Rucete ✏ Lehninger Principles of Biochemistry In a Nutshell
12.5 Multivalent Adaptor Proteins and Membrane Rafts
This chapter explains how cells organize signaling pathways through modular protein-binding domains, reversible phosphorylation, scaffold proteins, phosphatases, and specialized membrane microdomains called membrane rafts and caveolae. Signaling is not controlled only by enzymes, but also by spatial organization and assembly of multiprotein complexes.
Two Major Principles Learned from Signaling Systems
• Studies of signaling pathways reveal two broad principles.
• First, protein kinases that phosphorylate Tyr, Ser, and Thr residues—and phosphatases that remove those phosphates—are central regulators of signaling.
• These enzymes directly alter the activity of many substrate proteins.
• Second, reversible phosphorylation creates binding sites for other proteins.
• These protein-protein interactions generate indirect downstream effects in signaling pathways.
Multivalent Signaling Proteins
• Many signaling proteins are multivalent.
• Multivalent means one protein can interact simultaneously with several different partner proteins.
• This allows formation of multiprotein signaling complexes.
• Such complexes improve efficiency, specificity, amplification, and integration of signals.
Intrinsically Disordered Regions (IDRs)
• Many proteins involved in signaling contain intrinsically disordered regions (IDRs).
• IDRs are flexible segments lacking one fixed structure.
• Because of this flexibility, they can specifically bind more than one partner protein.
• Some IDRs may interact with many different proteins depending on context.
IDRs in Protein Kinases
• Protein kinases possess conserved catalytic and substrate-binding cores.
• During evolution, many kinases acquired extra sequences usually about 20–30 amino acids long.
• These added segments are often partially disordered.
• They help kinases participate in multienzyme regulatory networks.
Activation Loop as a Universal Regulator
• Most protein kinases contain an activation loop.
• The activation loop is an IDR.
• Phosphorylation of one or more residues changes its position.
• This movement turns kinase activity on.
• Therefore the activation loop is a universal regulator of kinase activity.
Other Kinase Regulatory Tails
• PKA, PKB, and PKC family kinases contain disordered carboxyl-terminal tails.
• Critical residues in these tails can be phosphorylated or dephosphorylated.
• Their phosphorylation state helps switch between active and inactive structures.
• In MAPK cascades, amino-terminal IDRs can act as docking regions within multienzyme complexes.
Huge Combinatorial Diversity
• Humans have about 1,000 protein kinase genes.
• Combined with many scaffold proteins and numerous IDR-based interactions, the number of possible signaling combinations is extremely large.
• This gives cells powerful control over metabolic regulation.
Protein Modules Bind Phosphorylated Residues
• Many signaling proteins contain domains specialized to recognize phosphorylated amino acids in partner proteins.
• These domains read phosphorylation signals and recruit proteins into complexes.
SH2 Domains Bind Phosphotyrosine
• Grb2 in insulin signaling is one example of an SH2-containing protein.
• Its SH2 domain binds proteins containing exposed phosphotyrosine residues.
• The human genome encodes at least 87 SH2-containing proteins.
• Many participate in signaling pathways.
How SH2 Binding Works
• The phosphotyrosine residue fits into a deep pocket of the SH2 domain.
• Phosphate oxygens participate in hydrogen bonds and electrostatic interactions.
• Two Arg residues commonly provide positive charge important for binding.
• Small structural differences among SH2 domains create specificity.
Sequence Specificity of SH2 Domains
• SH2 domains bind not only phosphotyrosine (position 0), but also nearby residues toward the carboxyl terminus.
• Different SH2 proteins prefer different neighboring amino acid sequences.
• Therefore each SH2 domain binds only selected phosphorylated targets.
Examples of SH2 Specificity
• Src, Fyn, Hck, and Nck favor negatively charged residues in certain adjacent positions.
• PLCγ1 and SHP2 contain hydrophobic grooves that prefer aliphatic residues in nearby positions.
• These structural differences create subclasses of SH2 domains.
PTB Domains
• Phosphotyrosine-binding (PTB) domains are another class of phosphotyrosine-recognition domains.
• PTB domains differ structurally from SH2 domains.
• The human genome encodes at least 24 PTB-containing proteins.
• IRS1 contains a PTB domain and functions in insulin signaling.
Creation and Removal of Binding Sites
• Tyr kinases create phosphotyrosine docking sites.
• Protein tyrosine phosphatases (PTPs) remove those phosphates.
• Thus cells can rapidly build or dismantle signaling complexes.
Domains that Bind Phosphoserine and Phosphothreonine
• Kinases such as PKA, PKC, PKG, and MAPK cascade members phosphorylate Ser or Thr residues.
• Some proteins then gain the ability to bind partner proteins through those phosphorylated residues.
• Many classes of phospho-Ser/phospho-Thr binding domains exist.
• More are likely to be discovered.
• Each recognizes specific surrounding sequences.
Autoinhibition by Internal Phosphorylation
• In some proteins, a phospho-binding region is blocked by binding to a phosphorylated residue within the same protein.
• This creates autoinhibition.
Example: Src Kinase
• Src is a soluble Tyr kinase.
• When phosphorylated on a specific Tyr residue, its SH2 domain binds that internal phosphotyrosine.
