5. The RasMAPK pathway

26.4 Protein tyrosine kinases induce phosphorylation cascades

Key terms defined in this section

Oncogenes are genes whose products have the ability to transform eukaryotic cells so that they grow in a manner analogous to tumor cells. Oncogenes carried by retroviruses have names of the form v-onc.

Growth factor receptors take their names from the nature of their ligands, which usually are small polypeptides (casually called growth factors, more properly called cytokines) that stimulate the growth of particular classes of cells. The factors have a variety of effects, including changes in the uptake of small molecules, initiation or stimulation of the cell cycle, and ultimately cell division. The ligands most usually are secreted from one cell to act upon the receptor of another cell. Examples of secreted cytokines are EGF (epidermal growth factor), PDGF (platelet-derived growth factor), and insulin. In some cases, ligands instead take the form of components of the extracellular matrix, or membrane proteins on the surface of another cell.

The receptors share a general characteristic structure: they are group I integral membrane proteins, spanning the membrane once, with an N-terminal protein domain on the extracellular side of the membrane, and the C-terminal domain on the cytoplasmic side. Some receptors, such as those for EGF or PDGF, consist of single polypeptide chains; others, such as the insulin receptor, are disulfide-bonded dimers (each dimer being a group I protein).

These receptors have a common mode of function. Their cytosolic domains have an enzymatic activity. They are protein kinases. There are many types of protein kinases, but they all have some features in common. The basic activity is the ability to add a phosphate group to an amino acid in a target protein. The phosphate is provided by hydrolyzing ATP to ADP. A protein kinase has an ATP-binding site and a catalytic center that can bind to the target amino acid (for review see Hunter and Cooper, 1985; Hunter, 1987).

Two groups of protein kinases are distinguished by their locations: the receptor protein kinases reside in the membrane; cytosolic protein kinases are free in the cytosol. Each group includes two major types of kinases, defined by the amino acids that they phosphorylate in the protein target:

• Protein tyrosine kinases are the predominant type of receptors with kinase activity, although there are also many cytosolic tyrosine kinases. More than 50 receptor tyrosine kinases are known.
  
• Protein serine/threonine kinases are the most common type of cytosolic kinases, and are responsible for the vast majority of phosphorylation events in the cell; there are some receptor kinases of the Ser/Thr type.
  
• A third type of kinase is found among the cytosolic enzymes: dual specificity kinases can phosphorylate target proteins on either tyrosine or serine/threonine.

There are phosphatases with specificity for the appropriate amino acids to match each type of kinase. Most phosphatases are cytosolic, although there are some receptor phosphatases. One way to terminate an activation event is for a phosphatase (typically a cytosolic phosphatase) to reverse the phosphorylation event caused by a receptor kinase (for review see Hunter, 1995).

The effector pathways that are activated by receptor tyrosine kinases (sometimes abbreviated to RTKs) fall into two groups:

Figure 26.12 Effectors for receptor tyrosine kinases include phospholipases and kinases that act on lipids to generate second messengers.

• An enzymatic activity is activated that leads to the production of a small molecule second messenger. The second messenger may be the immediate product of an enzyme that is activated directly by the receptor, or may be produced later in the pathway. Lipids are common second messengers in these pathways. The enzymes include phospholipases (which cleave lipids from larger substrates) and kinases that phosphorylate lipid substrates. Some common pathways are summarized in Figure 26.12. The second messengers that are released in each pathway act in the usual way to activate or inactivate target proteins.
  
• The effector pathway is a cascade that involves a series of interactions between macromolecular components. The most common components of such pathways are protein kinases; each kinase activates the next kinase in the pathway by phosphorylating it, and the ultimate kinases in the pathway typically act on proteins such as transcription factors that may have wide-ranging effects upon the cell phenotype.

The basic principle underlying the function of all types of effector pathway is that the signal is amplified as it passes from one component of the pathway to the next. When some components have multiple targets, the pathway branches, thus creating further diversity in the response to the original stimulus.

