Multivalent Scaffold Proteins and Membrane Rafts:- Protein Modules Bind Phosphorylated Tyr, Ser, or Thr Residues in Partner Proteins
We have seen that the protein Grb2 in the insulin signaling pathway (Fig. 12–6) binds through its SH2 domain to other proteins that contain exposed P–Tyr residues. The human genome encodes at least 87 SH2 containing proteins, many already known to participate in signaling. The P–Tyr residue is bound in a deep pocket in an SH2 domain, with each of its phosphate oxy gens participating in hydrogen-bonding or electrostatic interactions; the positive charges on two Arg residues figure prominently in the binding. Subtle differences in the structure of SH2 domains in different proteins account for the specificities of their interactions with various P–Tyr-containing proteins. The three to five residues on the carboxyl-terminal side of the P–Tyr residue are critical in determining the specificity of in teractions with SH2 domains (Fig. 12–22). PTB domains (phosphotyrosine-binding domains) also bind P–Tyr in partner proteins, but their critical sequences and three-dimensional structures distinguish them from SH2 domains. The human genome encodes 24 proteins that contain PTB domains, including IRS-1, which we have already met in its role as a scaffold protein in insulin-signal transduction (Fig. 12–6). Many of the signaling protein kinases, including PKA, PKC, PKG, and members of the MAPK cascade, phosphorylate Ser or Thr residues in their target proteins, which in some cases acquire the ability to inter act with partner proteins through the phosphorylated residue, triggering a downstream process. An alphabet soup of domains that bind P–Ser or P–Thr residues has been identified, and more are sure to be found. Each domain favors a certain sequence around the phosphorylated residue, so the domains represent families of highly specific recognition sites, able to bind to a specific subset of phosphorylated proteins.
FIGURE 12–22 Structure of an SH2 domain and its interaction with a P–Tyr residue in a partner protein. (PDB ID 1SHC) The SH2 domain is shown as a gray surface contour representation. The phosphorus of the phosphate group in the interacting P–Tyr is visible as an orange sphere; most of the residue is obscured in this view. The next few residues toward the carboxyl end of the partner protein are shown in red. The SH2 domain interacts with P–Tyr (which, as the phosphorylated residue, is assigned the index position 0) and also with the next three residues toward the carboxyl terminus (designated 1, 2, 3). The residues important in the P–Tyr residue are conserved in all SH2 domains. Some SH2 domains (Src, Fyn, Hck, Nck) favor negatively charged residues in the 1 and 2 positions; others (PLC-1, SHP-2) have a long hydrophobic groove that selects for aliphatic residues in positions 1 to 5. These differences define subclasses of SH2 domains that have different partner specificities.
In some cases, the domain-binding partner is internal. Phosphorylation of some protein kinases inhibits their activity by favoring the interaction of an SH2 domain with a P–Tyr in another domain of the same en zyme. For example, the soluble protein Tyr kinase Src, when phosphorylated on a critical Tyr residue, is ren dered inactive as an SH2 domain needed to bind to the substrate protein instead binds to an internal P–Tyr (Fig. 12–23). Glycogen synthase kinase 3 (GSK3) is in active when phosphorylated on a Ser residue in its auto inhibitory domain (Fig. 12–23b). Dephosphorylation of that domain frees the enzyme to bind and phosphory late its target proteins. Similarly, the polar head group of the phospholipid PIP3, protruding from the inner leaf let of the plasma membrane, provides points of attach ment for proteins that contain SH3 and other domains.
FIGURE 12–24 Some binding modules of signaling proteins. Each protein is represented by a line (with the amino terminus to the left); symbols indicate the location of conserved binding domains (with specificities as listed in the key; PH denotes plextrin homology; other abbreviations explained in the text); green boxes indicate catalytic ac C2 tivities. The name of each protein is given at its carboxyl-terminal end. These signaling proteins interact with phosphorylated proteins or phospholipids in many permutations and combinations to form integrated signaling complexes.
Most of the proteins involved in signaling at the plasma membrane have one or more protein- or phospholipid-binding domains; many have three or more, and thus are multivalent in their interactions with other signaling proteins. Figure 12–24 shows a few of the many multivalent proteins known to participate in signaling. A remarkable picture of signaling pathways has emerged from studies of many signaling proteins and the multiple binding domains they contain (Fig. 12–25). An initial signal results in phosphorylation of the receptor or a target protein, triggering the assembly of large multiprotein complexes, held together on scaffolds made from adaptor proteins with multivalent binding capacities. Some of these complexes have several protein kinases that activate each other in turn, producing a cascade of phosphorylation and a great amplification of the initial signal. Animal cells also have phosphotyrosine phosphatases (PTP ases), which remove the phosphate from P–Tyr residues, reversing the effect of phosphorylation. Some of these phosphatases are receptorlike membrane proteins, presumably controlled by extracellular factors not yet identified; other PTP ases are soluble and contain SH2 domains. In addition, animal cells have protein phosphoserine and phosphothreonine phosphatases, which reverse the effects of Ser- and Thr specific protein kinases. We can see, then, that signaling occurs in protein circuits, effectively hard-wired from signal receptor to response effector and able to be switched off instantly by the hydrolysis of a single phosphate ester bond. The multivalency of signaling proteins allows for the assembly of many different combinations of signaling modules, each combination presumably suited to particular signals, cell types, and metabolic circumstances. The large variety of protein kinases and of phosphoprotein binding domains, each with its own specificity (the consensus sequence required in its substrate), provides for many permutations and combinations and many different signaling circuits of extraordinary complexity. And given the variety of specific phosphatases that reverse
FIGURE 12–25 Insulin-induced formation of supramolecular signaling complexes. The binding of insulin to its receptor sets off a series of events that lead eventually to the formation of membrane-associated complexes involving the 12 signaling proteins shown here, as well as others. Phosphorylation of Tyr residues in the insulin receptor initiates complex formation, and dephosphorylation of any of the phospho proteins breaks the circuit. Four general types of interaction hold the complex together: the binding of a protein to a second phosphoprotein through SH2 or PTB domains in the first (red); the binding of SH3 domains in the first with proline-rich domains in the second (orange); the binding of PH domains in one protein to the phospholipid PIP3 in the plasma membrane (blue); or the association of a protein (RAS) with the plasma membrane through a lipid covalently bound to the protein (yellow). Two proteins shown here are not described in the text: 14-3-3, which binds a P–Ser in Raf and mediates its interaction with MEK; and MP1, a scaffold protein that cements the links between Raf, MEK, and ERK.
the action of protein kinases, some under specific types of external control, a cell can quickly “disconnect” the entire protein circuitry of a signaling pathway. Together, these mechanisms confer a huge capacity for cellular regulation in response to signals of many types.