• This prevents SH2 binding to substrate proteins.
• Src becomes inactive.
• Removal of the phosphate by a phosphatase reactivates Src.
Example: GSK3
• Glycogen synthase kinase 3 (GSK3) can be inactivated when a Ser residue in its autoinhibitory domain is phosphorylated.
• This internal phosphoserine blocks productive substrate binding.
• Dephosphorylation frees the enzyme to bind and phosphorylate targets.
A Fourth Phosphorylated Signaling Structure
• Besides Tyr, Ser, and Thr residues in proteins, another important phosphorylated signaling structure exists.
• It is the phosphorylated head group of membrane phosphatidylinositols.
• These lipids help nucleate supramolecular signaling complexes.
PH Domains and Membrane Recruitment
• Many signaling proteins contain PH (pleckstrin homology) domains.
• PH domains bind phosphorylated phosphatidylinositol head groups exposed on the cytoplasmic membrane surface.
• When PI3K generates these lipids, signaling proteins cluster at the membrane.
Many Membrane Signaling Proteins Are Multivalent
• Most signaling proteins at the plasma membrane contain one or more phosphoprotein- or phospholipid-binding domains.
• Many contain three or more such domains.
• This allows extensive simultaneous interactions.
Why Membrane Localization Helps
• Many signaling reactions occur on the inner surface of the plasma membrane.
• Molecules are confined to two-dimensional membrane space rather than three-dimensional cytosol.
• Collisions become much more likely.
• This increases signaling speed and efficiency.
Assembly of Large Signaling Complexes
• An initial signal often phosphorylates a receptor or target protein.
• This triggers assembly of large multiprotein complexes.
• These are often held together on scaffolds with multivalent binding capacity.
Kinase Cascades on Scaffolds
• Some complexes contain several kinases that activate one another sequentially.
• This produces phosphorylation cascades and large signal amplification.
• Scaffold proteins ensure kinases remain near each other.
Example: KSR in the MAPK Cascade
• In the MAPK pathway, scaffold protein KSR binds MAPKKK, MAPKK, and MAPK.
• This ensures correct proximity and orientation.
• KSR also provides allosteric effects on kinase interactions.
• As a result, serial phosphorylation becomes sensitive even to very small stimuli.
Feedback Regulation on Scaffolds
• Activated ERK can phosphorylate the Raf binding site on KSR.
• This conformational change displaces Raf.
• MEK phosphorylation then decreases.
• Thus scaffold proteins can regulate pathway timing and strength.
Protein Tyrosine Phosphatases (PTPs)
• Phosphotyrosine phosphatases remove phosphate from phosphotyrosine residues.
• They reverse effects of Tyr phosphorylation.
• At least 37 human genes encode PTPs.
Types of PTPs
• About half are receptor-like integral membrane proteins with one transmembrane domain.
• Their extracellular regulators are not fully identified.
• Other PTPs are soluble proteins.
• Many soluble PTPs contain SH2 domains that determine location and partners.
Ser/Thr Phosphatases
• Cells also contain Ser/Thr phosphatases such as PP1.
• These reverse actions of Ser/Thr-specific kinases.
Writers, Readers, and Erasers
• Signaling circuits can be viewed as information systems.
• Protein kinases are the writers because they add phosphate marks.
• Domains such as SH2 are the readers because they recognize phosphate marks.
• PTPs and other phosphatases are the erasers because they remove marks.
Lego-Like Signaling Modules
• Multivalent proteins allow assembly of many combinations of reusable signaling modules.
• Each combination can suit specific signals, cell types, and metabolic states.
• This creates signaling circuits of extraordinary complexity.
Membrane Rafts
• Membrane rafts are plasma membrane regions enriched in sphingolipids, sterols, and selected proteins.
• Many raft proteins are attached by GPI anchors.
• Rafts act as microdomains that concentrate signaling components.
β-Adrenergic Receptor in Rafts
• β-adrenergic receptors can localize in rafts containing G proteins, adenylyl cyclase, PKA, and PP2.
• Together these components form an integrated signaling unit.
• This allows a highly localized and brief puff of second messenger production.
RTKs in Rafts
• Some receptor tyrosine kinases such as EGFR and PDGFR are also found in rafts.
• Their localization likely has functional significance.
Caveolae
• Caveolae are specialized membrane rafts.
• In isolated fibroblasts, EGFR is commonly concentrated in caveolae.
EGF-Induced Receptor Movement
• When cells are treated with EGF, EGFR leaves the raft region.
• This separates it from other signaling components.
• The movement requires EGFR kinase activity.
• Mutant receptors lacking kinase activity remain in rafts even after EGF treatment.
Importance of Spatial Segregation
• Spatial organization of signaling proteins adds another level of regulation.
• Signaling depends not only on chemistry, but also on precise cellular location.
In a Nutshell
Cells organize signaling pathways through modular binding domains, phosphorylation marks, scaffold proteins, phosphatases, and membrane microdomains. SH2, PTB, PH, and related domains read phosphorylated proteins or lipids to assemble multiprotein complexes, while kinases write signals and phosphatases erase them. Membrane rafts and caveolae further concentrate receptors and enzymes, making signaling faster, more localized, and highly regulated.