Daniel Abankwa

PTRF-Cavin, a Conserved Cytoplasmic Protein Required for Caveola Formation and Function

Caveolae are abundant cell-surface organelles involved in lipid regulation and endocytosis. We used comparative proteomics to identify PTRF (also called Cav-p60, Cavin) as a putative caveolar coat protein. PTRF-Cavin selectively associates with mature caveolae at the plasma membrane but not Golgi-localized caveolin. In prostate cancer PC3 cells, and during development of zebrafish notochord, lack of PTRF-Cavin expression correlates with lack of caveolae, and caveolin resides on flat plasma membrane. Expression of PTRF-Cavin in PC3 cells is sufficient to cause formation of caveolae. Knockdown of PTRF-Cavin reduces caveolae density, both in mammalian cells and in the zebrafish. Caveolin remains on the plasma membrane in PTRF-Cavin knockdown cells but exhibits increased lateral mobility and accelerated lysosomal degradation. We conclude that PTRF-Cavin is required for caveola formation and sequestration of mobile caveolin into immobile caveolae.

Cells Respond to Mechanical Stress by Rapid Disassembly of Caveolae

The functions of caveolae, the characteristic plasma membrane invaginations, remain debated. Their abundance in cells experiencing mechanical stress led us to investigate their role in membrane-mediated mechanical response. Acute mechanical stress induced by osmotic swelling or by uniaxial stretching results in a rapid disappearance of caveolae, in a reduced caveolin/Cavin1 interaction, and in an increase of free caveolins at the plasma membrane. Tether-pulling force measurements in cells and in plasma membrane spheres demonstrate that caveola flattening and disassembly is the primary actin- and ATP-independent cell response that buffers membrane tension surges during mechanical stress. Conversely, stress release leads to complete caveola reassembly in an actin- and ATP-dependent process. The absence of a functional caveola reservoir in myotubes from muscular dystrophic patients enhanced membrane fragility under mechanical stress. Our findings support a new role for caveolae as a physiological membrane reservoir that quickly accommodates sudden and acute mechanical stresses.