Receptor tyrosine kinases have some common features. The extracellular domain often has characteristic repeating motifs. It contains a ligand-binding site. The catalytic domain is large (~250 amino acids), and often occupies the bulk of the cytoplasmic region. Certain conserved features are characteristic of all kinase catalytic domains. Sometimes the catalytic domain is broken into two parts by an interruption of some other sequence (which may have an important function in selecting the substrate).

When a ligand binds to the extracellular domain of a growth factor receptor, the catalytic activity of the cytoplasmic domain is activated. Phosphorylation of tyrosine is identified as the key event by which the growth factor receptors function because mutants in the tyrosine kinase domain are biologically inactive, although they continue to be able to bind ligand.

A key question in the concept of how a signal is transduced across a membrane is how binding of the ligand to the extracellular domain activates the catalytic domain in the cytoplasm. The general principle is that a conformational change is induced that affects the overall organization of the receptor. An important factor in this interaction is that membrane proteins have a restricted ability to diffuse laterally (in contrast with the continuous motion of the lipids in the bilayer). This enables their state of aggregation to be controlled by external events.

Figure 26.13 The principle underlying signal transduction by a tyrosine kinase receptor is that ligand binding to the extracellular domain triggers dimerization; this causes a conformational change in the cytoplasmic domain that activates the tyrosine kinase catalytic activity.
Animated figure

Lateral movement plays a key role in transmitting information from one side of the membrane to the other. Figure 26.13 shows that binding of ligand induces a conformation change in the N-terminal region of a group I receptor that causes the extracellular domains to dimerize. This causes the transmembrane domains to diffuse laterally, bringing the cytoplasmic domains into juxtaposition. The stabilization of contacts between the C-terminal cytosolic domains causes a change in conformation that activates the kinase activity. In some cases, phosphorylation also causes the receptor to interact with proteins present on the cytoplasmic surface of a coated pit, leading to endocytosis of the receptor. An extreme case of lateral diffusion is seen in certain cases of receptor internalization, when receptors of a given type aggregate into a "cap" in response to an extracellular stimulus.

Figure 26.14 Binding of ligand to the extracellular domain can induce aggregation in several ways. The common feature is that this causes new contacts to form between the cytoplasmic domains.
Multiple figure

Figure 26.14 shows that the dimerization can take several forms. A ligand binds to one or to both monomers to induce them to dimerize, a dimeric ligand binds to two monomers to bring them together, or a ligand binds to a dimeric receptor (one stabilized by extracellular disulfide bridges) to cause an intramolecular change of conformation. A major consequence of this mechanism is to allow transmission of a conformational change from the extracellular domain to the cytoplasmic domain without requiring a change in the structure of the transmembrane region (Cunningham et al., 1991; for review see Heldin, 1995).

The key event that triggers the signaling pathway in the cytoplasm is activation of the kinase activity when dimerization causes an "activation loop" to move into a tethered conformation. This transition causes an autophosphorylation, in which the kinase activity of one subunit phosphorylates the other subunit in the dimer. It is necessary for both subunits to have kinase activity for the receptor to be activated; if one subunit is defective in kinase activity, the dimer cannot be activated.

Autophosphorylation has two consequences. First, phosphorylation within the kinase region increases the catalytic activity, and therefore comprises a positive feedback. Second, phosphorylation at tyrosine residues elsewhere in the cytoplasmic domain provides the means for passing the signal to the next component in the pathway. The existence of the phosphorylated tyrosine(s) causes the cytoplasmic domain to associate with its target proteins (for review see Ullrich and Schlessinger, 1990; van der Geer et al., 1994).

We can distinguish three types of proteins with which the activated receptor may interact:

• The protein may be a target that is activated by its association with the receptor, but which is not itself phosphorylated. If the target in turn causes activation of an enzyme, which is usually the case, the pathway continues through an amplification step. Targets may be adaptor molecules which themselves have no catalytic activity (example: Grb2; see below), or may be enzymes that are activated by binding to the receptor (example: PI3 kinase; see Figure 26.12).
  
