Signalling scaffolds and local organization of cellular behaviour

Abstract

Cellular responses to environmental cues involve the mobilization of GTPases, protein kinases and phosphoprotein phosphatases. The spatial organization of these signalling enzymes by scaffold proteins helps to guide the flow of molecular information. Allosteric modulation of scaffolded enzymes can alter their catalytic activity or sensitivity to second messengers in a manner that augments, insulates or terminates local cellular events. This Review examines the features of scaffold proteins and highlights examples of locally organized groups of signalling enzymes that drive essential physiological processes, including hormone action, heart rate, cell division, organelle movement and synaptic transmission.

Figure 1. Properties of adaptor, docking and scaffold proteins

The figure depicts the distinguishing features of signal-organizing proteins and of scaffold proteins. Adaptor proteins, such as growth factor receptor-bound protein 2 (GRB2) (part a), and membrane-associated docking proteins, such as insulin receptor substrate 1 (IRS1) (part b), are both composed of protein-interaction modules that recruit signalling enzymes near transmembrane receptors. Essential features of scaffold proteins (shown in green) include the ability to hold in place successive members of a signalling cascade (part c), focus enzyme activity at a particular site of action (part d) and provide a molecular platform for the coordinated regulation of a particular effector protein by signal transduction and signal termination enzymes (part e). Phosphate groups are depicted as orange circles. PDK1, phosphoinositide-dependent kinase 1; PH, pleckstrin homology domain; PTB, phosphotyrosine-binding domain; SH2, SRC homology 2; SOS, son of sevenless.

Figure 2. Scaffold functions of pseudokinases and pseudophosphatases

Three prototypical examples of pseudokinase and pseudophosphatase function are shown. These non-catalytic proteins (shown in green) function as enzyme scaffolds and also allosterically modulate the activity of signalling enzymes. a | The tumour suppressor protein liver kinase B1 (LKB1) and the pseudokinase scaffold protein STRAD form a complex with MO25 (REF. 22). This heterotrimeric complex induces the ATP-bound STRAD to adopt a closed conformation that is reminiscent of an active protein kinase and that is able to activate LKB1. Interaction with MO25 stabilizes the active conformation of LKB1 (REF. 33). b | Kinase suppressor of RAS (KSR) is a multifunctional binding protein that brings together the three members of the conventional RAF–MEK–ERK–MAPK cascade^,. c | The pseudophosphatase serine/threonine/tyrosine-interacting protein (STYX) competes with the active dual-specificity phosphatase 4 (DUSP4) for binding to ERK1 and ERK2 in the nucleus. Through this mechanism, STYX traps active elements of the ERK cascade in the nucleus. Phosphate groups are depicted as orange circles.

Figure 3. Local coordination of second-messenger signalling by A-kinase anchor proteins

A-kinase anchor proteins (AKAPs) constrain protein kinase A (PKA) and other second-messenger-regulated signalling enzymes to form macromolecular units. a| A composite negative-stain electron microscopy image (class average) o f the intact type II PKA– AKAP18γ complex is shown. b | Three-dimensional reconstructions reveal that the complex has a heteropentameric protein assembly (containing one AKAP18 subunit, two regulatory type II (RII) subunits of PKA and two catalytic (C) subunits of PKA). The flexible tripartite configuration enables the associated catalytic subunits to have a radius of motion of up to 300 Å. c | Mouse AKAP150 is a multifunctional anchor protein that coordinates different combinations of second-messenger-regulate d enzymes. The figure shows an assembly of PKA and serine/threonine protein phosphatase 2B (PP2B) maintained by AKAP150. A structural model of the interface between AKAP150 and a pair of PP2B holoenzymes shows that protein–protein interactions occur through a modified PIXIT phosphatase-interaction motif^,. d | An established role for AKAP150 is the modulation of the phosphorylation events that control glutamate receptor ion channels. AKAP150-associated PKA and PP2B provide bidirectional regulation of glutamate receptor 1 (GluR1) phosphorylation at Ser845. Anchored PKA-mediated phosphorylation of GluR1 at Ser845 augments the membrane insertion of GluR1 at dendrites. PP2B–mediated dephosphorylation of GluR1 at this site reverses this process. e | In other cellular contexts, AKAP150 coordinates metabolic signalling events. AKAP150-associated PP2B activity modulates aspects of the insulin-responsive PI3K–phosphoinositide-dependent kinase 1 (PDK1)–AKT signalling cascade in skeletal muscle to control insulin sensitivity. Scaffold proteins are depicted in green; phosphate groups are depicted as orange circles. IRS1, insulin receptor substrate 1. Parts a and b are adapted from REF. , eLife Sciences Publications. Part c is adapted with permission from REF. , John Wiley & Sons.