Mechanism of Activation of Protein Kinase JAK2 by the Growth Hormone Receptor

Introduction Class I cytokines regulate key processes such as growth, lactation, hematopoiesis, and immune function and contribute to oncogenesis. Although the extracellular domain structures of their receptors are well characterized, little is known about how the receptors activate their associated JAK (Janus kinase) protein kinases. We provide a mechanistic description for this process, focusing on the growth hormone (GH) receptor and its associated JAK2. Receptor-JAK2 activation process. (Top) Cartoons of the GH receptor basal state (state 1, left) and the active state (state 2, right) with (Bottom) transmembrane helix alignments for these states derived by modeling. GHR, GH receptor. Rationale We tested whether the receptor exists as a dimer in the inactive state by homo-FRET [fluorescence resonance energy transfer (FRET) between the proteins labeled with the same fluorophore] and other means. Then, to define receptor movements resulting from activation, we attached FRET reporters to the receptor below the cell membrane and correlated their movement with receptor activation, measured as increased cell proliferation. We controlled the position of the transmembrane helices with leucine zippers and mutagenesis, and we again monitored FRET and receptor activation. We used cysteine cross-linking data to define the faces of the transmembrane helices in contact in the basal state and verified this with molecular dynamics, which allowed us to model the activation process. We also used FRET reporters to monitor the movement of JAK2, and we matched this with molecular dynamics docking of the crystal structures of the kinase and its pseudokinase domains to derive a model for activation, which we then verified experimentally. Results We found that the GH receptor exists predominantly as a dimer in vivo, held together by its transmembrane helices. These helices are parallel in the basal state, and binding of the hormone converts them into a left-hand crossover state that induces separation of helices at the lower transmembrane boundary (hence, Box1 separation). This movement is triggered by increased proximity of the juxtamembrane sequences, a consequence of locking together of the lower module of the extracellular domain on hormone binding. This movement is triggered by increased proximity of the juxtamembrane sequences , a Both this locking and the helix state transition require rotation of the receptors, but the key outcome is separation of the Box1 sequences. Because these sequences are bound to the JAK2 FERM (4.1, ezrin, radixin, moesin) domains, this separation results in removal of the pseudokinase inhibitory domain of one JAK2, which is blocking the kinase domain of the other JAK2, and vice versa. This brings the two kinase domains into productive apposition, triggering JAK2 activation. We verified this mechanism by kinase-pseudokinase domain swap, by changes in JAK2 FRET signal on activation, by showing association of pseudokinase-kinase domain pairs, and by docking of the crystal structures. An animation of our complete model of GH receptor activation is provided at http://web-services.imb.uq.edu.au/waters/hgh.html. Conclusion The proposed mechanism will be useful in understanding the many actions of GH, which include altered growth, metabolism, and bone turnover. We expect that it may extend to other members of this important receptor family. The mechanism provides a molecular basis for understanding the oncogenic JAK2 mutations responsible for polycythemia vera and certain other hematologic disorders and may thus be of value in the design of small-molecule inhibitors of clinical applicability. Signaling from JAK (Janus kinase) protein kinases to STAT (signal transducers and activators of transcription) transcription factors is key to many aspects of biology and medicine, yet the mechanism by which cytokine receptors initiate signaling is enigmatic. We present a complete mechanistic model for activation of receptor-bound JAK2, based on an archetypal cytokine receptor, the growth hormone receptor. For this, we used fluorescence resonance energy transfer to monitor positioning of the JAK2 binding motif in the receptor dimer, substitution of the receptor extracellular domains with Jun zippers to control the position of its transmembrane (TM) helices, atomistic modeling of TM helix movements, and docking of the crystal structures of the JAK2 kinase and its inhibitory pseudokinase domain with an opposing kinase-pseudokinase domain pair. Activation of the receptor dimer induced a separation of its JAK2 binding motifs, driven by a ligand-induced transition from a parallel TM helix pair to a left-handed crossover arrangement. This separation leads to removal of the pseudokinase domain from the kinase domain of the partner JAK2 and pairing of the two kinase domains, facilitating trans-activation. This model may well generalize to other class I cytokine receptors. A molecular mechanism for transmembrane signaling by the growth hormone receptor is elucidated. [Also see Perspective by Wells and Kossiakoff] The Hormones Message The receptor for growth hormone is a well-studied representative of a family of cytokine receptors through which binding of hormone molecules at the cell surface is converted into a biochemical signal within the cell. Brooks et al. (10.1126/science.1249783; see the Perspective by Wells and Kossiakoff) used a combination of crystal structures, biophysical measurements, cell biology experiments with modified receptors, and molecular dynamics and modeling to decipher how the receptor actually transmits the information that a hormone molecule is bound. The results suggest that the receptors exist in inactive dimeric complexes in which two associated JAK2 protein kinase molecules interact in an inhibitory manner. Binding of growth hormone causes a structural change in the receptor that results in movement of the receptor intracellular domains apart from one another. This relieves the inhibition of the JAK2 molecules and allows them to activate one another, thus initiating the cellular response to the hormone.

MURC/Cavin-4 and cavin family members form tissue-specific caveolar complexes

Polymerase I and transcript release factor (PTRF)/Cavin is a cytoplasmic protein whose expression is obligatory for caveola formation. Using biochemistry and fluorescence resonance energy transfer–based approaches, we now show that a family of related proteins, PTRF/Cavin-1, serum deprivation response (SDR)/Cavin-2, SDR-related gene product that binds to C kinase (SRBC)/Cavin-3, and muscle-restricted coiled-coil protein (MURC)/Cavin-4, forms a multiprotein complex that associates with caveolae. This complex can constitutively assemble in the cytosol and associate with caveolin at plasma membrane caveolae. Cavin-1, but not other cavins, can induce caveola formation in a heterologous system and is required for the recruitment of the cavin complex to caveolae. The tissue-restricted expression of cavins suggests that caveolae may perform tissue-specific functions regulated by the composition of the cavin complex. Cavin-4 is expressed predominantly in muscle, and its distribution is perturbed in human muscle disease associated with Caveolin-3 dysfunction, identifying Cavin-4 as a novel muscle disease candidate caveolar protein.