• If the protein is a substrate for the enzyme, it becomes phosphorylated. If the substrate is itself an enzyme, it may be activated by the phosphorylation (example: c-Src or PLCγ ; see Figure 26.12). Sometimes the substrate is a kinase, and the pathway is continued by a cascade of kinases that successively activate one another.
  
• Some substrates may be end-targets, such as cytoskeletal proteins, whose phosphorylation changes their properties, and causes assembly of a new structure.

Two motifs found in a variety of cytoplasmic proteins that are involved in signal transduction are used to connect proteins to the components that are upstream and downstream of them in a signaling pathway. The domains are named SH2 and SH3, for Src homology, because they were originally described in the c-Src cytosolic tyrosine kinase (see 28 Oncogenes and cancer; for review see Koch, 1991; Cohen et al., 1995).

Figure 26.15 Several types of proteins involved in signaling have SH2 and SH3 domains.

Their presence in various proteins is summarized in Figure 26.15. The cytoplasmic tyrosine kinases comprise one group of proteins that have these domains; other prominent members are phospholipase Cγ and the regulatory subunit (p85) of PI3 kinase (both targets for activation by receptor tyrosine kinases; see Figure 26.12). The extreme example of a protein with these domains is Grb2/sem5, which consists solely of an SH2 domain flanked by two SH3 domains (see below).

Figure 26.16 Phosphorylation of tyrosine in an SH2-binding domain creates a binding site for a protein that has an SH2 domain.

The SH2 domain is a region of ~100 amino acids that interacts with a target site in other proteins. The target site is called an SH2-binding site. Figure 26.16 shows an example of a reaction in which SH2 domains are involved. Activation of a tyrosine kinase receptor causes autophosphorylation of a site in the cytosolic tail. Phosphorylation converts the site into an SH2-binding site. So a protein with a corresponding SH2 domain binds to the receptor only when the receptor is phosphorylated.

An SH2 domain specifically binds to a particular SH2-binding site. The specificity of each SH2 domain is different (except for a group of kinases related to Src, which seem to share the same specificity). The typical SH2-binding site is only 3 V5 amino acids long, consisting of a phosphotyrosine and the amino acids on its C-terminal side. SH2 binding is a high-affinity interaction, as much as 10³ tighter compared to a typical kinase-substrate binding reaction (Songyang et al., 1993).

Figure 26.17 Autophosphorylation of the cytosolic domain of the PDGF receptor creates SH2-binding sites for several proteins. Some sites can bind more than one type of SH2 domain. Some SH2-containing proteins can bind to more than one site. The kinase domain consists of two separated regions (shown in blue), and is activated by the phosphorylation site in it.

Some proteins contain multiple SH2 domains, which increases their affinity for binding to phosphoproteins or confers the ability to bind to different phosphoproteins. A receptor may contain different SH2-binding sites, enabling it to activate a variety of target proteins. Figure 26.17 summarizes the organization of the cytoplasmic domain of the PDGF receptor, which has ~10 distinct SH2-binding sites, each created by a different phosphorylation event. Different pathways may be triggered by the proteins that bind to the various phosphorylated residues (Fantl et al., 1992).

Figure 26.18 The crystal structure of an SH2 domain (purple strands) bound to a peptide containing phosphotyrosine shows that the P-Tyr (white) fits into the SH2 domain, and the 4 C-terminal amino acids in the peptide (backbone yellow, side chains green) also make contact. Photograph kindly provided by John Kuriyan.

The SH2 domain has a globular structure in which its N-terminal and C-terminal ends are close together, so that its structure is relatively independent of the rest of the protein. The phosphotyrosine binds to a pocket in the SH2 domain, as illustrated in Figure 26.18.