Figure 4. Signal termination scaffolds

Scaffold proteins that target phosphatases and the enzymes that control protein ubiquitylation, acetylation and deacetylation are shown. a | A ribbon diagram of serine/threonine protein phosphatase 1 (PP1) catalytic subunit (turquoise) in a complex with a KVXF-motif peptide derived from the glycogen-targeting subunit G_M (green) is shown. Important regions for peptide binding are shown in red. The peptide-binding channel of PP1 lies at the interface of the two β-sheets of a β-sandwich. b | An example of a higher-order PP1 scaffold is depicted. A phosphatase–kinase scaffold tightly modulates smooth muscle contraction. The proximity of the activator protein kinase G (PKG) and the inhibitory RHO-associated protein kinase 1 (ROCK1) provides bidirectional control of PP1 when tethered to its scaffold protein, the myosin-targeting PP1 subunit MYPT1, and the myosin light chains (MLCs). c | A second example of a higher-order PP1 scaffold is shown. When in a complex with MRAS, the phosphatase-targeting subunit SHOC2 competes with another scaffold protein, SCRIB, for interaction with PP1. Thus, ERK signalling is antagonized by the SHOC2-mediated dephosphorylation of RAF and is facilitated when the phosphatase is sequestered by SCRIB. Ubiquitylation and acetylation are important molecular switches controlled by the protein–protein interactions within scaffolds. d | By recognizing the exposed hydrophobicity of misfolded proteins, the yeast E3 ligase San1 functions as a scaffold and is able to target misfolded proteins for ubiquitylation and proteasomal degradation. e | The scaffold formed by the phosphoenolpyruvate carboxykinase 1 (PEPCK1) efficiently responds to changes in glucose levels through its associated metabolic enzymes. In the presence of high levels of glucose, the associated acetyltransferase p300 acetylates PEPCK1, targeting it for ubiquitylation and subsequent degradation by the E3 ubiquitin protein ligase UBR5 that is recruited to the complex. When the glucose level is low, the deacetylase sirtuin 2 (SIRT2) blocks this process. Acetyl groups are depicted as red circles, phosphate groups are depicted as orange circles, and ubiquitin groups are depicted as blue circles. NO, nitric oxide. Part a is adapted with permission from REF. , John Wiley & Sons.

Figure 5. Scaffold proteins that function as molecular switches

MAPK scaffold proteins in yeast and mammals are depicted. a | The yeast scaffold protein Ste5 assembles a MAPK cascade that includes Ste11, Ste7 and Fus3 at the cell membrane. However, phosphorylation of Ste5 by cyclin-dependent kinase (Cdk) inhibits this process^,. b | The JUN amino-terminal kinase (JNK) and p38 MAPK scaffold proteins of the JNK-interacting protein (JIP) family associate with motor proteins to transport various cargo proteins along microtubules. Phosphorylation of Ser421 on JIP1 links the mixed lineage kinase 3 (MLK3)–MEK7–JNK cascade to the motor protein kinesin for forward motion along the microtubule, whereas dephosphorylation of this residue functions as a switch for retrograde movement facilitated by interaction with dynactin (not shown). Phosphate groups are depicted as orange circles. GPCR, G protein-coupled receptor.

Figure 6. Emergent technologies for the analysis of signalling scaffolds

Three emergent biophysical approaches have the potential to enhance the molecular dissection of macromolecular signalling scaffolds. Native mass spectrometry enables the investigation of intact protein complexes^,. This top-down mass spectrometry approach probes the quaternary structure of protein complexes suspended in volatile buffers, thus enabling accurate calculation of their mass. An A-kinase anchoring protein 79 (AKAP79)–kinase–phosphatase sub-complex was first assessed by SDS–PAGE (part a). Next, the molecular mass of the sub-complex was derived by native mass spectrometry (part b). The quantitative information that was obtained was used to determine a more refined model of the quaternary structure of this macromolecular complex (part c). Single-molecule pull-down photobleaching (SiMPull) is a sensitive new assay that combines a modified pull-down assay (part d) with single-molecule photobleaching of fluorescently tagged proteins (part e) to enable direct analysis of individual protein complexes. SiMPull can be used to derive the ratio of distinct protein complexes assembled on a specific population of scaffold protein. Electron microscopes have sufficient resolving power for structural studies of macromolecules. Recent technical innovations, including a new generation of direct-detection camera and the development of more sophisticated data-processing packages, have markedly increased the resolution of cryo-electron microscopy for higher-order macromolecular complexes^,. Smaller complexes (molecular weight (MW) <300,000 kDa) can be resolved by negative-stain electron microscopy (part f). Class averages of negative-stain electron microscopy particles show three perspectives of the AKAP18γ–protein kinase A (PKA) complex. The density map of the AKAP18γ–PKA holoenzyme complex can be overlaid with structural models from the RCSB Protein Data Bank (PDB) coordinates for AKAP18γ (PDB code: 3J4Q), and the type IIα regulatory (RIIα) and catalytic (C) subunits of PKA to enable pseudo-atomic modelling (part g). CaM, calmodulin; FLAG, DYKDDDDK polypeptide tag; m/z, mass-to-charge ratio; PEG, polyethylene glycol; PP2B, protein phosphatase 2B; TIRF, total internal reflection fluorescence; YFP, yellow fluorescent protein. Parts a and b are adapted from REF. , US National Academy of Sciences; part e is adapted from REF. , US National Academy of Sciences; and part g is adapted from REF. , eLife Sciences Publications.