A novel switch region regulates H‐ras membrane orientation and signal output

The plasma membrane nanoscale distribution of H‐ras is regulated by guanine nucleotide binding. To explore the structural basis of H‐ras membrane organization, we combined molecular dynamic simulations and medium‐throughput FRET measurements on live cells. We extracted a set of FRET values, termed a FRET vector, to describe the lateral segregation and orientation of H‐ras with respect to a large set of nanodomain markers. We show that mutation of basic residues in helix α4 or the hypervariable region (HVR) selectively alter the FRET vectors of GTP‐ or GDP‐loaded H‐ras, demonstrating a critical role for these residues in stabilizing GTP‐ or GDP‐H‐ras interactions with the plasma membrane. By a similar analysis, we find that the β2–β3 loop and helix α5 are involved in a novel conformational switch that operates through helix α4 and the HVR to reorient the H‐ras G‐domain with respect to the plasma membrane. Perturbation of these switch elements enhances MAPK activation by stabilizing GTP‐H‐ras in a more productive signalling conformation. The results illustrate how the plasma membrane spatially constrains signalling conformations by acting as a semi‐neutral interaction partner.

Ras membrane orientation and nanodomain localization generate isoform diversity

The structural elements encoding functional diversity among Ras GTPases are poorly defined. The orientation of the G domain of H-ras with respect to the plane of the plasma membrane is recognized by the Ras binding domain of C-Raf, coupling orientation to MAPK activation. We now show that two other proteins, phosphoinositide-3-kinase-α and the structurally unrelated galectin-1, also recognize G-domain orientation. These results rationalize the role of galectin-1 in generating active GTP-H-ras signaling nanoclusters. However, molecular dynamics simulations of K-ras membrane insertion and fluorescence lifetime imaging microscopy (FLIM)-Förster resonance energy transfer (FRET) imaging of the effector interactions of N-Ras, K-Ras, and M-ras suggest that there are two hyperactive, signaling-competent orientations of the Ras G domain. Mutational and functional analyses establish a clear relationship between effector binding and the amphilicities of helix α4 and the C-terminal hypervariable region, thus confirming that these structural elements critically tune the orientation of the Ras G domain. Finally, we show that G-domain orientation and nanoclustering synergize to generate Ras isoform specificity with respect to effector interactions.

ColonyArea: an ImageJ plugin to automatically quantify colony formation in clonogenic assays.

The clonogenic or colony formation assay is a widely used method to study the number and size of cancer cell colonies that remain after irradiation or cytotoxic agent administration and serves as a measure for the anti-proliferative effect of these treatments. Alternatively, this assay is used to quantitate the transforming potential of cancer associated genes and chemical agents. Therefore, there is a need for a simplified and standardized analysis of colony formation assays for both routine laboratory use and for parallelized automated analysis. Here we describe the freely available ImageJ-plugin “ColonyArea”, which is optimized for rapid and quantitative analysis of focus formation assays conducted in 6- to 24-well dishes. ColonyArea processes image data of multi-well dishes, by separating, concentrically cropping and background correcting well images individually, before colony formation is quantitated. Instead of counting the number of colonies, ColonyArea determines the percentage of area covered by crystal violet stained cell colonies, also taking the intensity of the staining and therefore cell density into account. We demonstrate that these parameters alone or in combination allow for robust quantification of IC50 values of the cytotoxic effect of two staurosporines, UCN-01 and staurosporine (STS) on human glioblastoma cells (T98G). The relation between the potencies of the two compounds compared very well with that obtained from an absorbance based method to quantify colony growth and to published data. The ColonyArea ImageJ plugin provides a simple and efficient analysis routine to quantitate assay data of one of the most commonly used cellular assays. The bundle is freely available for download as supporting information. We expect that ColonyArea will be of broad utility for cancer biologists, as well as clinical radiation scientists.