A protein that contains an SH2 domain is activated when it binds to an SH2-binding site. The reaction can take the form of activating enzymatic activities, typically kinases, phosphatases, and phospholipases. The activation may involve the SH2-containing protein directly (when it itself has enzymatic activity) or may be indirect. An example of a protein containing an SH2 domain that does not have a catalytic activity is provided by p85, the regulatory subunit of PI3 kinase; when p85 binds to a receptor, it is the associated PI3K catalytic subunit that is activated.

The SH3 domain provides the effector function by which some of the SH2-containing proteins bind to a downstream component. The case of the "adaptor" Grb2 strengthens this idea; consisting only of SH2 and SH3 domains, it uses the SH2 domain to contact the component upstream in the pathway, and the SH3 domain to contact the component downstream. SH3 binds the motif PXXP in a sequence-specific manner. It is the PXXP-containing target for Grb2 that is activated when Grb2 binds to the activated receptor (Booker et al., 1993).

A receptor tyrosine kinase can initiate a signaling cascade at the membrane. However, in many cases, the activation of the kinase is followed by its internalization, that is, it is removed from the membrane and transported to the interior of the cell by endocytosis of a vesicle carrying a patch of plasma membrane. The relationship between kinase activity and endocytosis is unclear. Phosphorylation at particular residues may be needed for endocytosis; whether the kinase activity as such is needed may differ for various receptors. It is possible that endocytosis of receptor kinases serves principally to clear receptor (and ligand) from the surface following the response to ligand binding (thus terminating the response). However, in some cases, movement of receptors to coated pits followed by internalization could be necessary for them to act on the target proteins.

Because growth factor receptors generate signals that lead to cell division, their activation in the wrong circumstances is potentially damaging to an organism, and can lead to uncontrolled growth of cells. Many of the growth factor receptor genes are represented in the oncogenes, a class of mutant genes active in cancers. The mutant genes are derived by changes in cellular genes; often the mutant protein is truncated in either or both of its N-terminal or C-terminal regions. The mutant protein usually displays two properties: the tyrosine kinase has been activated; and there is no longer any response to the usual ligand. As a result, the tyrosine kinase activity of the receptor is either increased or directed against new targets. The nature of these changes in generating tumorigenic phenotypes in cells is the subject of 28 Oncogenes and cancer.

Reviews

Cohen, G. B., Ren, R., and Baltimore, D. (1995). Molecular binding domains in signal transduction proteins. Cell 80, 237-248.

Heldin, C.-H. (1995). Dimerization of cell surface receptors in signal transduction. Cell 80, 213-223.

Hunter, T. (1987). A thousand and one protein kinases. Cell 50, 823-829.

Hunter, T. (1995). Protein kinases and phosphatases: the Yin and Yang of protein phosphorylation and signaling. Cell 80, 237-248.

Hunter, T. and Cooper, J. A. (1985). Protein-tyrosine kinases. Ann. Rev. Biochem 54, 897-930.

Koch, C. A. (1991). SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins. Science 252, 668-674.

Ullrich, A. and Schlessinger, J. (1990). Signal transduction by receptors with tyrosine kinase activity. Cell 61, 203-212.

van der Geer, P., Hunter, T., and Lindberg, R. A. (1994). Receptor protein-tyrosine kinases and their signal transduction pathways. Ann. Rev. Cell Biol. 10, 251-337.

Research

Booker, G. W. et al. (1993). Solution structure and ligand-binding site of the SH3 domain of the p85asubunit of phosphatidylinositol 3-kinase. Cell 73, 813-822.

Cunningham, B. C. et al. (1991). Dimerization of the extracellular domain of the human growth hormone receptor by a single hormone molecule. Science 254, 821-825.

Fantl, W. J. et al. (1992). Distinct phosphotyrosines on a growth factor receptor bind to specific molecules that mediate different signaling pathways. Cell 69, 413-423.

Songyang, Z. et al. (1993). SH2 domains recognize specific phosphopeptide sequences. Cell 72, 767-778.