Rapamycin-induced Dimerization Research Articles

Quantitative Description of Signaling Downstream of Gq-Coupled Receptors: Similarities and Differences in the Responses of IP3, Calcium, DAG, PKC, and PIP2

Gq-coupled plasma membrane receptors modulate cellular functions by activating phospholipase C (PLC), which hydrolyses the membrane lipid phosphatidylinositol (4,5)-bisphosphate (PIP2) into the second messengers inositoltrisphosphate (IP3) and diacylgycerol (DAG). To better understand the mechanisms that govern these partially independent signals we monitored in single, living tsA-201 cells levels of PIP2, IP3, calcium, DAG, and PKC by optical probes and current. We compared (i) activation of (low-abundance) endogenous purinergic receptors and overexpressed M1 muscarinic receptors, and (ii) different concentrations of the muscarinic agonist oxotremorine-M (oxo-M). Whereas the peak responses from reporters of IP3 (LIBRAvIII) and DAG (C1 domains of PKCγ) scale with abundance of receptor or agonist, downstream production of calcium (Fura4F) and PKC activation (CKAR) do not. Amplitude and duration of calcium signals elicited by 100 µM UTP, 10 nM oxo-M, or 10 µM oxo-M are almost identical. The only difference is a shorter latency with 10 µM oxo-M. These data suggest that a relatively low amount of IP3 is required for calcium release. This interpretation is supported by the finding that a full-size calcium response can still be elicited after PIP2 is depleted by recruiting a PI 5-phosphatase to the plasma membrane (by rapamycin-induced dimerization). Duration and late recovery time courses are different between IP3 (duration=68 s; τoff =55 s) and calcium (duration=110 s; τoff =34 s), suggesting that once a threshold of IP3 is reached, the calcium signal unfolds. Therefore we conclude that the IP3 requirement for calcium release must be low. The time point and IP3 level (from LIBRAvIII) at which the calcium response starts can provide an estimate of this IP3 threshold. Supported by NIH grants NS08174 & GM83913 and the HFSP.

Actin Comets Versus Membrane Ruffles: Distinctive Roles of Phosphoinositides in Actin Reorganization

Phosphoinositides, a group of lipids found in various cellular membranes, oversee diverse cellular functions including cell proliferation, differentiation, and cell migration. However, the question of how these lipids drive multiple cellular functions and differentially contribute to diseases mediated by aberrant phosphoinositide regulation remains unaddressed. Using a recently developed technique to rapidly manipulate the level of specific phosphoinositides, we aimed to elucidate a molecular mechanism underlying functional diversity achieved by a limited set of phosphoinositides. In particular, we developed techniques based on rapamycin-induced protein dimerization to rapidly change plasma membrane PIP2 in order to dissect the role of PIP2 in actin reorganization. We first increased plasma membrane PIP2 by rapamycin-induced recruitment of a PIP 5-kinase to the membrane, and found that cells form actin comets. Note that this manipulation increases PIP2 but depletes PI4P. We then developed a second technique that increases PIP2 without consuming PI4P, and found - surprisingly - that cells exhibit membrane ruffles. These distinctive phenotypes were critically regulated through two signaling events: 1) a crosstalk between the Rho GTPases Rac and RhoA, and 2) dynamin-mediated vesicular trafficking. We could convert actin comets into ruffles by perturbing these signaling events. As a proof-by-synthesis, we also demonstrated that actin comet formation can be reconstituted without changing the PIP2 level by combining manipulation of Rho GTPase activity with rapidly induced endocytosis. In summary, our results indicate that the role of PIP2 on actin reorganization depends on other phosphoinositides, such as PI4P. This combinatorial regulation may explicate the diversity of phosphoinositide functions.

Chemically Induced and Light-Independent Cryptochrome Photoreceptor Activation

The cryptochrome photoreceptors of higher plants are dimeric proteins. Their N-terminal photosensory domain mediates dimerization, and the unique C-terminal extension (CCT) mediates signaling. We made use of the human FK506-binding protein (FKBP) that binds with high affinity to rapamycin or rapamycin analogs (rapalogs). The FKBP-rapamycin complex is recognized by another protein, FRB, thus allowing rapamycin-induced dimerization of two target proteins. Here we demonstrate by bioluminescence resonance energy transfer (BRET) assays the applicability of this regulated dimerization system to plants. Furthermore, we show that fusion proteins consisting of the C-terminal domain of Arabidopsis cryptochrome 2 fused to FKBP and FRB and coexpressed in Arabidopsis cells specifically induce the expression of cryptochrome-controlled reporter and endogenous genes in darkness upon incubation with the rapalog. These results demonstrate that the activation of cryptochrome signal transduction can be chemically induced in a dose-dependent fashion and uncoupled from the light signal, and provide the groundwork for gain-of-function experiments to study specifically the role of photoreceptors in darkness or in signaling cross-talk even under light conditions that activate members of all photoreceptor families.

Small-molecule-mediated rescue of protein function by an inducible proteolytic shunt

Controlling protein function through posttranslational manipulations has emerged as an attractive complementary technology to existing genetic systems. Often these methods involve developing pharmacological agents to probe protein function without the need to generate a unique compound for each protein family. One common strategy uses small molecules that act as chemical inducers of dimerization by mediating the interaction of two proteins. Herein we report the use of a chemical inducer of dimerization for the development of a posttranslational technology for the manipulation of protein function. This system, split ubiquitin for the rescue of function (SURF), places the complementation of genetically split ubiquitin under the control of rapamycin-induced dimerization of FK506-binding protein and FKBP12-rapamycin-binding protein. Before complementation a "degron" dooms a protein of interest for destruction by the proteasome. Addition of rapamycin results in a proteolytic shunt away from degradation by inducing ubiquitin complementation and cleavage of the protein of interest from the degron. Importantly, the native protein is rescued. We characterized this system with firefly luciferase and went on to apply it to members of three important classes of proteins: proteases (caspase-3), kinases (v-Src), and transcription factors (Smad3). This general strategy should allow for inducible rescue of a variety of proteins in such a way that their native structure and function are maintained.