-41, Arg-47, and Asp-48, in addition to the previously identified second aryl phosphate-binding pocket.