Mechanisms of Ras membrane organization and signalling: Ras on a rocker.

Understanding the signalling function of Ras GTPases has been the focus of much research for over 20 years. Both the catalytic domain and the membrane anchoring C terminal hypervariable region (HVR) of Ras are necessary for its cellular function. However, while the highly conserved catalytic domain has been characterized in atomic detail, the structure of the full-length membrane-bound Ras has remained elusive. Lack of structural knowledge on the full-length protein limited our understanding of Ras signalling. For example, structures of the Ras catalytic domain solved in complex with effectors do not provide a basis for the functional specificity of different Ras isoforms. Recent molecular dynamics simulations in combination with biophysical and cell biological experiments have shown that the HVR and parts of the G domain cofunction with the lipid tails to anchor H-ras to the plasma membrane. In the GTP-bound state, H-ras adopts an orientation that allows read out by Ras effectors and translation into corresponding MAPK signalling. Here we discuss details of an analysis that suggests a novel balance model for Ras functioning. The balance model rationalizes Ras membrane orientation and may help explain isoform specific interactions of Ras with its effectors and modulators.

A FRET map of membrane anchors suggests distinct microdomains of heterotrimeric G proteins.

The standard model of heterotrimeric G protein signaling postulates a dissociation of Gα and Gβγ subunits after activation. We hypothesized that the different combination of lipid-modifications on Gα and Gαβγ subunits directs them into different microdomains. By characterizing rapidly and at high sensitivity 38 fluorescence resonance energy transfer (FRET) pairs of heterotrimeric-G-protein constructs, we defined their microdomains in relation to each other, free from the constraints of the raft/non-raft dualism. We estimated that in a cell ∼30% of these membrane-anchored proteins are mostly clustered in 3400-16,200 copies of 30-nm microdomains. We found that the membrane anchors of Gα and Gαβγ subunits of both the Gi/o and Gq family co-cluster differently with microdomain markers. Moreover, anchors of the Gαi/o and Gαq subunits co-clustered only weakly, whereas constructs that contained the anchors of the corresponding heterotrimers co-clustered considerably, suggesting the existence of at least three types of microdomain. Finally, FRET experiments with full-length heterotrimeric G proteins confirmed that the inactive, heterotrimerized Gα subunit is in microdomains shared by heterotrimers from different subclasses, from where it displaces upon activation into a membrane-anchor- and subclass-specific microdomain.

Quantitative Analysis of Prenylated RhoA Interaction with Its Chaperone, RhoGDI.

Background: RhoGDI is a key regulator and a chaperon of Rho GTPases. Results: RhoGDI strongly discriminates between GDP- and GTP-bound forms of prenylated RhoA, although both complexes are of high affinity. Conclusion: We provide direct evidence for the existence of two populations of the RhoGDI·RhoA complexes in the cell, characterized by different lifetimes. Significance: The obtained data allows us to formulate the model for membrane delivery and extraction of Rho GTPases. Small GTPases of the Rho family regulate cytoskeleton remodeling, cell polarity, and transcription, as well as the cell cycle, in eukaryotic cells. Membrane delivery and recycling of the Rho GTPases is mediated by Rho GDP dissociation inhibitor (RhoGDI), which forms a stable complex with prenylated Rho GTPases. We analyzed the interaction of RhoGDI with the active and inactive forms of prenylated and unprenylated RhoA. We demonstrate that RhoGDI binds the prenylated form of RhoA·GDP with unexpectedly high affinity (Kd = 5 pm). The very long half-life of the complex is reduced 25-fold on RhoA activation, with a concomitant reduction in affinity (Kd = 3 nm). The 2.8-Å structure of the RhoA·guanosine 5′-[β,γ-imido] triphosphate (GMPPNP)·RhoGDI complex demonstrated that complex formation forces the activated RhoA into a GDP-bound conformation in the absence of nucleotide hydrolysis. We demonstrate that membrane extraction of Rho GTPase by RhoGDI is a thermodynamically favored passive process that operates through a series of progressively tighter intermediates, much like the one that is mediated by RabGDI.